Molecular Biology Commons^™

Dissecting The Roles Of Human Smc Complexes In Transcription Regulation And Chromatin Organization, Ruoyu Wang

Dissertations and Theses (Open Access)

Metazoans utilize a constellation of distal regulatory elements to control gene transcription, and therefore they have to forge highly complex chromatin loops to spatially bridge these regulatory elements and genes in the three-dimensional (3D) genome. However, the hierarchy of chromatin contacts and their underlying mechanisms are not well-understood. SMC complexes including Cohesin complex and Condensin complex has been widely proposed to organize 3D genome structure, and further regulate metazoans’ gene transcription. Here, we aim to dissect the direct functions of SMC complexes (both Cohesin and Condensin) in transcriptional regulation and 3D genome organization, by utilizing an inducible protein degradation system. …

Antibiotic Drug Discovery Targeting Bacterial Metabolism, Miranda J. Wallace

Theses and Dissertations (ETD)

Over the last century, the use of antibiotics has enabled many advances in modern medicine, making life as we know it possible. In recent years, however, emerging bacterial resistance to virtually all major antibiotic classes has resulted in a worldwide increase in morbidity, mortality, and financial burden associated with drug resistant infections. The antimicrobial resistance crisis presents an urgent need for new antimicrobials with distinct mechanisms of action from existing drugs. The current pharmaceutical pipeline of new antibiotics is limited due to three obstacles: a lack of understanding of resistance mechanisms, a dearth of novel mechanisms of action among new …

Investigation Of Microbiota In Health And Disease Of Poultry, Bishnu Adhikari

Graduate Theses and Dissertations

The microbiotas play vital roles in health and diseases of both humans and animals. 16S rRNA genes sequence analysis is one of the most popular and commonly used methods in the analysis of microbiotas associated with hosts. In this dissertation, the microbiotas of chickens (broilers, breeders, and layers) and turkeys were evaluated by 16S rRNA gene sequencing. Characterization of the culturable subpopulations of Lactobacillus in the chicken gut can serve as a valuable resource for probiotic development. In Chapter 2, Lactobacillus subpopulations recovered on MRS from chicken gut were defined comprehensively for the first time using 16S rRNA gene profiling, …

Microrna Identification And Target Prediction In The Whitefly (Bemisia Tabaci), Alexis Aleman

Honors Theses

RNA interference, referred to as RNAi, is a biological phenomenon whereby knock-down of gene expression can be achieved through the use of RNA molecules, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). The use of miRNAs is an endogenous pathway that results in the degradation of the miRNA strand’s complementary messenger RNA (mRNA), preventing the translation of the mRNA and therefore the production of the proteins necessary for gene expression. While RNAi is a biological phenomenon that occurs naturally, it is also a method that can be used and manipulated in the laboratory to try to control the expression of …

The Dynamin-Like Protein Vps1 Stimulates Endosome-To-Golgi Fusion In Vitro, Jared Christopher Smothers

Graduate Theses/Dissertations

Intracellular membrane fusion events can be reconstituted by exploiting isolated organelles from cellular hosts or artificial membranes made of purified phospholipid components. Artificial construction of membranes provides two significant advantages. First, cellular isolation of the endosome-derived vesicles and TGN (trans-Golgi Network) compartments needed for the fusion assay would be extremely challenging. Second, reconstituting the membranes provides the added benefit of controlling size and lipid compositions to functionally mimic the individual membrane architectures and introduce only the purified proteins that are under investigation. For these reasons, I have developed the first simultaneous lipid and content mixing fusion assays that measures the …

Understanding The Molecular And Cellular Functions Of Odd-Skipped Related 1 In Outflow Tract Development, Menglan Xiang

Theses and Dissertations

The cardiac outflow tract (OFT) is a transient conduit that connects the embryonic heart chambers to the vascular network. Transcription factor Osr1 promotes the proliferation and cell cycle progression of second heart field (SHF), an essential cell population that contribute to the developing OFT. In this study, we investigated the role of Osr1 in OFT development on cellular and molecular levels using a systems biology approach. We observed OFT rotation and elongation defects, as well as double-outlet right ventricle and overriding aorta as a result of SHF-specific deletion of Osr1. Using genetic inducible fate mapping, we showed that Osr1-expressing SHF …

The Distinct Expressions Of Integrins Αdβ2 And Αmβ2 Differently Regulate Macrophage Migration In 3d Matrix In Vitro And In Tissue During Inflammation, Kui Cui

Electronic Theses and Dissertations

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins α_Dβ₂ and α_Mβ₂ to macrophage migration and development of chronic inflammation.

We found that integrin α_Dβ₂ is upregulated on M1-macrophages in vitro and …

Does Thermotolerance In Daphnia Depend On The Mitochondrial Function?, Rajib Hasan

Electronic Theses and Dissertations

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18^ᵒC and 25^ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP …

Translational Control Of Antibiotic Resistance, Anne Witzky, Rodney Tollerson Ii, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Many antibiotics available in the clinic today directly inhibit bacterial translation. Despite the past success of such drugs, their efficacy is diminishing with the spread of antibiotic resistance. Through the use of ribosomal modifications, ribosomal protection proteins, translation elongation factors and mistranslation, many pathogens are able to establish resistance to common therapeutics. However, current efforts in drug discovery are focused on overcoming these obstacles through the modification or discovery of new treatment options. Here, we provide an overview for common mechanisms of resistance to translation-targeting drugs and summarize several important breakthroughs in recent drug development.

Intrinsically Disorder Proteins And Liquid-Liquid Phase Transitions In Neurodegenerative Diseases, April Lynn Darling

USF Tampa Graduate Theses and Dissertations

Neurodegenerative diseases have a negative impact on health and economics, effecting more than 20 million people in the United States and costing around $800 billion in 2018. Additionally, most neurodegenerative diseases such as Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and several others are fatal and have no cure or effective treatment. Many of these diseases have a common theme of protein aggregates being present in disease tissue. These aggregates are thought to be toxic to the cell and prevention of pathological aggregation and elimination of these cellular aggregates may serve as a potential therapeutic approach to …

Novel Strategies On Characterizing Biologically Specific Protein-Protein Interaction Networks, Bi Zhao

USF Tampa Graduate Theses and Dissertations

The function, behavior, and environmental response of biological systems are essentially determined by the complex interaction and regulation of biomolecules inside the systems. Therefore, it is critical to characterize the inter-molecular interaction and regulation of biomolecules inside these systems. In this direction, many experimental techniques have been developed and these techniques have been used in many different model systems under various conditions. Consequently, a massive amount of data has been generated. These data cover multiple aspects of molecular interaction and regulation, such as protein-protein interaction, microRNA-RNA interaction, gene expression profiles, etc. While carrying rich information, these data may also contain …

Targeting Pten For Therapy In Cancer And Ptenopathies, Emily Palumbo

USF Tampa Graduate Theses and Dissertations

PTEN, a dual protein and lipid phosphatase, regulates a myriad of cellular functions including PI3K pathway signaling, cell migration, proliferation, invasion and apoptosis. PTEN mutations often lead to multiple malignancies, including prostate, breast, endometrial, skin and brain cancers, associated with hyperactive PI3K signaling. PTEN mutations have also been associated with a variety of other diseases, classified as PTEN Hamartoma Tumor Syndromes (PHTS). In addition, compromised function or reduced expression of PTEN due to non-genomic mechanisms are associated with many types of hyperproliferative diseases, such as restenosis and neoplastic diseases, including melanoma, lung, breast, prostate and colon cancers. Although PI3K pathway …

Roux-En-Y Gastric Bypass Attenuates Obesity-Related Liver Injury, Drew Alan Rideout

USF Tampa Graduate Theses and Dissertations

Obesity is a growing epidemic in the United States with significant co-morbidities. Non-Alcoholic Fatty Liver Disease (NAFLD) is a prevalent manifestation of obesity that can lead to cirrhosis. Roux-en-Y gastric bypass (RYGB) results in substantial long-term weight loss and resolution of obesity-related metabolic diseases. There appears to be a weight-independent molecular mechanism for the improvement of diabetes mellitus and NAFLD after RYGB, which is poorly understood. Obesity is associated with chronic inflammation that accompanies the hepatic steatosis. Through unknown mechanisms, RYGB in humans increases serum levels of the fat-derived adipocytokine, adiponectin. Adiponectin (an anti-inflammatory cytokine) is known to have a …

A High Throughput Assay For The Detection Of Stimulator Of Interferon Genes (Sting) Agonists, Michael J. Ingling

Graduate School of Biomedical Sciences Theses and Dissertations

The innate immune system includes a menagerie of different cell types, each with a different role in the process of monitoring the body for invaders and presenting gathered debris (antigen) to the adaptive immune system. Somatic cells have intracellular receptors for the same purpose. Cancer cells, however, have avoided these methods of detection despite, in many cases, the tumor’s immunogenic traits. Immuno-oncology is a field dedicated to the immunological traits of tumors, more recently finding ways of instigating an immune response against tumors. In this regard, STING, a receptor of cyclic dinucleotides (CDN), has come to the forefront of immuno-oncology. …

Characterization Of Human Dutpase, Shawna Marie Rotoli

Graduate School of Biomedical Sciences Theses and Dissertations

Deoxyuridine nucleotidyl transferase (dUTPase) is an enzyme found in all organisms that have thymine as a component of DNA. It catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate thus precluding the buildup of dUTP pools as well as providing the substrate, dUMP, for the de novo synthesis of thymidylate. In Homo sapiens, there are four isoforms: mitochondrial (mDut), nuclear (nDut), variant 3 and variant 4. This work is largely focused on nDut. Using structural and MS analyses of recombinant dUTPase constructs, an intermolecular disulfide bridge between cysteine-3 of each nDut monomer was discovered. It was found that these two …

Characterizing Novel Pathways For Regulation And Function Of Ataxin-2, Elise Spencer Melhado

Theses and Dissertations

Ataxin-2 is an RNA-binding protein that is involved in many crucial cellular processes such as R-loop regulation, mRNA stability, TOR signaling regulation, and stress granule formation. Ataxin-2 is highly conserved, found in organisms ranging from Saccharomyces cerevisiae to Caenorhabditis elegans and Homo sapiens. Recently, ataxin-2 has been linked to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal disease that causes loss of motor neurons. In addition to ataxin-2 interacting with known ALS risk factor proteins, research into the relationship between ataxin-2 and ALS shows that polyglutamine expansions in ataxin-2 are gain-of-function mutations that lead to overactivity of …

Snfing Glucose To Pass Mitochondrial Dysfunction: The Role Of Two Sensory Protein Kinases In Metabolic Diseases, Kai Li Ong

Theses and Dissertations

Mitochondria is no longer viewed as merely a powerhouse of the cell. It is now apparentthat mitochondria play a central role in signaling, maintaining cellular homeostasis and cell fate.Mitochondrial dysfunction has been linked to many human diseases caused by cellular metabolicderegulation, such as obesity, diabetes, neurodegenerative disease, cardiovascular disease andcancer. Eukaryotic organisms have evolved an efficient way in sensing, communicating andresponding to cellular stress and regulating mitochondrial activity correspondingly through acomplex network of intercommunicating protein kinases and their downstream effectors. Thisdissertation focuses on the interplay of two of the master metabolic regulators in the cell: AMPKand PASK, and characterization of …

Endogenous Force Transmission Between Epithelial Cells And A Role For Α-Catenin, Sandeep Dumbali

Mechanical & Aerospace Engineering Theses & Dissertations

In epithelial tissues, epithelial cells adhere to each other as well as to the underlying extra-cellular matrix (ECM). E-cadherin-based intercellular junctions play an important role in tissue integrity. These junctions experience cell-generated mechanical forces via apparent adaptor proteins such as beta (β) catenin, alpha (α) catenin and vinculin. Abnormalities in these junctions may result in skin related diseases and cancers. Here, I devised methods to determine the endogenous intercellular force within cell pairs as well as in large epithelial islands. I further ascertained the factors that affect the level of inter-cellular tension.

Experiments with pairs of epithelial cells exogenously expressing …

The Expression Of Connexin-43 By Cd11c+ Dendritic Cells Is Required To Maintain Cd4+ Foxp3+ Regulatory T Cell Population In Peripheral Lymphoid Organs, Caroline Titus Miller

Biological Sciences Theses & Dissertations

Foxp3⁺ regulatory T cells (T_R) are an immunosuppressive subset of CD4⁺ T cells that maintain homeostasis of the immune system. They are sustained by the interaction between the Major Histocompatibility Complex (MHC) molecules present on antigen presenting dendritic cells and the T Cell Receptor (TCR) expressed on T_R cells that is specific for the MHC loaded with an antigenic peptide. Here, we show that in addition to MHC/TCR interaction, Connexin-43 (Cx43) expression by dendritic cells is required to maintain the TR cell population. CD11c⁺ dendritic cells represent a major subset of antigen presenting cells. …

Knocking Out A Negative Regulator Of Hedgehog Signaling Blocks Differentiation Of Cells Into Neurons, Danielle Margaret Spice, Gregory M. Kelly Ph.D.

Western Research Forum

Hedgehog (Hh) signaling, one of many different protein signaling pathways found in mammals, is vital in many stage of neural development. A major negative regulator of Hh signaling is a protein known as Suppressor of Fused (SUFU), which acts to sequester the full length Gli transcription factors, proteins that can turn genes on and off, in the cytoplasm or facilitates its conversion to a repressive form. The P19 embryonal carcinoma cell line is a model of hind-brain neuronal differentiation and the involvement of Hh signaling, in particular the role of SUFU in this process has yet to be explored. We …