Directed cell migration plays a principal role in various aspects of important cellular phenomena such as wound healing, development and cancer metastasis. Although the mechanism of gradient stimulus leading to directed cell migration is well understood and exploited, the geometrical and topographical cues that cause directed migration has been largely unexplored. With the advent of accessible microfabrication techniques to precisely control the topography of the extracellular matrix (ECM) on substrates, researchers are just starting to study the complex mechanical signals that can alter directed cell motility. A key challenge now is to parse out the precise factors that affect directional movement of cells on certain micropatterns, use that understanding to design strategies to enhance the motility and bias of directed cell migration, and further apply these concepts to multiple cell types and higher-order cell systems.
\r\n\r\nHere, we investigate the tunability of directional bias through various geometrical manipulations using quantitative analysis of cell movement on micropatterns. We observe that MCF-10A epithelial cells in general jump with an unnaturally high bias between teardrop-based islands with specific gap distance, asymmetry and positional placement. Throughout the studies, we observe that lamellipodial protrusions and unilamellar morphology play a crucial role in dictating not only the directional bias of epithelial cells, but also their speed and persistence, and find that moderate alteration of Rac1 signal leads to an unexpected flip of bias. We further extend the concept of directional bias to design patterns to successfully control cell flux and effectively partition cell population, as well as induce unilamellar morphology in different cell types to promote directed cell motility. We also investigate the combinatorial effect of hybrid micropatterns in enhancing motility and unravel the unique properties and possible mechanisms behind directed cell motility on teardrop-based micropatterns.
\r\n\r\nOur results demonstrate a new type of directed cell motility using a micropattern that involves the use of physical constraints to stabilize the unilamellar morphology and guidance of the unilamella in the correct direction through purely geometrical cues. These studies offer multiple design strategies to modulate the cell motility and directional bias on micropatterns for various applications, such as tissue engineering.
\r\n", "doi": "10.7907/61Y2-AC28", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5826", "collection": "thesis", "collection_id": "5826", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05232010-172249275", "primary_object_url": { "basename": "Kim_JH_complete_thesis.pdf", "content": "final", "filesize": 2980059, "license": "other", "mime_type": "application/pdf", "url": "/5826/8/Kim_JH_complete_thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "State Diagram for Contact-Inhibition of Proliferation: A Quantitative Framework for Modulating Growth Patterns in Epithelial Cell Clusters", "author": [ { "family_name": "Kim", "given_name": "Jin-Hong", "orcid": "0000-0002-6480-1929", "clpid": "Kim-Jin-Hong" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Cell-cell contacts play a key role in the assembly and integrity of epithelial tissues. Cell-cell contact is not only a mere physical link between neighboring cells, but also a critical regulator of many cell behaviors including proliferation. Contact-inhibition of proliferation is a hallmark of normal epithelial tissues. Cancer development involves the loss of this key constraint. Both biochemical and physical mechanisms mediating contact-inhibition are emerging. A current, principal challenge is elucidating how the integrated performance of these mechanisms enforce or modulate contact-inhibition in a rich microenvironment that includes multiple, potentially conflicting cues such as soluble growth factors (GFs) and extracellular matrix (ECM).
\r\n\r\nHere, we propose a quantitative paradigm for contact-inhibition of proliferation. Our quantitative analysis of single cells within multicellular aggregates reveals that epithelial cells transition from a contact-inhibited to contact-independent mode of proliferation at a critical threshold EGF level. This transition point is a tunable property and can be modulated by varying the level of cell-cell contact. Furthermore, the proximity to this transition point is a quantitative gauge of \u201cdegree\u201d of contact-inhibition. Using this metric, we demonstrate that stiffening the adhesive matrix, a widely observed phenomenon during cancer development, leads to the quantitative, progressive reduction in the EGF threshold needed to induce contact-independent proliferation. Thus, stiffening the ECM moves an epithelial cell system closer to the transition to contact-independence, thereby quantitatively reducing the amount of EGF amplification needed to induce population-wide proliferation. Our results reveal that the potent effect of substratum compliance on contact-inhibition involves changes in contact-maturation and multicellular mechanics. The proposed quantitative model of contact-inhibition provides fundamental insights into our understanding of tissue morphogenesis and cancer progression in multicellular organisms. Furthermore, our findings provide design principles for engineering multicellular growth in applications such as tissue engineering.
\r\n", "doi": "10.7907/JV13-YS19", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5929", "collection": "thesis", "collection_id": "5929", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072010-151443763", "primary_object_url": { "basename": "FONG_thesis.pdf", "content": "final", "filesize": 3908813, "license": "other", "mime_type": "application/pdf", "url": "/5929/21/FONG_thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Wound Healing on Artificial Extracellular Matrix Proteins", "author": [ { "family_name": "Fong", "given_name": "Eileen", "clpid": "Fong-Eileen" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Collective cell migration is a key process in tissue repair, and in drawing parallels from complex multi-cellular events such as tumor morphogenesis and embryogenesis. Mechanisms of wound healing have been studied extensively in vitro. Extracellular matrix (ECM) is required to support cell migration and ensure rapid coverage of the wound area. The main challenge in designing biomaterials for tissue repair is to provide cells with the appropriate biological and mechanical cues. Hence, understanding key cell-ECM interactions during wound healing is necessary for effective biomaterial design.
\r\n\r\nGenetic engineering provides a convenient avenue to customize materials for any given application. The artificial protein-based biomaterials discussed in this work were derived from fibronectin and elastin. These proteins have a modular design, and have material properties that can be fine-tuned according to specific applications. The artificial extracellular matrix (aECM) proteins prepared by previous members of our laboratory have been shown to promote attachment of endothelial cells. In this work, we studied extensively epithelial and fibroblast wound healing behavior on these aECM biomaterials.
\r\n\r\nCrosslinked aECM protein films of varying RGD densities have been prepared by mixing aECM proteins with the RGD cell binding domain with aECM proteins containing the scrambled RDG sequence. Corneal epithelial wound healing was observed on aECM films with 100% RGD but not on aECM films with 2.5% RGD. Surprisingly, we found a five fold difference between the wound closure rates between these surfaces, but individual cell speeds did not increase significantly. We proposed that the five fold increase in wound closure rate was determined by the rate of crossing the boundary between the wound area and the area underneath the cell sheet. Both simulation and experimental data verified that the rate of boundary-crossing was sufficient to account for five-fold difference in wound closure rates between 100% RGD and 2.5% RGD surfaces.
\r\n\r\nFull-length fibronectin domains have also been incorporated to improve the overall cell binding properties of the aECM proteins. The aECM proteins containing full-length fibronectin domains were shown to facilitate rapid spreading of Rat-1 fibroblasts. The aECM protein containing both fibronectin domains 9 and 10 exhibited an increased binding affinity to the \u03b15\u03b21 integrin. More importantly, these aECM proteins also promoted rapid wound closure, which was comparable to that on fibronectin. We showed that aECM proteins containing full-length fibronectin domains also promoted higher phosphorylated levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), consistent with the faster cell migration and proliferation observed.
\r\n\r\nTo try to understand how cells select wound healing mechanisms, wound healing of Madin-Darby Canine Kidney (MDCK) epithelial cells were examined in vitro. On surfaces containing the aECM protein bearing the fibronectin domain 10, characteristic healing patterns were observed in MDCK wound healing. These patterns are defined by the formation of leader cells at regular intervals of actomyosin purse strings. The spacing between consecutive leader cell groups was also found to be independent of the wound diameter. This spacing however, was found to decrease with increasing myosin II inhibition. These observations could be explained using a simple force transmission mechanical model. Consistent with the model predictions, we demonstrated that wounds with a zigzag geometry biased the selection of the wound healing mechanism along the wound edge. These zigzag wounds also healed nearly eight fold faster than wounds with straight edges.
\r\n", "doi": "10.7907/8VQJ-DS58", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5830", "collection": "thesis", "collection_id": "5830", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242010-135950298", "primary_object_url": { "basename": "Melissa's_THESIS.pdf", "content": "final", "filesize": 1341037, "license": "other", "mime_type": "", "url": "/5830/4/Melissa's_THESIS.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Dynamics of Multicellular Aggregation and Disaggregation: Implications for Tissue Engineering and Cancer Metastasis", "author": [ { "family_name": "Pope", "given_name": "Melissa Davis", "clpid": "Pope-Melissa-Davis" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Epithelial tissues play an important physiological role. Tightly cohesive epithelial sheets form protective barriers that line organs, and in addition, fold into a wide variety of complex 3D architectures with specialized functions. A key facet of tissue morphogenesis involves the aggregation of similar cells into cohesive groups. Here, we have analyzed the dynamics of aggregation using quantitative imaging techniques (Chapter II). We show that multicellular aggregation dynamics adhere to a transport-reaction model that is broadly appreciated for physicochemical systems. This model of aggregation dynamics differs from the classical equilibrium paradigm of cell aggregation based on differential adhesivity of cells to neighboring cells versus the underlying substratum. Our findings reveal a previously unrecognized role for cell motility during developmental aggregation processes and provide design principles for promoting cell aggregation dynamics in contexts such as tissue engineering that are distinct from the currently accepted paradigm.
\r\n\r\nMulticellular aggregation is reversible. In fact, the break-up of multicellular clusters (\u201ccell scatter\u201d) is not only important for developmental processes, but also contributes to metastasis. However, current molecular genetics studies of cell scatter are predominantly qualitative and do not provide a quantitative assessment of the relative strengths of molecular signals in inducing cell scatter. By developing and implementing an automated image processing algorithm, we quantify two aspects of cell scatter \u2013 the breakdown of cell-cell adhesions and the dispersion of detached cells \u2013 in mammary epithelial cells treated with different combinations of biochemical cues (Chapter III). We demonstrate that our metrics of cell scatter identify the effects of individual cues and detect synergies between them. We envision that this approach will be useful for mapping the relative potencies of regulators of cell scatter and may guide therapeutic strategies.
\r\n\r\nMulticellular processes such as aggregation and scatter involve molecular-level changes within cell-cell adhesions. To complement imaging-based strategies at the cellular and multicellular levels, we developed a quantitative microtiter assay for examining the expression of cell-cell adhesion proteins and associations between them (Chapter IV). Using two case studies related to cancer biology, we demonstrate that our assay provides a more detailed quantitative picture of molecular changes within epithelial adhesive structures, which can provide added insight into the regulation of morphogenetic events.
\r\n", "doi": "10.7907/3Z4S-5676", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:1336", "collection": "thesis", "collection_id": "1336", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04102009-170632", "primary_object_url": { "basename": "00_Maskarinec_thesis.pdf", "content": "final", "filesize": 27675925, "license": "other", "mime_type": "", "url": "/1336/1/00_Maskarinec_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Protein-Based Biomaterials with Biological and Mechanical Cues to Direct Cellular Behavior", "author": [ { "family_name": "Maskarinec", "given_name": "Stacey Ann", "clpid": "Maskarinec-Stacey-Ann" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Ravichandran", "given_name": "Guruswami", "clpid": "Ravichandran-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biomaterials play critical roles in tissue engineering, reconstructive surgery, and regenerative medicine. Constructing biomaterials through protein engineering offers a unique and powerful solution to the challenges posed by the creation of well-defined, multi-functional materials that must provide structural support, as well as guide cell and tissue behavior. This thesis describes the construction, characterization, and application of protein-based biomaterials composed of photoreactive artificial extracellular matrix (aECM) proteins. These proteins consist of mechanical and biological motifs derived from natural proteins: an elastin-like backbone containing a photocrosslinking site dictates the mechanical properties of the material, and an RGD binding domain from fibronectin mediates cell attachment. aECM proteins were assembled at the genetic level and expressed in a bacterial host. Manipulation of an aminoacyl-tRNA synthetase of the expression strain permitted the incorporation of the photoreactive noncanonical amino acid, para-azidophenylalanine (pN3Phe), within the elastin subunit of the protein.
\r\n\r\nAn evaluation of the mechanical and biological properties of photoreactive aECM proteins has been performed. Nanoindentation experiments using atomic force microscopy (AFM) demonstrated that the elastic moduli of photocrosslinked protein films lie within the range of native elastins and that the mechanical properties of films can be tuned by altering the incorporation of pN3Phe in the protein or adjusting the irradiation dosage. Cell spreading experiments showed that cell attachment to protein films is sequence-specific to the presence of the RGD cell-binding domain. Patterning of protein films using standard photolithography methods allowed for the generation of cellular arrays.
\r\n\r\nA molecular characterization of cellular response to engineered aECM proteins has been initiated using mRNA microarrays and the proteomic technique, BONCAT (bio-orthogonal noncanonical amino acid tagging). These methods provide information regarding changes in the cellular expression of mRNA transcripts and proteins in response to the biochemical composition of protein-based materials.
\r\n\r\nCell-mediated deformation of the extracellular matrix in all three spatial dimensions was tracked and quantified using a newly developed method that combines time-lapse laser scanning confocal imaging and digital volume correlation. Analysis of the displacement profiles of migrating cells illustrates that, in addition to in-plane (x,y) forces, cells also exert significant normal (z) forces. A new \"push-pull\" aspect of cell migration was detected in the normal displacement profiles of monitored cells suggesting that cells simultaneously \"push\" and \u201cpull\u201d the underlying matrix while exploring their microenvironment.
\r\n", "doi": "10.7907/751E-J376", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:689", "collection": "thesis", "collection_id": "689", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02202009-142419", "primary_object_url": { "basename": "CompleteDocument.pdf", "content": "final", "filesize": 6742559, "license": "other", "mime_type": "application/pdf", "url": "/689/6/CompleteDocument.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Quantitative Performance and Tradeoffs in the MAP Kinase Signaling Module", "author": [ { "family_name": "Chapman", "given_name": "Stephen Allen", "clpid": "Chapman-Stephen-Allen" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Intracellular signal transduction networks propagate and integrate the information that cells sense from environmental stimuli. The quantitative performance of signaling networks regulates cell decisions, and aberrations in network performance lead to pathologies such as cancer. The mitogen-activated protein (MAP) kinase cascade is a highly-conserved signaling module that regulates diverse cellular processes, such as proliferation, differentiation, and apoptosis in eukaryotic species ranging from yeast to human. While the principal components and mechanisms that define the MAP kinase module are well established, our understanding of and ability to tune its quantitative performance is limited. Here, we probe more deeply how the quantitative properties of the MAP kinase module may be affected by variations in the expression levels of the key constituents of the cascade\u2014kinases, phosphatases and scaffolds.
\r\n\r\nUsing a computational approach, we delineate how four quantitative properties\u2014responsiveness to input, dynamic range of output, signal amplification, and signal lifetime\u2014depend on the relative abundances of the two core components of the MAPK module, kinases and phosphatases. We identify a reduced metric termed the \u2018resistance to activation\u2019 that predicts the quantitative properties of the module across a wide range of parameter values. Its predictive utility extends to dynamic properties such as signal lifetime, which often dictates the MAP kinase\u2019s effect on cell function. Our analysis highlights tradeoffs in design, as not all quantitative attributes of the module can be simultaneously optimized. Thus, the resistance to activation captures the fundamental principles that determine cascade behavior and can be exploited to guide quantitative redesign of the MAP kinase module.
\r\n\r\nIn addition to the expression levels of kinases and phosphatases, scaffolds play a key role in signal propagation through the MAP kinase module. Protein scaffolds bring together multiple components of a signaling pathway, thereby promoting signal flux along a common physical \u201cbackbone.\u201d Scaffolds figure prominently in natural signaling pathways and are emerging as a promising platform for synthetic circuits. To better understand how scaffolding quantitatively affects signal transmission, we conducted an in vivo experimental sensitivity analysis of MAP kinase response to broad perturbations in the expression level of Ste5, an exemplar scaffold of the yeast mating pathway. Our results demonstrate that the expression level of Ste5 significantly affects several quantitative aspects of signal propagation, including signal throughput, pathway ultrasensitivity, and baseline leakage. These new insights into the quantitative role of scaffolding in MAP kinase signaling suggest advantages and limitations in designing synthetic scaffold-based regulatory networks.
\r\n", "doi": "10.7907/YVHW-Z764", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4335", "collection": "thesis", "collection_id": "4335", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10302008-210903", "primary_object_url": { "basename": "KH_THESIS.pdf", "content": "final", "filesize": 1462797, "license": "other", "mime_type": "application/pdf", "url": "/4335/8/KH_THESIS.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Metabolic Engineering of Saccharomyces cerevisiae for the Production of Bensylisoquinoline Alkaloids", "author": [ { "family_name": "Hawkins", "given_name": "Kristy Michelle", "clpid": "Hawkins-Kristy-Michelle" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The engineering of synthetic metabolic pathways in microbial hosts holds much promise for the synthesis of new chemicals and materials, including a variety of natural and non-natural products. The benzylisoquinoline alkaloids (BIAs) represent a large and structurally diverse class of plant secondary metabolites that exhibit a broad range of pharmacological activities. The reconstitution of a BIA biosynthetic pathway in an engineered microbial host offers several advantages over isolation from plants, including the targeted production of key intermediate molecules, rapid biomass accumulation, ease of purification, and the availability of genetic tools for strain engineering and pathway optimization.
\r\n\r\nHere we describe the development of a synthetic BIA pathway in an engineered yeast host which incorporates heterologous enzymes from a variety of organisms. The BIA backbone is derived from two molecules of tyrosine and is assembled through a heterologous pathway comprising enzymatic activities from plants, bacteria, and humans. Simultaneous efforts have focused on the downstream portion of this pathway to convert a commercially available substrate to the major branch point intermediate reticuline. By synthesizing both stereoisomers of reticuline from a racemic substrate, we have demonstrated production of BIA metabolites along the diversified sanguinarine/berberine and morphinan branches. Further optimization, scale-up, and a combination of bioconversions and chemical synthesis will potentially revolutionize drug discovery and manufacturing of these compounds.
\r\n", "doi": "10.7907/BQF7-QZ35", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5264", "collection": "thesis", "collection_id": "5264", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05212009-144705", "primary_object_url": { "basename": "08-CLB-thesis.pdf", "content": "final", "filesize": 3984809, "license": "other", "mime_type": "", "url": "/5264/8/08-CLB-thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Engineering Ligand Control of RNA Interference", "author": [ { "family_name": "Beisel", "given_name": "Chase Lawrence", "orcid": "0000-0003-0650-9943", "clpid": "Beisel-Chase-Lawrence" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "RNA is a rich and versatile substrate for the construction of information processing devices. These devices detect the levels of specified intracellular biomolecules and control cellular behavior accordingly. With few superficial constraints on the identity of the recognized biomolecule or the targeted gene, RNA-based information processing devices can be rapidly implemented toward various applications in medicine and biotechnology. To advance the design and implementation of RNA-based information processing devices, we delineated general design principles and applied these principles to the construction of devices that operate through RNA interference (RNAi).
\r\n\r\nRNAi represents an endogenous enzymatic pathway present in humans and other eukaryotes that mediates targeted gene silencing. The pathway has garnered recent interest as a revolutionary biological research tool and as a targeted therapeutic strategy. While RNAi has left an indelible mark on the scientific community, exerting greater control would advance the applicability and safety of this already impressive gene silencing mechanism. Toward this goal, we engineered ligand control of three types of RNAi effectors in mammalian cells: small interfering (si)RNAs, small hairpin (sh)RNAs, and microRNAs (miRNAs). Engineering frameworks enabled facile replacement of the biomolecule sensory and gene targeting domains, thus lending to rapid implementation as biosensors or autonomous control devices. Experimental and computational characterization studies provided a comprehensive understanding of device behavior, thereby facilitating forward design.
\r\n\r\nNaturally-occurring analogs of RNA-based information processing devices are riboswitches. Riboswitches predominantly mediate dynamic feedback in metabolism and share many traits with current examples of engineered information processing devices. Various experimental characterization studies of riboswitches showed that kinetics underlying events such as conformational switching and ligand binding have a substantial impact on device performance, although these factors remain to be comprehensively evaluated or considered when formulating design principles for synthetic riboswitch construction. We explored the contribution of kinetic factors to riboswitch performance in silico, where model predictions matched experimental observations, including results from our ligand-responsive RNAi effectors. From our modeling results, we developed a general set of design principles that guide riboswitch assembly and performance tuning.
", "doi": "10.7907/CBRX-PG36", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1880", "collection": "thesis", "collection_id": "1880", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05192008-134942", "primary_object_url": { "basename": "00_FrontStuff.pdf", "content": "final", "filesize": 47525, "license": "other", "mime_type": "", "url": "/1880/1/00_FrontStuff.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Hydrophilic Polymers in Gels and Solutions: Surface Properties and Structure", "author": [ { "family_name": "Mackel", "given_name": "Michael John", "clpid": "Mackel-Michael-John" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Brady", "given_name": "John F.", "clpid": "Brady-J-F" }, { "family_name": "Flagan", "given_name": "Richard C.", "clpid": "Flagan-R-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Changes in the wetting properties of thin films of polyethyleneglycol (PEG) end-capped with fluoroalkyl groups are measured when the films are equilibrated at various relative humidity (RH). At high RH, the advancing contact angle on the surfaces is 20\u00ba higher than the advancing contact angle measured at low RH. The surprising transition to non-wetting character at high RH is attributed to fluoroalkyl groups ordering at the air-hydrogel interface when they are liberated by dissolution of PEG crystallites above 85% RH.
\r\n\r\nNext, the structure and tribology of a semi-interpenetrating hydrogel of agarose and an anionic polysaccharide (either hyaluronic acid or dextran sulfate) were studied. The porous structure of agarose allows incorporation of up to 2% dextran sulfate without weakening the gel\u2019s mechanical properties. Addition of the polyelectrolyte endows the gels with shape memory upon drying and reswelling; the gel can be dehydrated and rapidly swollen back to its original dimensions. The addition of both dextran sulfate and hyaluronic acid (HA) increases the lubricity of agarose when tested against hydrophilic clean glass or hydrophobic fluorinated glass. Migration of the polyelectrolytes out of the gel is believed to make the gels self-lubricating.
\r\n\t\r\nFinally, hydrophilic polymers were functionalized with photoresponsive hyrophobes in an attempt to control polymer self-assembly with light. Micelles of PEG end-capped with azobenzene molecules showed no change in size when the solutions were irradiated with UV light. PEG block copolymers were also functionalized with azobenzene and used to modify gold surfaces, but photoresponsive contact angles could not be measured. Finally, acrylamides copolymerized with the vinyl derivatives of malachite green were also studied, but the copolymers proved unsuitable for use at moderate pH.
\r\n", "doi": "10.7907/5ZVF-6156", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2212", "collection": "thesis", "collection_id": "2212", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282008-142750", "primary_object_url": { "basename": "Entire_Dissertation.pdf", "content": "final", "filesize": 2660266, "license": "other", "mime_type": "", "url": "/2212/7/Entire_Dissertation.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineering RNA Devices for Gene Regulation, Biosensing, and Higher-Order Cellular Information Processing", "author": [ { "family_name": "Win", "given_name": "Maung Nyan", "clpid": "Win-Maung-Nyan" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The proper regulation of gene expression is critical to many biological processes occurring in the cell. It is becoming increasingly apparent that post-transcriptional processing pathways play significant roles in regulating the expression of various genes in both prokaryotic and eukaryotic organisms, where they direct a variety of complex cellular functions. A striking example of a biological communication and control system directing sophisticated gene expression regulation through precise molecular recognition is the class of RNA regulatory elements, called riboswitches, comprised of distinct sensor (ligand-binding) and actuator (gene-regulatory) functions that control gene expression in response to changing levels of specific target ligand concentrations.
\r\n\r\nInspired by these natural examples, numerous synthetic riboswitch systems have been developed and have made profound contribution to the field of riboswitch engineering. However, these early examples of synthetic riboswitches pose one or more challenges, such as portability of the switch design across different cellular systems and modularity and programmability of the components comprising the switch molecule. Therefore, we set out to develop a modular and extensible RNA-based gene-regulatory platform that will provide a framework for the reliable design and construction of gene regulatory systems that can control the expression of specific target genes in response to effector molecules of interest. The platform is called the \u201cribozyme switch\u201d and composed of distinct functional components, which are modularly coupled and functionally independent of each other. Through this platform, ribozyme switch devices that enable up- or down-regulation of target gene expression were developed. Design modularity and response programmability of the switch platform were also demonstrated. We also exhibited the versatility of the platform in implementing application-specific control systems for small molecule-mediated regulation of cell growth and non-invasive in vivo sensing of metabolite production.
\r\n\r\nThrough the ribozyme switch platform, we further constructed higher-order RNA devices that enable complex cellular information processing operations, including logic control (AND, NOR, and NAND gates), advanced computation (bandpass filter and signal shift in the output swing), and cooperativity (signal gain). Finally, we extended the small ribozyme switch platform responsive to small molecules to a different class of ligand molecules, proteins, by developing protein-responsive gene regulators and cellular biosensors. In addition to engineering RNA devices for programming cellular function, we also developed a high-throughput method for functional characterization of small molecule-binding RNA aptamers, which enables robust, accurate, and rapid characterization of such RNA aptamers. This method can be very useful as we (and others) develop RNA aptamers for small molecules of specific interest, which can be subsequently integrated into the ribozyme switch platform as sensing elements for specific applications. Together, these research developments hold synergistic values for the reliable construction of \u2018designer\u2019 gene-regulatory systems for various biotechnological and medical applications.
", "doi": "10.7907/H6J4-P729", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4377", "collection": "thesis", "collection_id": "4377", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11022007-232906", "primary_object_url": { "basename": "complete_thesis.pdf", "content": "final", "filesize": 1946299, "license": "other", "mime_type": "application/pdf", "url": "/4377/15/complete_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Interplay Between Long-Range And Short-Range Interactions In Polymer Self-Assembly And Cell Adhesion", "author": [ { "family_name": "Zhang", "given_name": "Cheng-Zhong", "orcid": "0000-0001-8825-7158", "clpid": "Zhang-Cheng-Zhong" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Interplay between long-range and short-range interactions is a common theme in soft and biological matter, which results in complicated self-assembly behaviors. We study two examples of this interplay: reversible gelation of associating polymers and ligand-receptor interactions in membrane adhesion. In associating polymer solutions, the competition between the conformation flexibility of polymer chains and the enthalpic monomer interactions results in phase-separated micro-structures at the mesoscopic scale; both gelation and the microphase order-disorder transition are manifestations of this self-assembly. We further establish that reversible gelation is similar to the glass transition: both are characterized by ergodicity breaking, aperiodic micro-structures, and non-equilibrium relaxations over a finite temperature range. In the study of ligand-receptor interactions between surfaces, we emphasize the interplay between specific ligand-receptor binding, and generic physical interactions. We find that both the finite spatial extension of receptors and their mobilities affect their binding affinity. As a special case of the interplay between receptor binding and generic interactions, we study the dynamics of membrane adhesion that is mediated by receptor binding but fulfilled through membrane deformations. We calculate the energy barrier of the adhesion as a result of membrane bending deformations and the double-well adhesion potential, and analyze the different scenarios according to the shape of the adhesion potential by scaling arguments.\r\n", "doi": "10.7907/GCH8-4A59", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2483", "collection": "thesis", "collection_id": "2483", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062008-103127", "primary_object_url": { "basename": "CACThesis080606c.pdf", "content": "final", "filesize": 7455624, "license": "other", "mime_type": "application/pdf", "url": "/2483/1/CACThesis080606c.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Self-Assembled Monolayers for the Study of Biological Targets ", "author": [ { "family_name": "Canaria", "given_name": "Christie Anne", "clpid": "Canaria-Christie-Anne" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Lansford", "given_name": "Rusty", "clpid": "Lansford-R" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Understanding the interactions of biological molecules with solid supports is vital for the development of detection systems and assay platforms. These relationships are frequently quite complex, involving hydrophobic interactions, electrostatic interactions, van der Waals forces, and covalent chemical bonds. We can exploit these interactions in a solid support device by modifying the surface substrate with thin films and monolayers. Self-assembled monolayers (SAMs) are powerful tools for functionalizing and imparting chemical character to surfaces. In this thesis, alkylthiol reagents are utilized to build SAMs on gold (Au) substrates. This work characterizes and studies monolayer formation. In addition, I use SAMs to generate surfaces specific for binding proteins, DNA, and cells. The popular biotin-streptavidin motif is used to demonstrate protein binding, as well as characterize monolayer composition as a result of solvent effects. Novel reagent syntheses are presented for both biotinylated alkylthiols and triethylene-glycol alkylthiols. Together, these two reagents generate substrates which bind specific proteins, while repelling non-specific ones. An additional reagent, \u201cDMT-coated controlled porous glass (CPG),\u201d was designed and synthesized for the generation of custom sequence oligonucleotides. Phosphoramidite syntheses using this modified CPG yield oligos with a 3\u2019 alkylthiol modification. SAMs generated with this reagent demonstrate specific binding of complement strands. Both electrochemical techniques and restriction enzymes (where appropriate) provide methods for releasing monolayer-bound species. Lastly, I employ SAMs to generate substrates amenable to cell capture and cell adhesion. Binding B- and T-cell lymphocytes is achieved, demonstrating SAM-coated Au as a substrate for cell panning. Chemokine vascular endothelial growth factor (VEGF) is also bound to SAMs, generating surfaces amenable to cell adhesion and motility. Cells plated on higher surface concentrations of VEGF migrate faster, and I show the effect is specific to cells with VEGF receptors. Overall, this thesis explores the formation and utilization of SAMs for capturing and studying biological targets. The findings here may be transferred in the future into bio-sensing devices and arrays.", "doi": "10.7907/39DF-7V92", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5244", "collection": "thesis", "collection_id": "5244", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08022007-141737", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 4139235, "license": "other", "mime_type": "application/pdf", "url": "/5244/1/Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Quantitative Insights into Developmental Signals and Phenotypes in C. elegans", "author": [ { "family_name": "Giurumescu", "given_name": "Claudiu Adrian", "clpid": "Giurumescu-Claudiu-Adrian" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Design of biomaterials and cellular scaffolds for tissue-engineering applications and regenerative medicine requires a precise understanding of the principles underlying multicellular patterning. Adhesion, migration, division, differentiation, and apoptosis are characteristic cellular behaviors, the engineering of which has the potential to allow creation of custom, multicellular structures. These cellular events occur naturally during embryonic and postembryonic development of multicellular organisms. Development thus offers the opportunity to learn about the design principles and molecular mechanisms that guide cellular patterning.
\r\n\r\nA key finding in developmental biology is that a limited set of conserved molecular signaling pathways act at multiple times and locations throughout the embryo to introduce cell-fate asymmetries in homogenous populations of cells. In turn, these asymmetries serve as starting points for the patterning of new organs. These signaling pathways interact quantitatively at multiple levels, including signaling cues, post-translational regulation, and gene-regulatory networks, to guide multicellular patterning.
\r\n\r\nHow does the quantitative performance of these signaling networks ensure the intended phenotype pattern? How do changes in the quantitative performance of these networks, possibly over the course of evolution, give rise to new phenotypes? These are the central questions pursued in this thesis.
\r\n\r\nIn order to answer such questions, we used vulva formation in the nematode Caenorhabditis elegans as a model system of cellular patterning. We formulated a mathematical model of the molecular network underlying cellular-fate specification in this system. Computational analysis of this molecular network reveals that cell\u2013cell coupling through lateral LIN-12/Notch signaling amplifies the perception of the gradient in the epidermal-growth-factor-like soluble cue, LIN-3. Thus, the gradient in LIN-3 concentration produces an even steeper difference in LIN-3-mediated intracellular signals between adjoining cells. Such gradient amplification may be particularly important in converting a shallow, graded-specification signal into a spatial pattern of distinct fate choices.
\r\n\r\nThrough quantitative perturbations of interaction strengths between components of the vulval patterning network, we further show that our modeling approach can correctly predict phenotype patterns observed in C. elegans mutation studies. This study generated a framework for quantitative analysis of molecular networks that links quantitative molecular perturbations to patterning outcomes. This framework will prove useful in the analysis of other systems involving cellular fate decisions and in tissue engineering applications where the generation of precise cell patterns is needed. We demonstrate the generality of our approach through an application to evolutionary developmental biology. Since molecular connectivity of the vulva patterning network of several closely related Caenorhabditis species is preserved, we correctly predict the quantitative diversification that must have occurred in this network during species evolution.
", "doi": "10.7907/FVD0-R331", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:298", "collection": "thesis", "collection_id": "298", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01232007-144117", "primary_object_url": { "basename": "Zhang_Kechun_2007.pdf", "content": "final", "filesize": 2960289, "license": "other", "mime_type": "application/pdf", "url": "/298/11/Zhang_Kechun_2007.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineering Protein-Based Materials Through Coiled-Coil Motifs", "author": [ { "family_name": "Zhang", "given_name": "Kechun", "orcid": "0000-0002-7139-2799", "clpid": "Zhang-Kechun" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Natural biomaterials are highly organized from the molecular to the macroscopic level in a hierarchical manner, requiring synthetic technologies to achieve this level of complexity. A biosynthetic approach to material design has emerged as an attractive option. In particular, proteins represent a promising class of molecules for creating new materials due to their determined sequence and structure. The research described in this thesis focuses on engineering protein-based materials using coiled-coil motifs. The coiled coil is a common protein architecture consisting of two or more \u03b1-helices wrapped around one another to form a supercoil. Despite its simple conformation, the coiled-coil motif plays diverse roles in biological systems functioning as sensors, recognition elements, scaffolds, levers, rotating arms and springs.
\r\n\r\nFirst, a designed parallel heterodimeric leucine zipper pair was used as the protein capture domain to construct an artificial polypeptide scaffold for surface functionalization. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated. This protein polymer was spin-coated and photocrosslinked to octyltrichlorosilane-treated surfaces. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer. Furthermore, in conjunction with microfluidic chips that were specifically designed for on-chip mixing using laminar flow, gradients of leucine zipper tagged proteins were formed in the microchannels and immobilized on the engineered protein films. This provides a general technique for producing surface-bound multicomponent gradients. The adhesion of human umbilical vein endothelial cells cultured on a surface-bound gradient of cell binding ligands generated by this technique was examined. In addition, to generate protein walkers that have different lateral mobility rates on a surface, several variants of the leucine zipper pair with tunable heterodimerization affinities were designed and synthesized to allow diversity in the association strength of proteins linked to a surface.
\r\n\r\nThe coiled-coil motif was also used to construct protein hydrogels. Hydrogels formed from a triblock artificial protein bearing dissimilar helical coiled-coil end domains (P and A) erode more than one hundred fold slower than hydrogels formed from those bearing the same end domains (either P or A). The reduced erosion rate is a consequence of the fact that looped chains are suppressed because P and A tend not to associate with each other. Thus, by harnessing selective molecular recognition, discrete aggregation number and orientational discrimination of coiled-coil protein domains, the erosion rate of hydrogels can be tuned over several orders of magnitude.
\r\n\r\nFinally, a biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds using the heterodimeric leucine zipper pair. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.
", "doi": "10.7907/324A-2K76", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1477", "collection": "thesis", "collection_id": "1477", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04232007-111928", "primary_object_url": { "basename": "N_Graham_Complete_Thesis.pdf", "content": "final", "filesize": 12852475, "license": "other", "mime_type": "application/pdf", "url": "/1477/8/N_Graham_Complete_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Crosstalk Between Soluble Factors and Cell-Cell Interactions: Implications for Cell Cycle Control and Tumor Development", "author": [ { "family_name": "Graham", "given_name": "Nicholas Alexander", "orcid": "0000-0002-6811-1941", "clpid": "Graham-Nicholas-Alexander" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Precise and dynamic control of cell behaviors, including proliferation, adhesion, and migration, is required for proper tissue organization and homeostasis. A key element to understanding how these cellular functions are controlled lies in uncovering the topology of the molecular signaling networks that couple environmental signals to cellular responses. In this study, we have parsed the signaling networks involved in cell cycle regulation and tumor development and uncovered novel mechanisms of crosstalk between soluble factors and cell-cell interactions.
\r\n\r\nOur findings demonstrate that extracellular cues, including the epidermal growth factor (EGF), stimulate proliferative signaling through beta-catenin, an intracellular protein that participates in both cell adhesion at the plasma membrane and transcription of cell cycle genes in the nucleus. In fact, EGF-mediated beta-catenin transcriptional activity is an essential signal for proliferation of normal epithelial cells. Additionally, in a cancer cell system, we discover that EGF cooperates with Wnt 3a, a classical agonist of beta-catenin transcriptional activity, to induce greater signaling than either ligand alone. Notably, EGF and Wnt 3a activate transcription using different sub-cellular pools of beta-catenin. Because hyperactive beta-catenin signaling drives proliferation in cancer, this suggests that attenuation beta-catenin signaling may require different therapeutic strategies for EGF- and Wnt-driven tumors.
\r\n\r\nSince beta-catenin signaling can be antagonized by binding to the cell-cell contact protein E-cadherin at the plasma membrane, proliferative signals mediated by beta-catenin may regulate growth suppression at high density, a property of normal cells that is often lost during tumorigenesis. Indeed, in non-tumorigenic epithelial cells, we demonstrate that E-cadherin is upregulated in contexts where beta-catenin signaling and DNA synthesis are suppressed. Additionally, exogenous E-cadherin suppresses proliferation with a strict requirement for beta-catenin binding. Future studies to test the hypothesis that E-cadherin regulates the growth of normal cells will benefit from a quantitative assay developed to measure E-cadherin:beta-catenin complexes. Such quantitative measurements are likely to be important because contact-mediated growth suppression by E-cadherin is coupled with a density-dependent, ligand-depletion mechanism that concomitantly regulates proliferation.
\r\n\r\nFinally, we demonstrate that EGF and other soluble factors synergistically control cell-cell interactions governing organization of normal epithelial cells into multicellular structures. Notably, this behavior resembles the program initiated during metastatic cancer, thus illustrating the flexibility of the epithelial phenotype even in non-cancerous cells. Together, these studies illustrate how the topology of molecular signaling networks can couple environmental cues including soluble extracellular factors and cell-cell interactions to regulate fundamental cellular functions.
", "doi": "10.7907/DJ7K-MW95", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1456", "collection": "thesis", "collection_id": "1456", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04222007-221859", "primary_object_url": { "basename": "FinalThesis_2-6-07.pdf", "content": "final", "filesize": 957089, "license": "other", "mime_type": "application/pdf", "url": "/1456/1/FinalThesis_2-6-07.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Signaling in Context: Parsing the Adhesion-Dependence of Growth Factor Signaling", "author": [ { "family_name": "Galownia", "given_name": "Niki Chiyomi", "clpid": "Galownia-Niki-Chiyomi" } ], "thesis_advisor": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" } ], "thesis_committee": [ { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Intracellular signaling induced by adhesion and soluble growth factors is a significant contributor to cellular function. The serine/threonine kinase, extracellular signal-regulated kinase (Erk), is a prominent point of signaling crosstalk between adhesion and growth factors. Despite extensive effort, the effect of individual growth factors on adhesion-dependent Erk signaling remains unclear due to considerable protocol differences and qualitative analyses. To address these issues, we developed an experimental technique to compare systematically the crosstalk between adhesion and individual growth factors and a quantitative protocol for measuring the magnitude and dynamics of Erk signaling.
\r\n\r\nUsing these methods, we demonstrate that: (1) Adhesion to fibronectin desensitizes Erk activation for cells stimulated by either PDGF (platelet-derived growth factor) or bFGF (basic fibroblast growth factor), but not by EGF (epidermal growth factor); (2) EGF, but not PDGF or bFGF, induces adhesion-dependent Erk activation enhancement; and (3) for adherent cells, either EGF or PDGF stimulation generates transient Erk activation, while bFGF stimulation mediates sustained Erk activation. This data reveal that there are significant differences in the adhesion-dependence of growth factor signaling. The most striking observation was that adhesion desensitizes cells to low doses of specific growth factors (PDGF and bFGF). Studies conducted to uncover the underlying mechanism(s) revealed that adhesion-mediated desensitization of Erk activation exhibits rapid kinetics and occurs at or above the level of Ras activation, but does not involve Sos hyperphosphorylation.
\r\n\r\nTo further probe the mechanisms responsible for generating different Erk signaling dynamics, we constructed a simple coarse-grain model of Erk activation and deactivation pathways. These pathways are represented by four distinct motifs: activation, constitutive direct-deactivation, feedback-mediated direct-deactivation, and feedback-decoupling. Our model predicts that transient and sustained Erk signaling dynamics require specific combinations of the four signaling motifs. These predictions suggest that differences in the Erk activation motif are most likely responsible for the experimentally observed characteristics of adhesion-dependent EGF-mediated Erk signaling. Furthermore, the model indicates either feedback-decoupling deactivation or direct-deactivation as the mechanisms responsible for the observed transient-versus-sustained signaling dynamics induced by different growth factors.
", "doi": "10.7907/Q5RF-8K30", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1848", "collection": "thesis", "collection_id": "1848", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05172007-150701", "primary_object_url": { "basename": "Bartlett_Thesis.pdf", "content": "final", "filesize": 14914066, "license": "other", "mime_type": "application/pdf", "url": "/1848/13/Bartlett_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "An Engineering Approach to Cancer Therapy Using Systemically Delivered siRNA", "author": [ { "family_name": "Bartlett", "given_name": "Derek William", "clpid": "Bartlett-Derek-William" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Rossi", "given_name": "John J.", "clpid": "Rossi-J-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The next generation of cancer therapeutics will specifically target processes responsible for the growth and survival of cancer cells. Among the most promising of these molecularly targeted therapeutics are small interfering RNAs (siRNAs). These siRNAs serve as the effectors of RNA interference, a naturally occurring and highly specific mechanism for regulating gene expression through sequence-specific degradation of messenger RNA. While these siRNAs have shown potential in vitro and in preclinical animal models, safe and effective systemic delivery remains one of the greatest challenges hindering their clinical application. This thesis describes an engineering approach to address the challenge of systemic delivery of siRNAs for cancer therapy.
\r\n\r\nAnalysis of the kinetics of siRNA-mediated gene silencing reveals that gene inhibition by unmodified siRNAs can last for one week in rapidly dividing cells and up to one month in cells with minimal division. Additionally, chemical modifications to enhance siRNA nuclease stability do not prolong intracellular siRNA activity. These data, when used in combination with results from a mathematical model of siRNA function, demonstrate that dilution from cell division, and not intracellular nuclease stability, is the dominant factor governing the duration of gene inhibition by siRNAs.
\r\n\r\nCyclodextrin-containing polycations (CDP) can self-assemble with siRNAs to form nanoparticles with desirable properties for systemic application. Characterization of these nanoparticles demonstrates that they can contain several thousand siRNAs, protect the siRNA payload from nuclease degradation, and be modified with transferrin targeting ligands that show multivalent binding to cell surface receptors.
\r\n\r\nMultimodality in vivo imaging with positron emission tomography (PET) and bioluminescent imaging (BLI) is used to monitor the biodistribution and function of the siRNA nanoparticles after intravenous administration in live mice. Attachment of targeting ligands to the surface of the nanoparticles enhances gene inhibition within the tumor, although the biodistribution and tumor localization are not dependent on the amount of targeting ligand. The targeting ligand likely serves to augment nanoparticle uptake by the tumor cells. When the siRNA nanoparticles are used to deliver therapeutic siRNAs to achieve tumor growth inhibition in disseminated and subcutaneous murine cancer models, schedule-dependent anti-tumor effects are observed.
", "doi": "10.7907/TS5S-FD74", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3599", "collection": "thesis", "collection_id": "3599", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09172008-144626", "primary_object_url": { "basename": "Son_s_2007.pdf", "content": "final", "filesize": 7737840, "license": "other", "mime_type": "application/pdf", "url": "/3599/1/Son_s_2007.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Biosynthetic Approaches to Protein Engineering Using Fluorinated Amino Acids", "author": [ { "family_name": "Son", "given_name": "Soojin", "clpid": "Son-Soojin" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Non-canonical amino acids provide a new set of building blocks and a potential route to new chemistries that extend beyond those achieved through the 20 common amino acids. Among various methods developed for this purpose, residue-specific incorporation in vivo has the potential to economically engineer proteins of various sizes with altered physical and chemical behavior. The work presented in this thesis explored the incorporation of fluorinated aliphatic residues and their effect on protein structure, stability, and function.
\r\n\r\nIn Chapter 2, two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and 4,4,4-trifluorovaline (4TFV), were incorporated in vivo into mutant GCN4 proteins. Both 5TFI and 4TFV showed replacement levels higher than 88%, as indicated by quantitative amino acid and MALDI-MS analysis. The incorporation of 5TFI into a-positions of the coiled-coil protein raised the thermal denaturation temperature (Tm) by 27\u00b0C from that of its isoleucine counterpart. However, when valines were replaced by 4TFV in the same positions, Tm only increased by 4\u00b0C. Similar trends were observed in response to chemical denaturation by guanidine hydrochloride; \u0394\u0394Gfolding upon incorporation of 5TFI and 4TFV was -2.1 and -0.3 kcal/mol, respectively. Secondary and higher order structures as well as biological activity were retained in the presence of both 5TFI and 4TFV. These results indicate that, even when introduced into the same positions within the protein, the effect of fluorination differs depending on which amino acid is fluorinated.
\r\n\r\nIn Chapter 3, the stereochemical effects of 5,5,5-trifluoroleucine (5TFL) were studied using (2S, 4R)-5',5',5'-trifluoroleucine and (2S, 4S)-5',5',5'-trifluoroleucine. The results from in vitro activation assays correlated well with efficiency of their incorporation in vivo. The (2S, 4S) isomer, whose kcat/Km was 100-fold lower than that of leucine, was incorporated at high levels, with 91% replacement of the encoded leucine residues in a de novo engineered coiled-coil protein A1. The (2S, 4R) isomer exhibited 9- fold lower kcat/Km than the (2S, 45) isomer, resulting in a slightly lower level of incorporation, 80%. The secondary structure of A1 was undisturbed upon the incorporation of either isomer and their impact on thermostability was similar, with an increase of 11\u00b0C in Tm as compared to that of A1 containing leucine. However, the equimolar mixture of A1 containing (2S, 45)-TFL and A1 containing (2S, 4R)-TFL displayed a further increase in Tm of 3\u00b0C. Although this further enhancement in thermostability was modest, it may be attributed to the ability of the coils to pack more compactly into dimers due to the stereochemical differences.
\r\n\r\nIn Chapter 4, laboratory evolution was utilized to recover the catalytic activity of chloramphenicol acetyltransferase (CAT) after replacement of isoleucine by 5TFI. Upon global incorporation of 5TFI into CAT, the catalytic efficiency, k cat/Km, was reduced by more than 2-fold, from 10.2 \u00b1 0.8 \u03bcM-1 to 3.9 \u00b1 0.5 \u03bcM-1 min -1. Four rounds of random mutagenesis, enrichment, and screening were performed, yielding a 7-fold fluorinated mutant, tfi-G4, whose activity in fluorinated form was 2.8-fold higher than that of the fluorinated parent enzyme, tfi-WT. The total number of isoleucines decreased only by one in the 7-fold mutant, and the gap in activity between the hydrogenated (ile-G4) and fluorinated (tfi-G4) forms narrowed. Despite similar secondary structure, the incorporation of fluorinated amino acids decreased the stability of CAT for both the wild- type and G4 pairs based on both functional and structural analysis. Fluorinated forms were more sensitive towards thermal and chemical denaturation. However, both forms of G4 enzymes had increased stabilities as compared to their wild-type counterparts. This resulted in tfi-G4 exhibiting similar thermostability as that of ile-WT. Although structural changes were noted at both high and low pH, the pKa of the catalytically essential histidine (His-193) was not affected by the incorporation of 5TFI. Based on these results, the incorporation of 5TFI appears to be adversely affecting protein folding, which resulted in decreased activity and stability. However, laboratory evolution effectively recovered these losses and yielded a fluorinated enzyme that performed similarly to the wild-type.
\r\n\r\nFinally, in Chapter 5, the effect of fluorination on sensitivity to proteolytic degradation was explored. GCN4 proteins containing either 5TFI (tfi-INL) or 4TFV (tfv-VNL) as well as CAT proteins containing either 5TFI (tfi-G2) or 5TFL (tfl-L2A1) were treated with two proteases, trypsin and elastase. As evidenced by gel electrophoresis and densitometry analysis, the half life of tfi-INL in the presence of elastase was 4 times that of its hydrogenated counterpart INL. The increased resistance was less significant upon incorporation of 4TFV as well as in response to trypsin. The opposite trend was observed in the analysis of CAT mutants. Both of the fluorinated CAT mutants were more susceptible to elastase and trypsin as compared to their hydrogenated counterparts. Upon incorporation of fluorinated amino acids, two factors that affect proteolytic degradation are altered, the stability of the protein as well as substrate recognition and hydrolysis by the protease. It appears that changes in both of these factors contributed to the observed rates of proteolysis.
\r\n\r\nThe work explored in this thesis has expanded our understanding of fluorinated amino acids and their effects on proteins. Based on these insights, we are continuing to expand the use of fluorinated amino acids (and other non-canonical amino acids) to control protein structure, function, and stability.
\r\n\r\n\r\n", "doi": "10.7907/4dsq-m054", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1099", "collection": "thesis", "collection_id": "1099", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03242006-134157", "primary_object_url": { "basename": "00_Title.pdf", "content": "final", "filesize": 61997, "license": "other", "mime_type": "application/pdf", "url": "/1099/1/00_Title.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Endothelial Cell Response to Artificial Extracellular Matrix Proteins", "author": [ { "family_name": "Liu", "given_name": "Julie Chih-I", "clpid": "Liu-Julie-Chih-I" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Artificial extracellular matrix (aECM) proteins were designed originally for use in small-diameter vascular grafts. Current synthetic grafts fail primarily due to (i) a compliance mismatch between the prostheses and surrounding tissue and (ii) the inability to support the growth of an endothelial cell monolayer. To address these issues, biomimetic proteins were engineered with elastin-like repeats to confer elastomeric behavior and fibronectin cell-binding domains to promote endothelialization. Lysine residues or the non-canonical amino acid, para-azidophenylalanine (pN3phe), serve as crosslinking sites.
\r\n\r\nHuman umbilical vein endothelial cell (HUVEC) adhesion to aECM proteins was sequence-specific. Cells bind more strongly and exhibit faster spreading kinetics on aECM proteins containing the RGD versus the CS5 sequence. Furthermore, HUVECs on the former protein exhibited well-formed stress fibers and organized the alpha-v-beta-3 but not the alpha-5-beta-1 integrin into focal adhesions.
\r\n\r\nAlthough biomaterial design has focused on the sequences of cell-binding domains, elements remote to these bioactive sequences were found to affect cell response to aECM proteins. Proteins containing identical CS5 cell-binding domains differed in their placement of lysine residues that serve as crosslinking sites. Cell adhesion and spreading were more robust on proteins in which lysine residues were located at the termini versus within the elastin cassettes.
\r\n\r\nCrosslinked films of aECM proteins with RGD sequences adhered HUVECs in a sequence-specific manner. Poly(ethylene glycol) was covalently attached to films to reduce nonspecific cell interactions. Increasing the density of RGD in a film resulted in increased cell adhesion and spreading but did not have a significant effect on migration rates.
\r\n\r\naECM proteins were made photoreactive through the incorporation of pN3phe. Upon exposure to ultraviolet radiation through a patterned mask, proteins were patterned on a non-adhesive background. These two-dimensional patterns then served as templates for cell adhesion.
\r\n\r\nA new technique for studying cells on aECM proteins was developed. Cells were pulsed with homopropargylglycine. Newly synthesized proteins labeled with alkyne-containing amino acids were ligated to 3-azido-7-hydroxycoumarin. Fluorescence microscopy was used to visualize these proteins in a wide variety of mammalian cell types.
", "doi": "10.7907/ABQJ-TR83", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3692", "collection": "thesis", "collection_id": "3692", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09222005-123557", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 11991748, "license": "other", "mime_type": "application/pdf", "url": "/3692/1/thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Intracellular Considerations in the Development of Non-Viral Nucleic Acid Delivery Systems for Systemic Administration", "author": [ { "family_name": "Mishra", "given_name": "Swaroop", "clpid": "Mishra-Swaroop" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Webster", "given_name": "Paul", "clpid": "Webster-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Non-viral nucleic acid delivery systems must condense nucleic acids into small particles, confer protection from degrading factors in serum and in cells, achieve uptake to targeted cells, direct nucleic acids to appropriate intracellular destinations, release this cargo to permit its action, and exhibit minimal toxicity. The nature of synthetic vectors allows for facile addition of new features, but these modifications can affect performance in unanticipated ways. The effective combination of functional components necessitates a systems approach, where the materials design simultaneously considers the functional environment of and the various barriers to delivery. This thesis facilitates and promotes a systems approach by undertaking development of an improved mechanistic understanding of non-viral gene transfer in vitro, emphasizing elucidation of delivery vehicles\u2019 interactions with and behavior within cells. Special attention is given to the gene delivery behavior of cyclodextrin-containing polycations.
\r\n\r\nSimple modifications to delivery systems can have unanticipated consequences. In Chapter 2, it is shown that greater distance between toxicity-reducing cyclodextrin moieties and amidine charge centers increases both the transfection efficiency and toxicity of a polycationic vector. Chapter 3 shows data demonstrating that modification with poly(ethylene glycol) for extracellular salt-stabilization alters non-viral gene delivery particles\u2019 intracellular trafficking and resulting gene expression. Taken together, the results reveal that non-viral gene delivery vehicles behave as assembled, multifunctional systems.
\r\n\r\npH-buffering components exhibit complex behavior in non-viral gene delivery. In Chapter 4, the intracellular activity of such components is quantified using confocal microscopy. Analysis of chloroquine and its chemical analogues demonstrates in Chapter 5 that chloroquine improves non-viral gene transfer through pH-buffering as well as through enhanced nucleic acid unpackaging and its own interactions with nucleic acids. Chapter 6 gives results that characterize delivery behavior of analogous vectors with and without pH-buffering capacity and show that factors beyond buffering activity contribute to improved transfection efficiency. Collectively, these results emphasize consideration of new system components\u2019 effects on all functions of a non-viral gene delivery system.
\r\n\r\nA systems approach requires comprehensive consideration of the gene delivery process. Chapter 7 reviews current understanding of intracellular barriers to non-viral gene delivery, and Chapter 8 offers recommendations for future work.
\r\n", "doi": "10.7907/7MP8-JJ24", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3201", "collection": "thesis", "collection_id": "3201", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08232005-174620", "primary_object_url": { "basename": "00-Tobias_Whole_Thesis.pdf", "content": "final", "filesize": 5273145, "license": "other", "mime_type": "application/pdf", "url": "/3201/1/00-Tobias_Whole_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Directed Evolution of Biosynthetic Pathways to Carotenoids with Unnatural Carbon Backbones", "author": [ { "family_name": "Tobias", "given_name": "Alexander Vincent", "orcid": "0000-0002-5866-5254", "clpid": "Tobias-Alexander-Vincent" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Over the course of evolution, nature continually discovers new small molecules through the alteration of biosynthetic enzymes and pathways by mutation and gene transfer. Hundreds of these natural products have proven indispensable to medicine, culture, and technology, greatly contributing to increases in the length and quality of human lives. Chemists have found that the \"chemical space\" surrounding natural products is especially rich in functional molecules, and synthesis of natural product analogs has uncovered many with new or improved properties.
\r\n\r\nInspired by nature's search algorithm, we and others have conducted our own evolution of carotenoid biosynthetic pathways in the laboratory. Chapter 1 comprehensively reviews the motivations, accomplishments, and challenges of this research area as of early 2005, and describes in detail how biosynthetic routes to dozens of new carotenoids have been established.
\r\n\r\nTo expand the number of carotenoid backbones beyond the C30 and C40 carbon scaffolds that give rise to the ~700 known natural carotenoids, we subjected a carotenoid synthase, the enzyme responsible for carotenoid backbone synthesis, to directed evolution. Chapter 2 describes the evolution of the C30 carotenoid synthase CrtM from Staphylococcus aureus for the ability to synthesize C40 carotenoids. This work also resulted in novel carotenoids with C35 backbones. We later found that some of the CrtM mutants generated in this laboratory evolution experiment, as well as several second-generation variants, are also capable of synthesizing unnatural C45 and C50 carotenoid backbones when supplied with appropriate prenyl diphosphate precursors.
\r\n\r\nChapter 3 describes the creation of full-fledged pathways to carotenoid pigments based on the C45 and C50 scaffolds. Coexpression of the carotenoid desaturase CrtI from Erwinia uredovora resulted in the biosynthesis of at least 10 new C45 and C50 carotenoids with different systems of conjugated double bonds. We also present evidence of an unnatural asymmetric C40 carotenoid pathway beginning with the condensation of farnesyl diphosphate (FPP, C15PP) and farnesylgeranyl diphosphate (FGPP, C25PP). In addition to clarifying how CrtM and CrtI achieve their product specificities, this work also sheds light on the molecular mechanisms used by evolution to access new chemical diversity and the selective pressures that have shaped natural product biosynthesis.
", "doi": "10.7907/WF0Q-2J98", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2090", "collection": "thesis", "collection_id": "2090", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262005-102319", "primary_object_url": { "basename": "Jones_EAV_011305.pdf", "content": "final", "filesize": 6736077, "license": "other", "mime_type": "application/pdf", "url": "/2090/9/Jones_EAV_011305.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Blood Flow and The Mammalian Embryo", "author": [ { "family_name": "Jones", "given_name": "Elizabeth Anne Vincent", "clpid": "Jones-Elizabeth-Anne-Vincent" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Hemodynamics, or blood fluid dynamics, is of great importance in vascular biology and its role is well recognized in events ranging from atherosclerosis to wound healing. The importance of hemodynamics during embryonic development, however, is less clear. The early vertebrate vasculature is established through two processes; vasculogenesis, which is the de novo formation of vessels and angiogenesis, which is the sprouting of new vessels from existing vessels and the remodeling of existing vessels. The latter process, angiogenesis and vascular remodeling, is dependent on blood flow and does not occur if cardiac output is blocked. As well, if blood flow is altered, such as with mutations that affect cardiac contraction, the early vessels also fail to remodel. Flowing blood imparts a physical force, called shear stress, on the endothelial lining of the blood vessels. Many genes known to be regulated by shear stress are important for vascular remodeling in the embryo. In this work, we investigate the role of shear stress on the remodeling process.
\r\n\r\nStudying the role of shear stress in embryos requires the ability to measure changes in both fluid dynamics and vascular morphology as well as methods to alter shear stress levels. In this work, we use an optical technique for the quantitative analysis of hemodynamics during early organogenesis in the mouse embryo. We established the morphological changes that occur in the vasculature during remodeling and link these to the fluid dynamics that are present. We establish the mechanical cues that are available to the endothelial cells and the type of flow present at various stages of development. In order to understand how these mechanical cues affect embryonic development, we examine altered shear stress during development using a mutant mouse model in which the atrial cardiac contraction is lacking as well as inducing specific changes in shear stress through chemical manipulation of the embryonic cardiovascular system. These studies establish a link between the pattern of blood flow within the vasculature and the stage of cardiovascular development and enable analysis of the influence of mechanical forces during development.
", "doi": "10.7907/TTW7-YY07", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1202", "collection": "thesis", "collection_id": "1202", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03302005-120327", "primary_object_url": { "basename": "Popielarski_Final_Submitted_Thesis.pdf", "content": "final", "filesize": 7067218, "license": "other", "mime_type": "application/pdf", "url": "/1202/1/Popielarski_Final_Submitted_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "I. Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. II. Development of a Nanoparticle-Based Model Delivery System to Guide the Rational Design of Gene Delivery to the Liver\r ", "author": [ { "family_name": "Popielarski", "given_name": "Stephen R.", "clpid": "Popielarski-Stephen-R" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Linear cationic beta-cyclodextrin (b-CD)-based polymers can bind with plasmid DNA to form colloid-sized composite particles that transfect cultured cells. In the first part of this thesis, synthetic variations of the b-CD structure are used to probe structure-function gene delivery properties. The type of cyclodextrin and its functionalization are investigated by synthesizing numerous 3A,3B-dideoxy-3A,3B-diamino-b- and g-CD monomers, which are polymerized with dimethyl suberimidate to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When complexed with plasmid DNA, polycations with longer alkyl regions between the CD and the charge centers show increased transfection efficiency and toxicity in BHK-21 cells. More hydrophilic spacers resulted in lower toxicity, and g-CD-based polycations were less toxic than otherwise identical b-CD-based polycations.
\r\n\r\nIn the second part of this thesis, a model delivery system is developed that can mimic the size and surface properties of the cyclodextrin-based gene-delivery particles, and this system is used to define design constraints that should be applied to next generation gene delivery particles targeted to the liver. Gal-50 and Gal-140 are galactosylated 50 nm and 140 nm nanoparticles that have the same surface galactose density, while MeO-50 and MeO-140 are methoxy-terminated 50 nm and 140 nm nanoparticles. All four particles have the same surface charge and resist aggregation in serum.
\r\n\r\nIn freshly isolated hepatocytes, Gal-50 nanoparticles are taken up to a greater extent than are MeO-50, but both 50 nm beads are taken up to a much greater extent than are either of the 140 nm nanoparticles. TEM and immunohistochemistry confirm that Gal-140 nanoparticles are primarily internalized by Kupffer cells, though isolated examples of a few Gal-140 in hepatocytes can also be found. On the other hand, Gal-50 nanoparticles are overwhelmingly found in vesicles throughout the cytoplasm of hepatocytes, with only isolated examples of Kupffer cell uptake. As such, it is clear that slightly anionic, galactose-PEGylated nanoparticles should be about 50 nm in diameter to preferentially target hepatocytes while they should be about 140 nm in diameter to selectively target Kupffer cells.
", "doi": "10.7907/rskv-rf12", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2723", "collection": "thesis", "collection_id": "2723", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06252004-101813", "primary_object_url": { "basename": "JFM_Thesis_Complete.pdf", "content": "final", "filesize": 2117681, "license": "other", "mime_type": "application/pdf", "url": "/2723/1/JFM_Thesis_Complete.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Methods for Collection and Processing of Gene Expression Data", "author": [ { "family_name": "Murphy", "given_name": "John Frank", "clpid": "Murphy-John-Frank" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Examination of the transcriptional messages encoded in the manifold of mRNA molecules within a cell is a central task of molecular biology and functional genomics. This examination can be broken down into two parts: collection of gene expression data, and analyses of those data. Here, a new method for collecting gene expression data, and two new methods for analyzing those data are presented.
\r\n\r\nA new method for quantifying gene expression denoted as the Mass-spectrometric Analysis of Gene Expression (MAGE) is developed. MAGE relies on novel conjugates of DNA oligonucleotide 30-mers; each unique sequence is conjugated via photolabile linker to an N-substituted glycine oligomer (peptoid) of unique mass. Deuterated bromoacetic acid is incorporated into some peptoids yielding two chemically identical probe conjugates of different molecular weights for each nucleic acid sequence of interest. Mixtures of these probes, along with 3' adjacent biotin-labeled oligonucleotides, are used to interrogate a target mixture of cDNA. Following hybridization, the two adjacent probes are ligated to enhance the specificity of the identification, and to enable the use of a biotin-affinity column for removal of confounding peptoid tags. The resulting mixture is exposed to longwave ultraviolet light to release the peptoid tags, that are quantified using MALDI-TOF mass spectrometry using the isotopically labeled peptoids as internal standards. These individual components of MAGE are demonstrated.
\r\n\r\nA strategy for simplification and visualizing of high-dimensional gene expression data, as well as a strategy for inferring the presence of clusters within those data, is formulated and implemented. In order to visualize high-dimensional gene expression data, principle components analysis is used with subsequent mapping of the data onto an orthogonal set of basis functions known as Andrews curves. This analysis method is demonstrated by visualizing of breast cancer tumor data and yeast sporulation data. In order to cluster gene expression data, the expectation-maximization algorithm is employed to optimize the parameters of a mixture model of Lorentzian distributions. The difference between Lorentzian and Gaussian mixture models is first demonstrated with artificial data, and then applied to yeast sporulation data. The results indicate that mixtures of Lorentzian distributions may have significant utility for gene expression analysis.
\r\n\r\nThe tools demonstrated here offer unique advantages when compared to the current suite of experimental and analytical tools employed by investigators of functional genomics.
\r\n", "doi": "10.7907/8p8e-2147", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2052", "collection": "thesis", "collection_id": "2052", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252005-114817", "primary_object_url": { "basename": "heidel_thesis.pdf", "content": "final", "filesize": 4815091, "license": "other", "mime_type": "application/pdf", "url": "/2052/1/heidel_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Targeted, Systemic Non-Viral Delivery of Small Interfering RNA in vivo", "author": [ { "family_name": "Heidel", "given_name": "Jeremy David", "clpid": "Heidel-Jeremy-David" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rossi", "given_name": "John J.", "clpid": "Rossi-J-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Armed with the complete sequence of the human genome and an ever-increasing array of biological techniques, researchers continue to learn more about the genetic basis of diseases. For two decades, scientists and physicians have been developing therapeutic strategies for treating many diseases at the genetic level, creating the field of \"gene therapy.\" For those diseases caused by loss-of-function mutations in a specific gene, delivery of a wild-type copy of that gene to affected cells can reduce or eliminate the disease phenotype. Viruses, having evolved to be extremely effective at delivering nucleic acids (i.e., their own genes for viral production) to cells, have been modified to include therapeutic genes of interest. While such viral gene therapy vectors are the most efficient vectors developed, concerns about their safety and immunogenicity have prompted many to investigate non-viral vector alternatives. Cationic polymers and lipids have emerged as leading non-viral vector materials. Our laboratory has developed a class of cyclodextrin-containing polycations (CDPs) that condense DNA into complexes that can be endocytosed by cells, achieve expression of their genetic payload in those cells, and may be modified to target particular cell types within an animal.
\r\n\r\nIn the past five years, scientists have discovered a new mechanism for the reduction of gene expression in mammalian cells via sequence-specific cleavage of a particular messenger RNA (mRNA); this phenomenon is known as RNA interference (RNAi). Since RNAi is triggered by nucleic acids (small interfering RNA (siRNA) duplexes), I hypothesized that CDPs may be suitable vectors for the delivery of siRNA. In my thesis work, the safety of synthetic siRNA duplexes is examined both in cultured cells and in vivo. Using a number of different siRNA sequences, two different strains of mice, and three different methods of administration, I fail to observe any cytokine (IL-12 or IFN-a) responses, morphological changes, or alterations in complete blood counts (CBCs) or liver enzyme levels.
\r\n\r\nThe ability of CDP to serve as a delivery vehicle for siRNA is also explored. I demonstrate that CDP/siRNA complexes can be formed that are small enough to be endocytosed, can be modified to ensure stability in physiological fluid, and protect the siRNA payload from serum nuclease degradation. Finally, down-regulation of specific target genes, including genes implicated in disease, is shown in vitro and in mice. An endogenous reporter gene (luciferase) in the livers of transgenic mice is down-regulated by galactosylated CDP/siRNA formulations that target hepatocytes. The level of a chimeric oncogene, EWS-Fli1, is reduced by polyplex formulations in cultured Ewing\u2019s sarcoma cells and by transferrin-targeted formulations in tumor-bearing mice; this in vivo down-regulation corresponds to an inhibition of tumor growth. These results suggest that CDP-containing siRNA formulations have the potential for development into therapeutics.
", "doi": "10.7907/F5AX-3Y24", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1991", "collection": "thesis", "collection_id": "1991", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05242004-103633", "primary_object_url": { "basename": "01_title.pdf", "content": "final", "filesize": 267594, "license": "other", "mime_type": "application/pdf", "url": "/1991/1/01_title.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Design and Characterization of Artificial Extracellular Matrix Proteins for Use as Small-Diameter Vascular Grafts", "author": [ { "family_name": "Heilshorn", "given_name": "Sarah Christine", "orcid": "0000-0002-9801-6304", "clpid": "Heilshorn-Sarah-Christine" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic small-diameter vascular grafts often fail within three years of implantation. The underlying causes of graft failure are thought to be i) a mismatch in the mechanical properties between the graft and host material and ii) an inability of the graft to support the adhesion of endothelial cells. To address these two issues, artificial extracellular matrix (aECM) proteins contain elastin-like regions to provide physical integrity and cell-binding domains derived from fibronectin to promote endothelial cell attachment. Using recombinant protein technology, a family of artificial proteins was created with differing ratios of elastin-like regions to cell-binding domains, with variable placement of amino acid crosslinking residues, and with differing identity of cell-binding domain.
\r\n\r\nHuman umbilical vein endothelial cells (HUVEC) adhere in a sequence-specific manner to aECM proteins, secrete basal levels of key fibrinolytic regulators, and are capable of resisting a physiologically relevant detachment force. HUVEC spread more quickly and adhere more firmly to aECM proteins that contain the RGD versus the CS5 cell-binding domain. Decreasing the density of RGD cell-binding domains results in decreased HUVEC adhesion. Furthermore, amino acid selection even at sites up to 16 residues away from the cell-binding domain impacts HUVEC spreading and adhesion. HUVEC also adhere more strongly to stiffer aECM films. Therefore, the identity, density, and context of the cell-binding domain as well as the elastic modulus of the substrate are all important variables in influencing cell-substrate interactions.
\r\n\r\nProper amino acid sequence choice also influences the susceptibility of aECM proteins to elastase proteolysis; modifying 3% of the amino acid side chains results in a 7-fold reduction in degradation rate. An alternative strategy to decrease degradation involves incorporation of the noncanonical amino acid, 5,5,5-trifluoroisoleucine, into the favored proteolytic cut site, isoleucine. Replacing 82% of the isoleucines results in a twofold reduction in degradation rate without compromising sequence-specific HUVEC adhesion. Incorporation of another noncanonical amino acid, para-azidophenylalanine, allows synthesis of photoreactive proteins that can be patterned using photolithography. These protein patterns retain their ability to adhere HUVEC and produce stable cell patterns after 48 hours in medium supplemented with serum.
\r\n", "doi": "10.7907/BX95-3X10", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" } ]