Includes bibliographical references (leaves 103-110). / Forhead box G1 (FoxG1) is a winged-helix transcription factor that plays a crucial role in the development of the telecephalon, the most rostral region of the brain Here, FoxG1 acts as a transcriptional repressor and maintains the population of cortical progenitor cells by promoting their proliferation and inhibiting differrentiation. Vertebrate FoxG1 orthologs have highly conserved DNA-binding and corbosy-terminal domains that have functional roles. Conversely, no functional role has yet been assigned to the N-terminal domain which shows a high degree of variability across vertebrates, with a remarkable stretch of consecutive histidine, proline and glutamine (HPQ) residues in the mammalian orthologs. In this study it was tested whether differences in FoxG1 sequence amongst vertebrates might account for the increased cortex size of mammals compared to non-mammals. Furthermore, changes in the sub-cellular localization of FoxG1 in response to fibroblast growth factor 2 (FGF-2) were investigated in a neural precursor cell line.

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CACNA1A encodes for the voltage-gated calcium channel CaV2.1. Mutations to CACNA1A are known to cause an assortment of disorders, including episodic ataxia 2, spinocerebellar ataxia 6, familial hemiplegic migraine 1, and more. The pathogenic nature behind these mutations is still unknown, but recent studies have given new information that points toward certain conclusions. In particular, episodic ataxia 2 appears to be caused (in some mutations) by misfolding and improper trafficking of both wild-type and mutant CaV2.1 channels. Spinocerebellar ataxia 6 is more complicated, as recent findings of the bicistronic nature of CACNA1A may mean that the disorder is more akin to a polyglutamine disease than a channelopathy. The goal of this thesis is to review the literature available that addresses these disorders and their progression, as well as future treatment strategies.

Over half of the proteins found in nature are anticipated to be glycosylated, yet less than 2% of the protein structures in the protein data bank are glycoproteins. Clearly, we are behind in our structural understanding of glycoproteins. One reason for the lack of structural information for glycoproteins is due to their oligosaccharide heterogeneity. The two main methods for determining detailed three-dimensional protein structures are X-ray crystallography and nuclear magnetic resonance. Oligosaccharide heterogeneity hinders crystallization for X-ray crystallography analysis and complicates resonance assignment of nuclear magnetic resonance data. Another challenge is expressing high enough yields from eukaryotic cells for X-ray crystallography and nuclear magnetic resonance analysis. Additionally, unlike bacteria, eukaryotic cell lines typically cannot synthesize amino acids from glucose and ammonium chloride. Thus, eukaryotic media must contain amino acids for cell growth and protein production. Structural analysis of glycoproteins by nuclear magnetic resonance requires that the amino acids are labeled with 13C- and 15N and that the oligosaccharides are labeled with 13C. Media containing 13C- and 15N-labeled amino acids is extremely expensive. This dissertation explains methods that address each of these challenges utilizing the glycoprotein Thy-1. An economic approach for labeling recombinant protein expressed from insect cells for NMR is explained. A method for labeling oligosaccharides on glycoproteins expressed from mammalian cells is also explained. Changes in previously established expression and purification protocols for Thy-1 derived from Lec1 cells, which produce relatively homogeneous glycoforms, have increased yields to levels suitable for nuclear magnetic resonance studies. In addition, the biological relevancy of recombinant Thy-1, which is expressed with truncated oligosaccharides and without it's glycosylphosphatidylinositol anchor, was confirmed by receptor-binding analysis. / A Dissertation Submitted to the Program in Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Spring Semester, 2006. / December 9, 2005. / Isotopic Enrichment, NMR, Receptor-Binding Analysis, Thy-1, Glycoprotein Structure / Includes bibliographical references. / Timothy M. Logan, Professor Directing Dissertation; Richard L. Hyson, Outside Committee Member; Thomas C. S. Keller, III, Committee Member; Hong Li, Committee Member; Alan Marshall, Committee Member.

The mechanism by which muscle generates force has been the subject of considerable study. Key to our understanding of this mechanism is the conformational changes occurring in the myosin "head" as it interacts with the thin filament. Each myosin head individually binds to the thin filament, hydrolyzes ATP and generates force. This study has used electron paramagnetic resonance (EPR) spectroscopy to study the structure and function of the regulatory domain located on the distal portion of the myosin head. Skeletal myosin S1 was labeled with a variety of lysine targeting spin labels, under conditions optimized to label the reactive lysine residue Lys-83, to determine if spin label suitable for EPR studies could be found. Two spin labels (HO-226 and HO-2095T) had ordered populations. Competitions, of the two labels with selective blocking agents, determined that they were labeling both lysine and cysteine residues. The anisotropy for both labels was found to derive from labeled cysteine residues not Lys-83, rendering both unsuitable for EPR studies of myosin S1. The mobility of the essential light chain (ELC) and the regulatory light chain (RLC) subdomains of the regulatory domain was individually measured utilizing saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy. Their mobilities were found to be similar, and that similarity persisted under conditions, which increase the overall mobility of the myosin head: upon RLC phosphorylation, an increase of pH or the presence of divalent cations. Modeling of this mobility enabled calculation of the persistence length of the regulatory domain, which at 1.5 µm, is adequate for it to serve as a lever arm. This is consistent with theories of force generation where the regulatory domain serves as a lever to amplify movement of the catalytic domain during the power stroke of striated muscle. / A Dissertation Submitted to the Program in Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Spring Semester, 2003. / December 16, 2002. / muscle, force, mechanism / Includes bibliographical references. / Tim Logan, Professor Directing Dissertation; David Van Winkle, Outside Committee Member; Peter Fajer, Committee Member; Nancy L. Greenbaum, Committee Member; Kenneth A. Taylor, Committee Member.

Interactions between charged residues are known to have significant effects on protein folding stability and binding properties. The contributions of different types of non-covalent interactions are altered by mutating one or more residues, resulting in change in the protein stability to considerable extent. The main goal of this dissertation is to understand contributions, specifically from electrostatic interactions, to the protein folding stability and also, to devise strategies in order to enhance protein stability. We have introduced single and double mutations in FKBP12 (FK506 binding protein) modeled after its close homolog FKBP12.6, and later accumulated the most stabilizing mutations in order to create a hyper stable mutant. Similar experimental study was carried out, where insertion, deletion and systematic introduction of ion-pair clusters in FKBP12 were modeled after the thermophilic homolog MtFKBP17 and the results suggest that charge residues can modulate the folding stability significantly. This experimental study has allowed rigorous testing of computational models developed by our group for predicting electrostatic contributions to protein folding stability, which does not match closely and need further refinement. To further broaden our research in understanding biophysical properties of proteins in living cells, we have investigated the effects of macromolecular crowding on the folding stability of one of the less stable mutant of FKBP. We show that there is an optimal size of crowder at which stability increase is maximum and also, the stabilization effect of mixture (different crowders) is greater than the sum of constituent crowding agents. These findings may have profound implications for understanding crowding effects inside cells. The main aim of my dissertation is to understand protein folding and stability, which are the fundamental problems in biophysics. Experiments are underway to understand effects of crowding on protein binding and their quantitative information will provide key insights to the biological relevance of experimental results obtained in vitro. Overall my research aims for understanding fundamental study on protein folding stability and binding which will serve as a valuable tool for designing therapies for human diseases. / A Dissertation Submitted to the Department of Physics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Spring Semester, 2009. / March 31, 2009. / Binding Stability, Protein Stability, Macromolecular Crowding, Protein Folding, Electrostatic Interactions, FKBP / Includes bibliographical references. / Huan-Xiang Zhou, Professor Directing Dissertation; Michael Blaber, Outside Committee Member; Peng Xiong, Committee Member; Per Arne Rikvold, Committee Member; Vladimir Dobrosavljevic, Committee Member.

Adeno-associated virus type-2 (AAV-2) is a leading candidate vector for gene therapy. My PhD is a part of the ongoing research project towards the long-term goals of engineering AAV to evade immune neutralization when repeated doses are required, and of modulating the specificity of cellular targeting. The specific aim of my PhD research is the structural analysis of AAV-2 using X-ray crystallography and cryo-Electron Microscopy (cryo-EM). The two parts of this thesis follow my participation in the crystallographic structure determination of AAV-2 at 3Å resolution. In the first part, cryo-EM has been used to visualize the wild type AAV-2 (wt-AAV2) and its structure determined using the principles of icosahedral three-dimensional reconstruction. A difference map has been calculated by subtracting the atomic structure of the major component, viral protein 3 (VP3), from theEM density of the wt-AAV2. The results indicate the probable location of the viral nucleic acid and the other minor viral proteins, VP1 and VP2, not imaged in the crystallographic structure. This research lays the foundation for the future studies of AAV-2-antibody complexes that will be studied by the same techniques. The second part of the research involves the analysis of crystal contacts of the virus in the crystallographic unit cell. Surface features such as molecular shape and chemical character determine interactions of a molecule with its surroundings in vivo or in crystalline forms. Amino acids involved in particle interactions have been identified from buried surface area calculations. This research addresses the question of what drives a virus to choose one packing scheme over another and whether the crystal contacts carry any biological information. The residues buried in inter-particle contacts play a stabilizing role in the packing and are often a part of a surface with demonstrated biological function such as immunogenic stimulation and receptor recognition. The results of this research will form the groundwork for the study of virus-receptor and virus-antibody complexes. / A Dissertation Submitted to the Institute of Molecular Biophysics in Partial FulfiLlment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2003. / July 15, 2003. / Molecular Packing, 3D Reconstruction, Virus Structure, Cryo-Em, AAV-2 / Includes bibliographical references. / Michael S. Chapman, Professor Directing Dissertation; Robert H. Reeves, Outside Committee Member; Kenneth A. Taylor, Committee Member; Timothy A. Cross, Committee Member; Nancy L. Greenbaum, Committee Member.

Glucocorticoids (GC) are used widely for the treatment of a large number of inflammatory conditions. A loss in muscle mass and increases in muscle weakness are common complications of GC therapy. Androgen therapy has been suggested to reverse GC-associated muscle loss (GAML), but evidence of its effectiveness is inconsistent. Herein, I established a mouse model of GAML. Young adult male mice receiving 10 mg/(kg day) of the GC dexamethasone (Dexa) s.c. daily, for a week, lost 3% of their total body weight. Based on NMR lean body mass quantification and muscle dissection, more than 10% of their muscle mass was lost. More than half of the Dexa-induced muscle loss could be reversed by co-administration of 35 mg/(kg day) of testosterone (Testo). To my knowledge, this is the first mouse model of GAML demonstrating alleviation by Testo. Dexa-upregulated intramuscular atrogene expression and proteasome catalytic activity were suppressed by Testo co-administration. Dexa downregulated cathepsin L enzymatic activity and beclin expression, indicating that lysosome was not a major effector of GAML. Changes in calpain 1 and in translation factors 4E-BP, eIF3f and eIF2, following Testo treatment, were inconclusive. The changes in proteasome activity and atrogene expression were correlated with changes in expression of Foxo 1, 3a, and 4. Pro-catabolic factors REDD1 and Klf15 were repressed by Testo co-administration. C2C12 differentiated myotubes were used to model GAML in vitro. Myotube diameter and total protein were reduced by Dexa, and restored by Testo co-administration. Changes in C2C12 total protein were correlated with changes in protein degradation. Dexa-induced proteolysis was inhibited by the proteasome inhibitor MG132. In vivo, Dexa reduced intramuscular IGF-I expression, an effect reversed by Testo co-administration. In C2C12, inhibition of IGF-1R signaling with picropodophyllin did not modify Testo protective effect. Mechanisms potentially explaining these observations are discussed. In summary, my model demonstrates that Testo protective effect in GAML is mainly anti-catabolic, through the reversal of proteasome upregulation induced by Dexa. In vivo, Testo stimulates a potentially protective intramuscular IGF-I response. The roles of protein synthesis and IGF-I in anabolic myoprotection could not be addressed in these models, and require further investigations.

The developments of the polymerase chain reaction (PCR) and the short tandem repeat multiplex kits increased the ease and lowered the time and sample quantity required for deoxyribonucleic acid (DNA) typing compared to previous methods. However the amplification of low mass of DNA can lead to increased stochastic effects, such as allele drop-out (ADO) and heterozygous peak height (PH) imbalance, which make it difficult to determine the true donor profile. These stochastic effects are believed to be due to: 1) pre-PCR sampling from pipetting and sample transferal of dilute samples prior to amplification resulting in unbalanced heterozygous allele templates in the amplification reaction, and 2) the kinetics of the PCR process where, when few target templates are available, there is uneven amplification of heterozygous alleles during early PCR cycles. This study looks to examine the contribution of PCR chemistry and pre-PCR sampling errors on stochastic effects by utilizing a single-tube DNA extraction and direct amplification method. Cells were collected into tubes using the McCrone and Associates, Inc. cell transfer method, which allowed for approximation of DNA mass without quantification. The forensicGEM® Saliva Kit was used to lyse the cells and inactivate nucleases without inhibiting downstream amplification. The samples were then directly amplified with the AmpFLSTR® Identifiler® Plus PCR Amplification Kit. These samples should only show the effects of PCR chemistry since pipetting and tube transferal steps prior to amplification were removed with the expectation that equal numbers of heterozygous alleles are present in the sample pre-amplification. Comparisons of PH imbalance were made to samples extracted with forensicGEM® but had one or more pipetting and tube transferal steps prior to amplification. These samples were either created through the dilution of stock DNA or from the cell transfer method where aliquots were then taken for amplification; thus these samples would exhibit the effects of both pre-PCR sampling and PCR chemistry errors and inefficiencies. The use of carrier ribonucleic acid (cRNA) was also added to cell transfer samples prior to the amplification of samples to see if it assisted with amplification and increased signal. Results show that the samples with only PCR chemistry generally have significantly higher mean peak height ratios (PHRs) than samples with both pre-PCR sampling and PCR chemistry except in cases where there were large numbers of ADOs. When compared to the diluted samples, the cell transfer samples had significantly higher mean PHR at 0.0625 ng and 0.125 ng, and higher mean PHR at 0.0375 ng when PHs from ADOs are included. Average peak heights (APHs) in the cell transfer samples were also significantly higher in these comparisons. When compared to aliquots taken from cell transfer samples, mean PHR was significantly higher at 0.0625 ng in cell transfer samples with only PCR chemistry than cell transfer samples with both pre-PCR sampling and PCR chemistry; however APH for the samples with only PCR chemistry was also significantly higher in one experiment and not significantly different in another. In a third experiment, the difference in mean PHR was not significant while APH was significantly higher in the samples with pre-PCR sampling and PCR chemistry; however there were also a large numbers of ADOs. Our results also found quantification of dilute samples unreliable but cell counting through the cell transfer method is an appropriate alternative for DNA mass approximation. Also there were no significant changes in PHR or APH in the presence or absence of cRNA.