Biochemistry, Biophysics, and Structural Biology Commons^™

Hepatic Nutrient And Hormonal Regulation Of The Pancreatic-Derived Factor (Pander) Promoter, Whitney Ratliff

USF Tampa Graduate Theses and Dissertations

PANcreatic-DERived factor (PANDER, FAM3B) has been shown to regulate glycemic levels via interactions with both pancreatic islets and the liver. Although PANDER is predominantly expressed from the endocrine pancreas, recent work has provided sufficient evidence that the liver may also be an additional tissue source of PANDER production. At physiological levels, PANDER is capable of disrupting insulin signaling and promoting increased hepatic glucose production. As shown in some animal models, strong expression of PANDER, induced by viral delivery within the liver, induces hepatic steatosis. However, no studies to date have explicitly characterized the transcriptional regulation of PANDER from the liver. …

Novel Enzyme Perspectives: Arylalkylamine N-Acyltransferases From Bombyx Mori & 1-Deoxy- D-Xylulose-5-Phosphate Synthase From Plasmodium Falciparum And Plasmodium Vivax, Matthew R. Battistini

USF Tampa Graduate Theses and Dissertations

This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction.

Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding …

Structure Based Drug Design Targeting Bacterial Antibiotic Resistance And Alzheimer's Disease, Eric Michael Lewandowski

USF Tampa Graduate Theses and Dissertations

Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, …

Characterization And Diurnal Measurement Of Oral Inflammation In Association With Glycemic Control, Periodontal Status, & Glucose Stimulation, Melanie N. Kuehl

USF Tampa Graduate Theses and Dissertations

Diabetes has afflicted 8.3%, approximately 25.8 million, of the United States population and is the seventh leading cause of death [1]. Type I diabetes (T1D) accounts for 5 to 10% of all diagnosed cases of diabetes in the United States [2]. If present trends continue, the rate of T1D incidence among children under the age of 14 will increase by 3% globally [3]. T1D is an autoimmune disorder in which the β-cells of the pancreatic islets are destroyed, leading to high blood sugar. Hyperglycemia and loss of immunological tolerance to self-antigens are common associations of T1D [4]. Periodontal disease impacts …

Photoacoustic Calorimetry Studies Of The Earliest Events In Horse Heart Cytochrome-C Folding, Tarah A. Word

USF Tampa Graduate Theses and Dissertations

The protein folding problem involves understanding how the tertiary structure of a protein is related to its primary structure. Hence, understanding the thermodynamics associated with the rate-limiting steps for the formation of the earliest events in folding is most crucial to understanding how proteins adopt native secondary and tertiary structures. In order to elucidate the mechanism and pattern of protein folding, an extensively studied protein, Cytochrome-c (Cc), was chosen as a folding system to obtain detailed time-resolved thermodynamic profiles for the earliest events in the protein folding process. Cytochrome-c is an ideal system for understanding the folding process for several …

A Forward Genetic Screen Identifies Factors Associated With Fever Pathogenesis In Plasmodium Falciparum, Phaedra J. Thomas

USF Tampa Graduate Theses and Dissertations

Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go …

Function Of Long Noncoding Rnas In Breast Cancer, Edward J. Richards

USF Tampa Graduate Theses and Dissertations

Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., …

Promoting Genome Stability Via Multiple Dna Repair Pathways, Scott Cukras

USF Tampa Graduate Theses and Dissertations

Maintaining genome integrity is indispensible for cells to prevent and limit accruement of deleterious mutations and to promote viable cell growth and proliferation. Cells possess a myriad of mechanisms to detect, prevent and repair incurred cellular damage. Here we discuss various proteins and their accompanying cellular pathways that promote genome stability. We first investigate the NEDD8 protein and its role in promoting homologous recombination repair via multiple Cullin E3 ubiquitin ligases. We provide specific mechanisms through which, UBE2M, an E2 conjugating enzyme, neddylates various Cullin ligases to render them catalytically active to degrade their substrates by the proteasome. We show …

A Functional Chlorophyll Biosynthesis Pathway Identified In The Kleptoplastic Sea Slug, Elysia Chlorotica, Julie A. Schwartz

USF Tampa Graduate Theses and Dissertations

The sacoglossan sea slug, Elysia chlorotica, feeds upon and sequesters plastids from the heterokont alga, Vaucheria litorea, and maintains the metabolically active organelles for up to nine months under starvation conditions while utilizing the photosynthate to survive and reproduce. The photosynthetic pigment, chlorophyll a (Chla), is found in all oxygenic photosynthetic organisms and is responsible for capturing photons of light and converting them into chemical energy. Chlorophyll and its associated proteins involved in the light capturing process are subject to photo oxidative damage and must be continually replaced for ongoing photosynthesis to continue; however, genes encoding these proteins are present …

Gamma-Aapeptides As A New Class Of Peptidomimetics: Synthesis, Structures, And Functions, Haifan Wu

USF Tampa Graduate Theses and Dissertations

Peptidomimetics are synthetic oligomers that resemble the activities of peptides. Their advantages over peptides include high stability towards proteolysis and enormous chemical diversity. Over the past two decades, there have been extensive efforts to develop peptide mimics, such as beta-peptides, peptoids, D-peptides, etc. The research on peptidomimetics have led to many important applications in both medicinal and material science. In order to explore new functions, the discovery of peptidomimetics with novel frameworks is essential. We reported the synthesis and evaluation of a new class of peptidomimetics, termed as gamma-AApeptides. Previous studies of gamma-AApeptides have revealed that gamma-AApeptides are highly resistant …

Identification And Characterization Of N-Acyltransferase Enzymes That Are Involved In The Biosynthesis Of Fatty Acid Amides, Daniel Robert Dempsey

USF Tampa Graduate Theses and Dissertations

Fatty acid amides are an emerging family of bioactive lipids that consists of N-acylethanolamines, N-acylarylalkylamides, N-acylglycines, N-acyl amino acids, N-monoacylpolyamides, and primary fatty acid amides. Short chain fatty acid amides are products of inactivated biogenic amines such as dopamine, histamine, octopamine, and serotonin, whereas long chain fatty acid amides have been implicated in a number of physiological process such as the perception and inhibition of chronic pain through binding to their specific receptors. The most famous; therefore, the most studied long chain fatty acid amide is anandamide or also known as N-arachidonylethanolamine. The biosynthesis …

Regulation And Targeting Of The Fancd2 Activation In Dna Repair, Valentina Celeste Caceres

USF Tampa Graduate Theses and Dissertations

Fanconi anemia (FA) is a genome instability syndrome that is clinically manifested by bone marrow failure, congenital defects, and elevated cancer susceptibility. The FA pathway is known to regulate the repair of DNA interstrand crosslinks in part through DNA homologous recombination (HR) repair. Up to today 16 FA proteins have been discovered that may participate in the common pathway. Cells that have mutations in the FA genes are hypersensitive to DNA damaging agents and display chromosome instability. A key regulatory event in the FA pathway is monoubiquitination of FANCD2-FANCI heterodimer that is mediated by a multi-component E3 ubiquitin ligase complex …

Functional Analysis Of The Ovarian Cancer Susceptibility Locus At 9p22.2 Reveals A Transcription Regulatory Network Mediated By Bnc2 In Ovarian Cells, Melissa Buckley

USF Tampa Graduate Theses and Dissertations

GWAS have identified several chromosomal loci associated with ovarian cancer risk. However, the mechanism underlying these associations remains elusive. We identify candidate functional Single Nucleotide Polymorphisms (SNPs) at the 9p22.2 ovarian cancer susceptibility locus, several of which map to transcriptional regulatory elements active in ovarian cells identified by FAIRE-seq (Formaldehyde assisted isolation of regulatory elements followed by sequencing) and ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) in relevant cell types. Reporter and electrophoretic mobility shift assays (EMSA) determined the extent to which candidate SNPs had allele specific effects. Chromosome conformation capture (3C) reveals a physical association between Basonuclin 2 (BNC2) and …

High-Throughput Screening Of Age-Related Changes In Caenorhabditis Elegans, Neil Copes

USF Tampa Graduate Theses and Dissertations

This project was developed to identify novel methods for high-throughput culturing and screening of C. elegans to investigate age-related metabolic changes and to survey the proteomic and metabolomic factors associated with age-related changes. To accomplish these goals we developed a novel way to grow C. elegans in liquid culture in 96-well microplates for several weeks without suffering significant fluid loss due to evaporation and without needing to shake or unseal the plates for aeration. We also developed methods for assaying the total volume of live C. elegans in microplate cultures using a fluorescence microplate reader and for performing RNAi experiments …

Strategies For Preventing Age And Neurodegenerative Disease-Associated Mitochondrial Dysfunction, Vedad Delic

USF Tampa Graduate Theses and Dissertations

Mitochondrial dysfunction plays a pivotal role in the development of aging phenotypes and aging-associated neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS). Strategies that restore mitochondrial dysfunction may rescue the deficits of central metabolism in these disorders and improve cell survival. For example, we found that modulating the mTOR signaling pathway in a tissue culture model of aging-induced mitochondrial DNA mutation enhanced mitochondrial function as evidenced by increased oxygen consumption. Our previous melatonin studies also led us to hypothesize that caloric restriction and the hormone melatonin would reverse brain mitochondrial dysfunction in animal …

Genomic Aberrations At The 3q And 14q Loci: Investigation Of Key Players In Ovarian And Renal Cancer Biology, Punashi Dutta

USF Tampa Graduate Theses and Dissertations

Genomic aberrations are primary contributors to the pathophysiology of cancer [11]. Dysregulated expression of genes located within these aberrations are important predictors of chemoresistance, disease prognosis, and patient outcome [12]. This dissertation is focused on understanding the regulation and/or functions of specific genes located at dysregulated genomic regions such as 3q26 and 14q32 in the biology of ovarian and renal cancer, respectively.

Serous epithelial ovarian cancer (EOC) manifest amplification at the 3q26.2 locus [2], an observation consistent with the cancer genome atlas (TCGA) [13]. The most amplified gene in this region is EVI1 which has been extensively studied in hematological …

The Effects Of Supplemented Metabolites On Lifespan And Stress Response Pathways In Caenorhabditis Elegans, Clare B. Edwards

USF Tampa Graduate Theses and Dissertations

Understanding how metabolites contribute to anaplerosis, antioxidant effects, and hormetic pathways during aging is fundamental to creating supplements and dietary habits that may decrease age-associated disease and decline, thus improving the quality of life in old age. In order to uncover metabolic pathways that delay aging, the effects of large sets of metabolites associated with mitochondrial function on lifespan were investigated.

Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in C. elegans. Addition of fumarate and succinate also extended lifespan and all three metabolites activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced …

Minocycline Treatment And The Necessity To Develop A Novel Outcome Measure For Children With Angelman Syndrome, Joseph Christopher Grieco

USF Tampa Graduate Theses and Dissertations

Angelman syndrome (AS) is a rare genetic disorder affecting 1/10,000 to 1/20,000 births. This disorder arises through the genetic disruption of the maternal UBE3A allele, which when coupled with epigenetic silencing of the paternal allele UBE3A allele, gives rise to an absence of UBE3A protein in the central nervous system. Clinical manifestations of the syndrome vary in severity and include poor motor function, deficits in language and severe intellectual impairments. Previous research in the Angelman syndrome mouse model revealed abnormalities in dendritic spine density and morphology of hippocampal pyramidal cells. As seen in humans with AS, mice show abnormal behavioral …

Immunological Characterization Of Duffy Binding Protein Of Plasmodium Vivax, Miriam Thankam George

USF Tampa Graduate Theses and Dissertations

Plasmodium vivax Duffy binding protein (DBP) is an essential ligand for reticulocyte invasion making it a premier asexual blood stage vaccine candidate. However, strain-specific immunity due to DBPII allelic variation may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target immune responses to conserved epitopes that are potential targets of strain-transcending neutralizing immunity. Anti DBPII monoclonal antibodies, which were previously characterized by COS7 cell binding assay as inhibitory and non-inhibitory to DBPII-erythrocyte binding, were mapped to DBPII gene fragment libraries using phage display. Inhibitory mAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3 while non-inhibitory …

Biosynthesis Of Long-Chain Fatty Acid Amides, Kristen A. Jeffries

USF Tampa Graduate Theses and Dissertations

The vast variety of long-chain fatty acid amides identified in biological systems is intriguing. The general structure of a fatty acid amide is R-CO-NH-X, where R is an alkyl group and X is derived from an immense variety of biogenic amines. Although structurally simple, the bioactivities of these molecules as signaling lipids are very diverse and have just recently begun to emerge in the literature. Interest in the long-chain fatty acid amides dramatically increased following the identification and characterization of one specific N-acylethanolamine, N-arachidonoylethanolamine (anandamide), as the endogenous ligand for the cannabinoid receptors in the mammalian brain. Since …

Exploring The Role Of Intracellular Aminopeptidases In Staphylococcus Aureus Pathogenesis, Devon Nicole Marking

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus is a remarkably pathogenic bacterium that is widely prevalent among the human population. It is the leading agent of skin and soft tissue infections, and is also responsible for causing an array of severe and life threatening diseases. The invasiveness of the pathogen, coupled with increasing antibiotic resistance seen for S. aureus infections, makes this bacterium a prominent public health concern. The extended pathogenicity of S. aureus is largely due to its repertoire of virulence factors, which are typically characterized by being bound to the cell wall, or secreted into the extracellular environment. Previously, our lab identified a …

A Novel Role For Nf-Κb In Proximal T Cell Signaling, Crystina Bronk Watson

USF Tampa Graduate Theses and Dissertations

The interrogation of T cell signaling over the past fifty years has led to the discovery of amazingly intricate cascade networks and elaborate descriptions of individual proteins' domains and functions. A complex landscape has been rendered in which proteins relay messages from the extracellular ligation of the TCR by a cognate peptide loaded MHC via changes in sub-cellular location, phosphorylation, and binding affinities and partners to enact nuclear localization of three key transcription factors required for cellular effector function and proliferation: AP-1, NF-AT, and NF-κB. Dogma has favored activation of each of these transcription regulating elements to be a linear …

Mechanisms And Molecular Biology Of Major Tumor Suppressors, Brienne E. Engel

USF Tampa Graduate Theses and Dissertations

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively.

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes …

Role And Regulation Of Snon/Skil And Plscr1 Located At 3q26.2 And 3q23, Respectively, In Ovarian Cancer Pathophysiology, Madhav Karthik Kodigepalli

USF Tampa Graduate Theses and Dissertations

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian …

Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski

USF Tampa Graduate Theses and Dissertations

Unraveling the microbiological processes that occur as water travels from a river's mouth into the ocean is critical to understanding the role of river plumes in global biogeochemical cycles. Metranscriptomics, the gene expression of a whole community of organisms, was utilized to examine six stations along the Amazon River Plume (ARP) in 2010 to test thehypothesis that there were measurable differences in gene expression for key biogeochemical genes along the ARP. This body of work focuses on methods developed to identify which genes are biogeochemically important for a particular environment along extreme salinity, nutrient and community gradients in the ARP, …

The Interrelationship Of Brca1 185delag, Interleukin-1Β, And Ovarian Oncogenesis, Kamisha Woolery

USF Tampa Graduate Theses and Dissertations

While the etiology of ovarian cancer (OC) is not completely understood, evidence suggests that chronic inflammation may promote malignant transformation. However, familial history remains the strongest risk factor for developing OC and is associated with germline BRCA1 mutations, such as the 185delAG mutation. Normal human ovarian surface epithelial cells expressing the 185delAG mutant, BRAT, exhibit molecular and pathological changes that may contribute to OC oncogenesis. In the current study, I sought to determine whether BRAT could promote an inflammatory phenotype by investigating BRAT's impact on the expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Using a culture model system of normal …

Rna Detection Technology For Applications In Marine Science: Microbes To Fish, Robert Michael Ulrich

USF Tampa Graduate Theses and Dissertations

The accurate identification of taxa from mixed assemblages using genetic analysis remains an important field of molecular biology research. The common principle behind the development of numerous documented genetic detection technologies is to exploit specific nucleotide sequences inherent to each taxon. This body of work focuses on practical applications of real-time nucleic acid sequence-based amplification (RT-NASBA) in marine science, and is presented in four case studies. Each study represents novel work in the genetic identification of respective taxa of interest using RT-NASBA. Two case studies documented the development of an assay targeting mitochondrial 16S rRNA to discern legally salable grouper …

Synthesis, Characterization And Mechanistic Studies Of Biomolecules@Mesomofs, Yao Chen

USF Tampa Graduate Theses and Dissertations

Encapsulation of biomolecules is of great interest to research advances related to biology, physiology, immunology, and biochemistry, as well as industrial and biomedical applications such as drug delivery, biocatalysis, biofuel, food and cosmetics. Encapsulation provides functional characteristics that are not fulfilled by free biomolecules and stabilizes the fragile biomolecules. In terms of biocatalysis, solid support can often enhance the stability of enzymes, as well as facilitate separation and recovery for reuse while maintaining activity and selectivity. Various kinds of materials have been used for encapsulation of biomolecules, among which, porous materials are an important group. Metal-organic frameworks (MOFs) have attracted …

Novel Cell Cycle Proteins In Apicomplexan Parasites, Carrie Butler

USF Tampa Graduate Theses and Dissertations

Apicomplexans are responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii (T. gondii) and the deadliest form of malaria caused by Plasmodium falciparum (P. falciparum). The genomes of these pathogens are now sequenced ushering in a new era of drug development. A major hurdle to exploiting this genome resource is that a large number of the encoded genes are "hypotheticals" and have yet to be characterized. Hypothetical proteins comprise roughly half of the predicted gene complement of T. gondii and P. falciparum and represent the largest class of uniquely functioning proteins in these parasites.

Following the idea …

Genetic Basis For The Virulence Of Enterohemorrhagic Escherichia Coli Strain Tw14359, Jason Kyle Morgan

USF Tampa Graduate Theses and Dissertations

Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak …