Biochemistry, Biophysics, and Structural Biology Commons^™

Regulation Of The Kras Promoter In Pancreatic Cancer By Proteins And Small Molecules, Harshul Batra

Electronic Theses and Dissertations

DNA-binding proteins play a pivotal role in cell biology. The major class of DNA-binding proteins are transcription factors (TFs). TFs are central to almost every fundamental cellular process such as cell development, differentiation, cell growth, and gene expression. They account for 10% of the genes in eukaryotes. In mammals, more than 700 TFs are identified to be DNA-binding TFs. They bind to the TF binding sites (TFBSs) in the genome and regulate the expression of their target genes. kRAS is a proto-oncogene with intrinsic GTPase activity, that contributes to cell proliferation, division, and apoptosis. kRAS mutations are observed in >95% …

Studies Of Sumoylation In Regulating Mif Stability And Rangap1 Nucleo-Cytoplasmic Shuttling In Controlling Its Sumo Modification, Progga Sen

Wayne State University Dissertations

SUMOylation is an essential post-translational modification that regulates a variety of critical cellular pathways ranging from nuclear transport to protein stability. Accumulating lines of evidence have shown that a perturbation of the SUMOylation pathway is associated with human diseases, especially various types of cancer. Our recent proteomic studies revealed a drastic increase in levels of SUMO2/3 modification on the proinflammatory cytokine MIF in the metastatic breast cancer cell line compared to the non-metastatic control cell line. Interestingly, the increase in levels of both MIF and global SUMO-2/3 modification in the metastatic cells are positively correlated to that of unmodified MIF …

Structural Basis Of Membrane Protein Scaffolding And Signaling In Human Disease, Joshua Holcomb

Wayne State University Dissertations

Protein structural elucidation by means of X-ray crystallography is a powerful approach for both insight into a proteins biophysical properties and function. To date X-ray crystallography remains the gold standard in high resolution structural determination and serves as the basis for rational drug design for the purpose of combating a number of human diseases. Such an approach also allows for the exploitation of how various proteins interact with their substrates providing a molecular basis for their physiological function. In this dissertation, using X-ray crystallographic analysis along with other biophysical characterization methods, we seek to understand the mechanistic foundation for which …

Role Of Alström Syndrome 1 (Alms1) In Nkcc2 Endocytosis, Thick Ascending Limb Function, Blood Pressure Regulation And Metabolic Function, Ankita Bachhawat Jaykumar

Wayne State University Dissertations

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na+/K+/2Cl- co-transporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to salt sensitive hypertension in rodents and humans. NKCC2 endocytosis is important for maintaining surface NKCC2 such that blocking NKCC2 endocytosis increased NKCC2 surface abundance and NKCC2-mediated NaCl reabsorption. Despite its importance, NKCC2 endocytosis has been poorly studied and a part of the reason may be attributed to the lack of availability of methods with good time resolution. Hence, we developed a method to image apical NKCC2 to monitor its endocytosis in real-time by Total Internal …

Identifying Multiple Predictors Of Physical Rehabilitation Outcomes, Jacklyn M. Sullivan, Elizabeth B. Fauth, Sydney Y. Schaeffer

Research on Capitol Hill

Previous studies have suggested that disability in activities of daily living (ADLs) is an outcome of cognitive impairment, physical limitations, psychosocial factors, and environmental constraints. We hypothesis that cognitive abilities and psychosocial factors measured at admission can be used to predict a patients physical therapy progression in a transitional care facility. Environmental conditions were held constant for all patients and physical limitations were not used as a predictor of therapy progression, as the test for therapy progression is based on physical ability. In a sample of adults (n=93) aged 38-98 receiving physical rehabilitation treatment, we collected Minimum Data Set (MDS) …

Imaging Live Drosophila Brain With Two-Photon Fluorescence Microscopy, Syeed Ehsan Ahmed

Open Access Theses & Dissertations

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance …

Rad Gtpase: Identification Of Novel Regulatory Mechanisms And A New Function In Modulation Of Bone Density And Marrow Adiposity, Catherine Nicole Kaminski Withers

Theses and Dissertations--Molecular and Cellular Biochemistry

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not …

Viral Fusion Protein Tm-Tm Interactions: Modulators Of Protein Function And Potential Antiviral Targets, Stacy Webb

Theses and Dissertations--Molecular and Cellular Biochemistry

Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning …

Platelet Endocytosis: Roles In Hemostasis And Innate Immunity, Meenakshi Banerjee

Theses and Dissertations--Molecular and Cellular Biochemistry

Endocytosis is key to fibrinogen (Fg) uptake, receptor trafficking of integrins (α_IIbβ₃, α_vβ₃) and purinergic receptors (P2Y₁, P2Y₁₂), and thereby for normal platelet function. However, platelet endocytosis could potentially be critical for actively sensing changes in vascular micro-environments and responding accordingly to what is being taken up. This is a more dynamic view of platelets as active surveyors of the vasculature; extending the importance of platelet endocytosis beyond granule biogenesis and perhaps even hemostasis. The mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required …

Micrornas Modulate Pathogenesis Resulting From Chlamydial Infection In Mice, Laxmi Yeruva, S. Pouncey, Michael R. Eledge, Sudeepa Bhattacharya, Chunqiao Luo, Erin W. Weatherford, David M. Ojcius, Roger G. Rank

All Dugoni School of Dentistry Faculty Articles

Not all women infected with chlamydiae develop upper genital tract disease, but the reason(s) for this remains undefined. Host genetics and hormonal changes associated with the menstrual cycle are possible explanations for variable infection outcomes. It is also possible that disease severity depends on the virulence of the chlamydial inoculum. It is likely that the inoculum contains multiple genetic variants, differing in virulence. If the virulent variants dominate, then the individual is more likely to develop severe disease. Based on our previous studies, we hypothesized that the relative degree of virulence of a chlamydial population dictates the microRNA (miRNA) expression …

Characterization Of Staphylococcal Nuclease And Tudor Domain Containing Protein 1 (Snd1) As A Molecular Target In Hepatocellular Carcinoma And Non-Alcoholic Steatohepatitis, Nidhi H. Jariwala

Theses and Dissertations

CHARACTERIZATION OF STAPHYLOCOCCAL NUCLEASE AND TUDOR DOMAIN CONTAINING PROTEIN 1 (SND1) AS A MOLECULAR TARGET IN HEPATOCELLULAR CARCINOMA AND NON-ALCOHOLIC STEATOHEPATITIS

Nidhi Jariwala, PhD

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Life Sciences

Virginia Commonwealth University, 2017

Devanand Sarkar, M.B.B.S., PhD.

Associate Professor, Department of Human and Molecular Genetics

Virginia Commonwealth University

Richmond, Virginia

SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). …

Study Of Molecular Interactions Of Glycosaminoglycans And Glycosaminoglycan Mimetics With Their Protein Targets, Daniel K. Afosah

Theses and Dissertations

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE).

Anticoagulants are very important for the treatment of thrombotic diseases. The …

Role Of Erk3 In Regulating Rhogdi1-Paks Signaling Axis, Hitham Abdulrahman Aldharee

Browse all Theses and Dissertations

Extracellular signal-regulated kinase 3 (ERK3) is an atypical protein kinase of the mitogen-activated protein kinase (MAPK) family. In comparison to well-investigated ERK1/2 (classical) MAPKs, much less has been discovered about ERK3 signaling and its cellular functions. Recent studies have shown that ERK3 is overexpressed in various types of cancers such as lung cancer and breast cancer and that ERK3 promotes cancer cell migration and invasion. How ERK3 regulates cancer cell motility and invasiveness, however, is still largely unknown. RhoGTPases, including Rho, Cdc42 and Rac1, play critical roles in regulating cell motility and invasiveness through activating downstream effectors such as p21-activated …

Dnp63a Suppresses Cell Invasion By Targeting Rac1 Through Mir-320a, Amjad Ahmed Aljagthmi

Browse all Theses and Dissertations

DNp63a, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and known to play a role in proliferation, differentiation, migration and invasion. DNp63a has been shown to regulate several microRNAs that play a role in both development and cancer, but to date there has not been a global analysis of p63- regulated miRNA. Using next-generation sequencing of small RNA from wild type and sip63 transfected HaCaT cells, our laboratory recently identified a number of DNp63a- regulated miRNAs by RNA-Seq studies which may serve as biomarkers of cancer progression. We identified a novel miRNA, …

Exploring The Role Of Lipin1 In Mitophagy Process Using Lipin1 Deficient-Egfp Tagged Lc3 Transgenic Mice, Abdullah Ali Alshudukhi

Browse all Theses and Dissertations

Lipin1 (phosphatidic acid phosphatase) is a key molecule in the cells with two functions: first, it converts phosphatidic acid into diacylglycerol in the cytosol which in turn makes triglycerides. Second, in nucleus lipin1 acts as a transcriptional factor which regulates the expression of genes involved in the fatty acid oxidation and lipid metabolism. Clinically, Lpin1 gene mutations have been detected in patients with severe rhabdomyolysis accompanied with aggregated and dysfunctional mitochondria in their type II muscle fiber. Previously, we have observed that mice with lipin1 deficiency had aggregated mitochondria and abnormal autophagosomes formations by electron microscopy. The mechanism underlying the …

Methane Fluxes And Porewater Dissolved Organic Carbon Dynamics From Different Peatlands Types In The Pastaza-Marañon Basin Of The Peruvian Amazon., Jhon Del Aguila-Pasquel

Dissertations, Master's Theses and Master's Reports

Relevant components of the carbon budget such as methane fluxes and porewater dissolved organic carbon (DOC) are poorly characterized in the Peruvian Pastaza Marañon basin (PMB). PMB is associated with a tropospheric hotspot for methane and therefore it may represent a significant methane source. We aimed to quantify the magnitude of CH₄ fluxes and porewater DOC; and to determine their predictors across different peatland types in the PMB (open peatland, palm swamp, and pole forest). The study was conducted in two peatland sites covering the three main vegetation types in PMB. We collected gas and water samples using chamber-based …

Investigation Of The Physiological Role Of Rin Gtpase In Cell Death, Axonal Injury, And Inflammation Following Traumatic Brain Injury, Megan Pannell

Theses and Dissertations--Molecular and Cellular Biochemistry

Traumatic brain injury (TBI) is a progressive disorder, in which the primary injury results in the initiation of a complex cascade of secondary biochemical and metabolic changes resulting in lasting neurological dysfunction and cognitive impairment. The heterogeneous nature of the disease has complicated the development of pharmacological agents to improve the outcomes of TBI; to date, no therapeutic treatment has been shown to be effective in clinical trials. Treatments targeting multiple secondary outcomes (cell death, axonal degeneration, and inflammation) may provide enhanced therapeutic efficacy following TBI.

Small Ras family GTP-binding proteins govern diverse cellular processes by directing the relay of …

Rapid Typing Of Mannheimia Haemolytica Major Genotypes 1 And 2 Using Maldi-Tof Mass Spectrometry, John Dustin Loy, Michael L. Clawson

School of Veterinary and Biomedical Sciences: Faculty Publications

Genotype 2 M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate genotype 2 strains in veterinary diagnostic laboratories.

Efficacy Of Urtoxazumab (Tma-15 Humanized Monoclonal Antibody Specific For Shiga Toxin 2) Against Post-Diarrheal Neurological Sequelae Caused By Escherichia Coli O157:H7 Infection In The Neonatal Gnotobiotic Piglet Model, Rodney A. Moxley, David H. Francis, Mizuho Tamura, David B. Marx, Kristina Santiago-Mateo, Mojun Zhao

School of Veterinary and Biomedical Sciences: Faculty Publications

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 x 10⁹ colony-forming units of EHEC O157:H7 strain EDL933 (Stx1⁺, Stx2⁺) when 22–24 h old. At 24 h post-inoculation, piglets were intraperitoneally …

Branched Chain Α-Ketoacid Dehydrogenase Kinase 111–130, A T Cell Epitope That Induces Both Autoimmune Myocarditis And Hepatitis In A/J Mice, Bharathi Krishnan, Chandirasegara Massilamany, Rakesh H. Basavalingappa, Arunakumar Gangaplara, Guobin Kang, Qingsheng Li, Francisco A. Uzal, Jennifer L. Strande, Gustavo A. Delhon, Jean-Jack Riethoven, David J. Steffen, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

Introduction: Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread.

Methods: In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKD_k) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver.

Results: We demonstrate that BCKD_k protein contains at least nine immunodominant epitopes, three of which, BCKD_k 71–90, BCKD_k 111–130 and BCKD_k 141–160, …

Β1-Adrenergic Receptor Contains Multiple IaK And IeK Binding Epitopes That Induce T Cell Responses With Varying Degrees Of Autoimmune Myocarditis In A/J Mice, Rakesh H. Basavalingappa, Chandirasegaran Massilamany, Bharathi Krishnan, Arunakumar Gangaplara, Rajkumar A. Rajasekaran, Muhammad Z. Afzal, Jean-Jack Riethoven, Jennifer L. Strande, David J. Steffen, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is β1-adrenergic receptor (β₁AR). Previous reports indicate that animals immunized with a β₁AR fragment encompassing, 197–222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified β₁AR 171–190, β₁AR 181–200, and β₁AR 211–230 as the major T cell epitopes that bind major histocompatibility complex class II/IA^k or IE^{k …}

The Effect Of Feed Form On Diet Digestibility And Cecal Parameters In Rabbits, Isabella Corsato Alvarenga, Charles Gregory Aldrich, Micah Kohles

School of Veterinary and Biomedical Sciences: Faculty Publications

Fifteen New Zealand rabbits were randomly assigned to one of 3 dietary treatment groups of 5 animals each and fed pelleted, extruded, or muesli diets in a completely randomized design experiment. Rabbits were placed in individual cages with ad libitum access to water and food for 45 days acclimation followed by 30 days experimental period. Feed intake of rabbits fed pelleted and extruded diets was greater (p < 0.05) than rabbits fed the muesli diet (125.6 and 130.4 vs. 91.9 g/d), but weight change and feed efficiency were not affected by treatment. Diet digestibility among the treatments was inconsistent when comparing results obtained from total fecal collection and AIA (please define) as an internal marker. Rabbits fed extruded and pelleted diets had lower (p < 0.05) cecal pH (6.42 and 6.38 vs. 7.02, respectively), and higher (p < 0.05) production of SCFA (18.5 and 19.0 vs. 11.7 mM, respectively) than those fed muesli. The fermentation products from rabbits fed pelleted and extruded diets had a greater proportion of butyrate and less propionate than rabbits fed muesli. The results of this study indicate that the basal dietary composition had a greater impact on diet utilization and cecal fermentation than food form.

Mechanistic Understanding Of N-Glycosylation In Ebola Virus Glycoprotein Maturation And Function, Bin Wang, Yujie Wang, Dylan A. Frabutt, Xihe Zhang, Xiaoyu Yao, Dan Hu, Zhuo Zhang, Chaonan Liu, Shimin Zheng, Shi-Hua Xiang, Yong-Hui Zheng

School of Veterinary and Biomedical Sciences: Faculty Publications

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP₁ and the fusion-mediating GP₂ subunits and incorporated into virions to initiate infection. GP₁ and GP₂ form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP₁ NGSs are not critical, the two GP₂ NGSs, Asn⁵⁶³ and Asn⁶¹⁸, are essential for GP function. Further analysis uncovered that Asn⁵⁶³ and Asn^{618 …}

A Novel Role Of Silibinin As A Putative Epigenetic Modulator In Human Prostate Carcinoma, Ioannis Anestopoulos, Aristeidis P. Sfakianos, Rodrigo Franco, Katerina Chlichlia, Mihalis I. Panayiotidis, David J. Kroll, Aglaia Pappa

School of Veterinary and Biomedical Sciences: Faculty Publications

Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin’s pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, …

Pathogenesis, Molecular Genetics, And Genomics Of Mycobacterium Avium Subsp. Paratuberculosis, The Etiologic Agent Of Johne’S Disease, Govardhan Rathnaiah, Denise K. Zinniel, John P. Bannantine, Judith R. Stabel, Yrjo T. Grohn, Michael T. Collins, Raul G. Barletta

School of Veterinary and Biomedical Sciences: Faculty Publications

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants causing chronic diarrhea, malnutrition, and muscular wasting. Neonates and young animals are infected primarily by the fecal–oral route. MAP attaches to, translocates via the intestinal mucosa, and is phagocytosed by macrophages. The ensuing host cellular immune response leads to granulomatous enteritis characterized by a thick and corrugated intestinal wall. We review various tissue culture systems, ileal loops, and mice, goats, and cattle used to study MAP pathogenesis. MAP can be detected in clinical samples by microscopy, culturing, PCR, and an enzyme-linked immunosorbent assay. There are commercial …

Ccpa Affects Infectivity Of Staphylococcus Aureus In A Hyperglycemic Environment, Markus Bischoff, Bodo Wonnenberg, Nadine Nippe, Naja J. Nyffenegger-Jann, Meike Voss, Christoph Beisswenger, Cord Sunderkotter, Virginie Molle, Quoc Thai Dinh, Frank Lammert, Robert Bals, Mathias Herrmann, Greg A. Somerville, Thomas Tschernig, Rosmarie Gaupp

School of Veterinary and Biomedical Sciences: Faculty Publications

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the …

Significant Enhancement Of Fatty Acid Composition In Seeds Of The Allohexaploid, Camelina Sativa, Using Crispr/Cas9 Gene Editing, Wen Zhi Jhang, Isabelle M. Henry, Peter G. Lynagh, Lucia Comai, Edgar B. Cahoon, Donald P. Weeks

Department of Biochemistry: Faculty Publications

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. …