abstract: Membrane protein structure is continuing to be a topic of interest across the scientific community. However, high resolution structural data of these proteins is difficult to obtain. The amino acid transport protein, Outer Envelope Protein, 16kDa (OEP16) is a transmembrane protein channel that allows the passive diffusion of amino acids across the outer chloroplast membrane, and is used as a model protein in order to establish methods that ultimately reveal structural details about membrane proteins using nuclear magnetic resonance (NMR) spectroscopy. Methods include recombinant expression of isotope enriched inclusion bodies, purification and reconstitution in detergent micelles, and pre-characterization techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and high pressure liquid chromatography (HPLC). High resolution NMR spectroscopy was able to assign 99% of the amide backbone and the chemical shifts provided detailed secondary structure of OEP16 on a per residue basis using the software TALOS+. Relaxation studies explored the intramolecular dynamics of OEP16 and results strongly support the resonance assignments. Successful titration studies were able to locate residues important for amino acid binding for import into the chloroplast as well as provide information on how the transmembrane helices of OEP16 are packed together. For the first time there is experimental evidence that can assign the location of secondary structure in OEP16 and creates a foundation for a future three dimensional structure. / Dissertation/Thesis / Ph.D. Biochemistry 2012

The purpose of this study was to determine how a soy-based diet modifies tissue-specific adipogenesis and the impact on the development of type 2 diabetes mellitus and non-alcoholic fatty liver disease in obese male lean and obese Zucker rats. Animals were randomly assigned to three diet groups for 17 weeks: casein control, whey control and soy protein. Physiological data were collected throughout the study and at week 14 animals were subjected to an OGTT. As previously demonstrated, obese soy-fed animals had greater final body weights and adiposity, and exhibited an increased food intake. Despite hypertrophic adipocytes in all obese animals, those fed soy protein presented with a benign obesity phenotype. Soy diets attenuated obesity-induced hepatic lipid accumulation and markers of inflammation. The obese soy-fed rats had greater adipocyte hypertrophy without an increase in adipocyte density (number per area), suggesting adipocyte hyperplasia in this group. This corresponded with maintenance of glucose tolerance and serum lipid profiles in the obese soy-fed group despite the greater adiposity. Transcript abundance of adipogenic regulatory genes revealed no significant diet effect at 17 weeks in adipose tissue, but did show greater modification of Wnt/β-catenin signaling in the liver. These results demonstrate benefits of a soy protein diet in amelioration of obesity-related pathologies such as non-alcoholic fatty liver and impaired insulin sensitivity. Furthermore, these outcomes may be mediated through an interaction of soy with the Wnt/β-catenin signaling.

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Antibodies are capable of high specificity interactions with a virtually infinite range of substrates (antigens). This property has lead to a number of scientific and medical applications. The extreme variability is essentially confined to 6 hypervariable loops or 'CDRs' which constitute the antigen combining site. To make intelligent modifications to antibody affinity and specificity, by methods such as site directed mutagenesis, requires an understanding of the relationship between primary sequence and three dimensional structure of the combining site. A new 'combined algorithm' which makes use of both knowledge-based and ab initio (conformational search) modelling approaches is presented. It is routinely and reliably able to predict the conformation of all six CDRs and requires no arbitrary decisions by the user. All known protein structures are searched for loops of conformation similar to known antibody structures. These are positioned onto the conserved framework and the loops are processed into a form suitable for conformational search using the program CONGEN (Bruccoleri and Karplus, Macromolecules 18(1985),2767--2773). The midsection of each loop is deleted and reconstructed by conformational search. The conformations generated are screened using a solvent-modified potential and, from the low energy conformations, a final choice is made on the basis of structurally determining residues. The method presented provides a route by which to model modifications to known antibody structures or to model complete antibody combining sites - either in combination with other less computer intensive methods, or alone. The procedure has been tested by the individual modelling of the 6 CDR's of two antibodies, in the presence of the crystal structure of the other 5 loops. In addition, it has been applied to modelling CDR's which are difficult to model by other methods and to the construction of a complete antibody combining site. In all cases the algorithm performed very well.

Extracellular vesicles (EVs) are small nano-sized particles released constantly by cells into body fluids like blood, saliva, urine, plasma and milk. Depending in the size, EVs are divided into exosomes (30–100 nm), microvesicles (100–1000 nm) and apoptotic bodies (50–5000 nm). During this work, I extracted exosomes from kidney cancer cells using two different centrifugation methods (sequential centrifugation and sucrose gradient ultracentrifugation) under two different conditions (hypoxia and normoxia). The characterization was done by electron microscopy, western blot and nanoparticle tracking analysis (NTA). The effect of exosomes in normal kidney cells was studied in vitro by treating metanephric mesenchyme (MM) cells with exosomes for cell proliferation and motility. The exosomes were injected in chicken embryos in vivo together with renal carcinoma (Renca YFP) cells to see if they have some role in tumor growth. The protein and RNA contents of the exosomes were also analyzed. The cells release more vesicles when they are exposed to hypoxic conditions. Electron microscopy and western blot showed the presence of exosomes expressing CD63 and CD81 markers. Cell proliferation and motility was found to enhance when cells were treated with exosomes. Chicken embryos showed formation of bigger Renca YFP tumor after treatment with exosomes. Different proteins and miRNAs were detected in exosomes which may play active roles in biological processes. In summary, I successfully purified exosomes from kidney cancer cells and characterized them. We concluded that these vesicles play important roles in cell activity and have ability to enhance the tumor growth. The presence of proteins and miRNAs suggest the potential role in cellular communication.

In previous studies we have found that urinary fibrinogen-derived peptides are potential tumor markers for renal cell carcinoma. These peptides occur at low concentrations in urine. Identification of a low-abundant tumor marker requires optimal sample preparation and a highly sensitive analyzer. In this work different chromatographic and mass spectrometric methods were compared and evaluated for tumor marker discovery. We used urine samples from patients with renal cell carcinoma and prostate cancer. Our main targets were peptides derived from fibrinogen beta with unknown sequence that are produced by differential proteolysis. With our optimized workflow we discovered 26 fibrinogen beta derived peptides that have not been identified in urine previously.

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