A multifacted approach to the imaging of biological structures by scanning force microscopy is described. The major problems addressed are the distortion of biological samples by excessive forces applied by the cantilever stylus and sample motion relative to the imaging substrate.
\r\n\r\nThe first two chapters discuss the design of digital signal processor based scanning force microscope control electronics and a novel microscope head that eliminates the application of excessive forces to the sample caused\r\nby electronic or vibrational noise.
\r\n\r\nThe third chapter presents a novel use of chemical vapor deposition for application of heterofunctional alkoxysilanes to scanning force microscopy imaging subsrates. This technique provides imaging substrates which have chemical groups that can be used for sample immobilization without compromising substrate smoothness. The use of the chemically derivatized substrates for scanning force microscopy is also explored.
\r\n\r\nThe final chapter presents high resolution images of bovine liver catalase micro-crystals. The images of the protein micro-crystals show resolution on the order of 2 to 3 nanometers allowing the visualization of individual catalase tetramers. To our knowledge this is the first report of images of protein micro-crystals taken by scanning force microscopy which have resolution comparable to that of electron microscopy.
\r\n", "doi": "10.7907/0vhp-pk35", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:2673", "collection": "thesis", "collection_id": "2673", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-075739", "primary_object_url": { "basename": "Hoh_jh_1991.pdf", "content": "final", "filesize": 16024320, "license": "other", "mime_type": "application/pdf", "url": "/2673/1/Hoh_jh_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies on the structure and molecular diversity of the gap junction", "author": [ { "family_name": "Hoh", "given_name": "Jan Hakan", "clpid": "Hoh-Jan-Hakan" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed.\r\n\r\nThe structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after \"force dissection,\" which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible.\r\n\r\nLow stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels.", "doi": "10.7907/8n7d-2t98", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:11812", "collection": "thesis", "collection_id": "11812", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10112019-091636747", "type": "thesis", "title": "Biochemical and Genetic Studies of Peripheral Myelination in Normal Development and in the Mouse Mutant Trembler", "author": [ { "family_name": "Fryxell", "given_name": "Karl Joseph", "orcid": "0000-0002-2975-7897", "clpid": "Fryxell-Karl-Joseph" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "I developed a radioimmunoassay for P0, the major peripheral myelin protein, and adapted an existing radioimmunoassay for myelin basic protein. Results from these assays showed that Schwann cells do not make either protein before myelination begins, and accumulate P0 and myelin basic protein with the same time course during development. Schwann cells cultured in the absence of neurons do not express detectable levels of either protein, but they do continue to synthesize sulfatide, a myelin sulfolipid, apparently indefinitely, as shown by biosynthetic labeling.
\r\n\r\nThe trembler (Tr/+) mouse mutant has a pronounced reduction in peripheral myelin, while central myelin and peripheral unmyelinated nerves appear normal. This myelin deficiency is caused by an autonomous Schwann cell defect. Since the Trembler phenotype is expressed in heterozygotes, most previous studies were confined to Tr/+ mice. Using two alleles, I show here that the six possible genotypes can be ordered as follows: +/+ > Trj/+ > Tr/+ > Trj/Trj > Trj/Tr > Tr/Tr, based on myelin basic protein radioimmunoassays of sciatic nerve extracts. The amount of compact myelin in each genotype, as shown by electron microscopy, is in good agreement with the radioimmunoassay results, with two important provisos. First, Tr/Tr mice have 1% of wild-type myelin basic protein levels but essentially no compact myelin. Secondly, the first steps in the above genetic series reduce both the average myelin sheath area and the number of myelin sheaths by similar amounts, while the last steps primarily reduce the number of myelin sheaths. These results suggest that, if the activity of the Tr+ gene product is reduced below a certain point, myelin protein synthesis can be induced without forming a myelin sheath. Visualization of P0 by indirect immunofluorescence shows that Schwann cells without compact myelin express much lower levels of P0 than adjacent Schwann cells, associated with the same axon, that do form compact myelin. Finally, although Trembler Schwann cells proliferate excessively in vivo, their proliferation is not affected by Tr gene dose. In vitro Trj/Trj Schwann cell proliferation is apparently normal. Thus, it is likely that the function of the Tr+ gene product is not directly concerned with Schwann cell proliferation.
", "doi": "10.7907/eahp-bx93", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11842", "collection": "thesis", "collection_id": "11842", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283", "type": "thesis", "title": "Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens", "author": [ { "family_name": "Nicholson", "given_name": "Bruce John", "orcid": "0000-0003-1649-7173", "clpid": "Nicholson-Bruce-John" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
\r\n\r\nComparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
\r\n\r\nThe extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
", "doi": "10.7907/bhjv-8053", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" } ]