Biochemistry, Biophysics, and Structural Biology Commons^™

Key Residues Of Human Cytoplasmic Protein Tyrosine Phosphatase-A And -B For Substrate Binding And Specificity, Byunghyun Park

Open Access Theses

Reversible tyrosine phosphorylation plays an important role in signaling pathways that are essential for regulating cellular growth, differentiation and metabolism. Moreover, several human diseases such as diabetes, obesity and cancers are associated with the deregulation of protein tyrosine phosphatases (PTPs). Several studies provide evidence that PTPs not only contribute to cellular differentiation, but over-expression of these molecules also leads to transformation of non-transfomed cells as well. Based on these results, designing specific PTP inhibitors may ultimately function as potential therapeutic agents to treat various diseases including cancer, diabetes, and autoimmune diseases. EphA2 is a receptor tyrosine kinase which is hypo-phosphorylated …

Experimental And Computational Analysis Of The Synucleins, Agatha Munyanyi

Theses and Dissertations in Biomedical Sciences

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson's disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we …

Branching Into Rnai: Synthesis, Characterization And Biology Of Branch And Hyperbranch Sirnas, Anthony Muriithi Maina

Seton Hall University Dissertations and Theses (ETDs)

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new …

Conformationally Stabilized Hiv Envelope Immunogens, Peter Kwong, John Mascola, Gary Nabel, Richard Wyatt, Barna Dey, Ling Xu, Tongqing Zhou, Joseph Sodroski, Wen Yuan, Shi-Hua Xiang

School of Veterinary and Biomedical Sciences: Faculty Publications

Isolated immunogens including a HIV-1 gp120 polypeptide or immunogenic fragment thereof stabilized in a CD4 bound confirmation by crosslinked cysteines, and methods of their use are disclosed. The immunogens are useful, for example, for generating an immune response to HIV-1 gp120 in a Subject.

Defining The Sites Of Interaction Of The Fancd2, Fance, And Fancl Proteins, Joseph Mcclanaghan

Senior Honors Projects

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure and increased cancer susceptibility. FA is caused by mutations in any one of 16 genes. These genes encode for proteins that function in the FA-BRCA pathway to repair damaged DNA. Because of its important r­­­ole in DNA repair, this pathway is considered a major cellular tumor suppressor pathway, i.e. is critical for the prevention of cancer. Underscoring this fact, several of the FA genes - including BRCA2, BRIP1, PALB2, and RAD51C - are bona fide breast and ovarian cancer susceptibility genes.

My …

The Development Of A Comprehensive Antifungal Susceptibility Testing Assay For Vulvovaginal Candidiasis Therapy, David Hardaker

Graduate School of Biomedical Sciences Theses and Dissertations

Vulvovaginal candidiasis is the most common fungal infection of the female urogenital tract, commonly caused by Candida albicans and C. glabrata. However, treatment can be difficult when caused by non-albicans strains due to resistance to the oral antifungal drug fluconazole. The mechanism through which strains of Candida, particularly C. glabrata, develop resistance to different antifungal classes has not been completely characterized. The ergosterol biosynthesis pathway, an important component of the cell membrane, is the major target of antifungals such as fluconazole, an inhibitor of the ERG 11 gene. Current research shows that an upregulation of specific genes in C. glabrata …

Molecular Chaperone Tools For Use Against Neurodegenerative Diseases, Matthew Tinkham

Senior Honors Projects

A noted characteristic found in several neurodegenerative disorders, including Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease and bovine spongiform encephalopathy, is the accumulation of amyloid plaques in the brain. Amyloid plaques contain deposits of fibrillar aggregates of misfolded proteins that disrupt normal functionality in neurons. Certain variants of these misfolded proteins are self-replicating; these self-replicating amyloids are termed prions (for infectious protein). We are interested in how protein misfolding contributes to amyloid formation and how molecular chaperone proteins can change the formation of amyloid deposits. Chaperone proteins function by catalyzing the proper folding of other proteins, the refolding of misfolded proteins, …

Phthalates And Phthalate Alternatives: Effects On Proliferative And Estrogenic Target Genes In Ishikawa Cells, Ranjani Sundar '15, Ping Yin, Serdar E. Bulun

Student Publications & Research

Phthalates are used as plasticizers in many of the products found in medical, household, and industrial applications. Much research has not been completed on the effects of these phthalates as potential endocrine disrupting chemicals (EDCs). As these chemicals are ingested, the mechanism by which they affect the reproductive system is largely unknown. The purpose of this study was to observe how 2 phthalates, Di-n-butyl phthalate (DBP) and Diisononyl phthalate (DINP), and 2 phthalate alternatives, Dioctyl terephthalate (DOTP) and BHT (butylated hydroxytoluene)affect uterine cells in comparison to a vehicle treatment and 17β-Estradiol treatment. Changes in expression of mRNA were observed using …

The Effect Of Small Molecule 390 On Cxcr4 Receptors, Selam B. Zenebe-Gete '14, Shruti R. Topudurti '14, Shum Andrew, Richard J. Miller

Student Publications & Research

CXCR4 is the chemokine receptor which aids in chemotaxis of stem cells, such as those in the bone marrow or the brain. SDF-1 is the natural ligand for the CXCR4 receptor. Similarities between novel molecule 390 synthesized by the Miller Lab and SDF-1 make this novel small molecule a possible agonist of the CXCR4 receptor. To determine whether 390 is an agonist to the CXCR4 receptor, we transfected cells with CXCR4 and exposed them to no agonist [vehicle control], SDF-1, or varying concentrations of our agonist drug. Next, we took calcium images using the dye fura-2, which indicates changes in …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast, Jennifer Lynn Abrams

Dissertations and Theses (Open Access)

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the …

Characterization Of Ftsa-Ftsn Interaction During Escherichia Coli Cell Division, [email protected] K. Busiek

Dissertations and Theses (Open Access)

Division of a bacterial cell into two equal daughter cells requires precise assembly and constriction of the division machinery, or divisome. The Escherichia coli divisome includes nearly a dozen essential cell division proteins that assemble at midcell between segregating sister chromosomes. FtsZ, a homolog of eukaryotic tubulin, is the first essential cell division protein to localize at midcell where it polymerizes into a ring-shaped scaffold (Z ring). Establishment of the Z ring is required for recruitment of downstream cell division proteins including FtsA, a cytoplasmic protein that tethers the Z ring to the inner membrane. Following localization of FtsA and …

Diabetes And Obesity Induce Transcriptomic And Metabolomic Changes Enhancing Pancreatic Cancer Aggressiveness, Guermarie Velázquez Torres

Dissertations and Theses (Open Access)

Pancreatic cancer is one of the most aggressive types of cancer, with poor prognosis that lacks effective diagnostic markers and therapies. It is expected that in 2014 the incidence and the mortality of pancreatic cancer in the United States will be 46,420 and 39,590 respectively. Diabetes and obesity are modifiable risk factors associated with accelerated pancreatic carcinogenesis and tumor progression, but the biological mechanisms are not completely understood. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating these epidemiologic phenomena. Our hypothesis is that obesity and diabetes mellitus type 2 (DM2) accelerate pancreatic cancer and …

The Regulation Of Microrna Biogenesis By Ribosome-Interacting Proteins, Brian Pickering

Dissertations and Theses (Open Access)

MicroRNA (miRNA) are small, non-coding RNAs that affect gene expression through degradation of complementary mRNA targets or inhibition of translation. As they affect approximately 50% of all cellular processes, miRNA are tightly regulated by the cell through transcriptional and post-transcriptional mechanisms. Transcribed miRNA are capped and polyadenylated (referred to as pri-miRNA) which are cleaved by Drosha and DGCR8 to generate 60-90 nucleotide precursor miRNA. The precursors are cleaved again by Dicer and loaded into the RNA-induced silencing complex (RISC) of which Argonaute 2 is the functional component. Many of the proteins involved in miRNA biogenesis share a common role in …

Structural And Functional Analysis Of Harv1 Lipid Binding, Jamie Francisco

Graduate School of Biomedical Sciences Theses and Dissertations

Cardiovascular disease (CVD) has the highest mortality rate worldwide and is considered epidemic in the United States. Statins as the primary and almost sole method of treatment for cholesterol-related CVD, but they have limitations. Proper sterol transport is needed to maintain normal blood lipid levels. Studies have shown that misregulation of lipid trafficking aids in the progression of CVD and in the activation of the unfolded protein response (UPR). Misregulation of lipid and sterol homeostasis also results in the loss of lipid rafts, known cellular signaling hubs and major components of ER associated membrane contact sites. Arv1 is a highly …

Cd8Α Dendritic Cells Drive Establishment Of Hsv-1 Latency, Kevin R. Mott, Sariah J. Allen, Mandana Zandian, Bindu Konda, Behrooz G. Sharifi, Clinton Jones, Steven L. Wechsler, Terrence Town, Homayon Ghiasi

School of Veterinary and Biomedical Sciences: Faculty Publications

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wildtype (WT) C57BL/6 versus CD8α^‒/‒ (lack functional CD8 T cells and CD8α⁺ DCs), CD8β^‒/‒ (have functional CD8α⁺ T cells and CD8α⁺ DCs), and β2m^‒/‒ (lack functional CD8 T cells but have CD8α^{+ …}

Neurotrophins And Their Effects On Breast Cancer Cell Proliferation And Migration, Kayla Elise Minser

Open Access Theses

Cancer is a large health issue in all parts of the world. In the United States alone, approximately 1 in 4 deaths are cancer related. Breast cancer is a particularly prevalent form, accounting for a little over 14 percent of all cancer incidence. The largest obstacle to overcome for breast cancer morbidity is metastasis. Over 90 percent of all breast cancer related deaths are due to metastasis. Because metastasis is a complex, multi-step process, it is difficult to treat. A recent observation in the Kirshner lab has revealed a type of phenotypic plasticity, where migratory cancer cells have a neuronal-like …

Overexpression Of Thioredoxin Binding Protein (Tbp-2) Increases Oxidation Sensitivity And Apoptosis In Human Lens Epithelial Cells, Yibo Yu, Kuiyi Xing, Rilwan Badamas, Charles A. Kuszynski, Hongli Wuand, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multi-physiological functions in cells and its bioavailability is negatively controlled through active site binding to a specific thioredoxin binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx, and the effect of overexpression of TBP-2 in the human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a 7- fold increase of TBP-2, and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day …

Exploration Of Mutations In Erythroid 5-Aminolevulinate Synthase That Lead To Increased Porphyrin Synthesis, Erica Jean Fratz

USF Tampa Graduate Theses and Dissertations

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed …

Evaluation Of Novel Oral Vaccine Candidates And Validation Of A Caprine Model Of Johne’S Disease, Murray E. Hines Ii, Sue E. Turnquist, Marcia R.S. Ilha, Sreekumari Rajeev, Arthur L. Jones, Lisa Whittington, John P. Bannantine, Raul G. Barletta, Yrjö T. Gröhn, Robab Katani, Adel M. Talaat, Lingling Li, Vivek Kapur

School of Veterinary and Biomedical Sciences: Faculty Publications

Johne’s disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn’s disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne’s Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne’s disease go at challenge model. …

Molecular Pap Biomarkers For Cervical Cancer Risk Assessment In Thinprep® Clinical Specimens, Jennifer Taylor

Graduate School of Biomedical Sciences Theses and Dissertations

Approximately 500,000 patients are diagnosed with cervical cancer worldwide each year, thus making it the second most common cause of cancer-related deaths in women. Persistent high-risk human papillomavirus (HR-HPV) infections induce cervical dysplasia by increasing the expression of two viral oncogenes, E6 and E7. Utilization of the Papanicolaous smear test (Pap test) has contributed to a decrease in morbidity and mortality of cervical cancer; however, subjective analysis of cell morphology with a limited amount of cells has led to a substantial rate of false positive and false negative diagnoses. A more objective and sensitive diagnostic tool for the detection of …

Immature Myeloid Cells Promote Tumor Formation Via Non-Suppressive Mechanism, Myrna Lillian Ortiz

USF Tampa Graduate Theses and Dissertations

ABSTRACT

Although there is ample evidence linking chronic inflammation with cancer, the cellular mechanisms involved in early events leading to tumor development remain unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells represented by macrophages, dendritic cells and granulocytes and a population of Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to mature cells. There is a substantial expansion of IMC in cancer and many other pathological conditions which is associated with pathologic activation of these cells. As a result, these cells acquire the ability to suppress immune responses and are …

Polymerase Alpha Components Associate With Telomeres To Mediate Overhang Processing, Raffaella Diotti

Dissertations, Theses, and Capstone Projects

Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a …

The Sex Of The Cell Dictates Its Response, Carlos Ganesh Penaloza

Dissertations, Theses, and Capstone Projects

Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of …

Lipid Dependence In Ras-Driven Tumors, Darin Salloum

Dissertations, Theses, and Capstone Projects

Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. …

Metabolic Checkpoints In Cancer Cell Cycle, Mahesh Saqcena

Dissertations, Theses, and Capstone Projects

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from …

Identification And Characterization Of Protein Phopshatases Regulating The Sma/Mab Pathway In C. Elegans, Sheng Xiong

Dissertations, Theses, and Capstone Projects

TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved …

The Effects Of Lipoxin A4 (Lxa4) On Neutrophil Biology In Sepsis, Benedict Wu

Graduate School of Biomedical Sciences Theses and Dissertations

During sepsis, neutrophils are inappropriately activated and have prolonged lifespans, thus becoming dysfunctional. Excessive neutrophil activation can lead to tissue injury while neutrophil dysfunction can lead to decreased free radical production and reduced phagocytosis, preventing the host from clearing preexisting infections. Lipoxin A4 (LXA4) is a specialized pro-resolution mediator which reduces neutrophil migration and expression of proinflammatory mediators. Intact neutrophil functions are critical for the host to efficiently clear invading pathogens. The effects of LXA4 on neutrophil function in sepsis have not been established. Using the cecal ligation and puncture (CLP) model of sepsis, LXA4 administered 1 h after sepsis …

The Role Of Mammalian Cyclin C In Stress Response And Thyroid Disease Progression, Kun Wang

Graduate School of Biomedical Sciences Theses and Dissertations

The cellular response to stress involves changes in gene expression programs as well as the remodeling of subcellular organelles including the mitochondria. Important for this study, the mitochondria undergo extensive fragmentation when the cell is exposed to a variety of stressors. The cyclin C-Cdk8 kinase is a highly conserved transcription factor that associates with the RNA polymerase II holoenzyme. Using cyclin C knockout (CCNC^-/-) mouse embryonic fibroblast (MEF) cells, I revealed that cyclin C is required for the extensive mitochondrial fragmentation following treatment with pro-oxidants or the anti-cancer drug cisplatin. However, Cdk8 is not required for this process …