Molecular Biology Commons^™

Expression Of Trichoderma Reesei Beta]-Mannanase In Tobacco Chloroplasts And Its Utilization In Lignocellulosic Woody Biomass Hydrolysis, Pankaj M. Agrawal

Electronic Theses and Dissertations

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plantbased production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β- mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific …

Quantifying Agonist Activity At G Protein-Coupled Receptors, Frederick J. Ehlert, Hinako Suga, Michael T. Griffin

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence …

Bio-Functionalized Clay Nanoparticles For Wound Healing Applications, Christopher Anthony Vaiana

Browse all Theses and Dissertations

Wound healing is a complex, multi-step process that can be summarized into three stages, namely hemostasis and inflammation, proliferation, and finally tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we first functionalize three types of alumoinosilicate …

Interrogation Of The Distal Gut Microbiota Of Healthy Adolescents And Those With Irritable Bowel Syndrome, Laura J. Rigsbee

Browse all Theses and Dissertations

The human-associated microbiota has been the focus of much current research, with the microbiota inhabiting the gastrointestinal tract of particular interest. These organisms play many roles in human health and well-being. However, shifts in the composition of the intestinal microbiota have been associated with diseases such as irritable bowel syndrome, inflammatory bowel disease, and colon cancer. Several recent studies have reported on the distal gut microbiota composition of healthy adults and those with IBS, while there is a lack of studies devoted to adolescents. This study utilized a custom-designed Affymetrix Microbiota Array capable of detecting 775 phylo-species of intestinal bacteria …

Functional Metabolomics' Enhances Assessment Of Tissue Dysfunction As Demonstrated In A Rat Model Of Sub-Acute D-Serine Exposure, Isaie Sibomana

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We describe a methodology that combines urinary metabolomics with a tissue-specific stressor administration to enhance assessment of tissue function. Kidney function in rats was mildly compromised with a sub-acute dose of D-serine and stressed with furosemide. NMR-based metabolomics analyses showed no detectable effects due to D-serine alone; but furosemide or D-serine + furosemide groups, classified separately from each other, and from control. Furosemide alone caused a ca. 2-fold increase in glucose, lactate, choline, and a 30% decrease in TCA intermediates (p≤0.05). D-serine suppressed these effects and produced a 1.7-fold increase in a p-phenolic acid-derivative of tyrosine (PAdY) relative to control …

Structural And Functional Alteration Of Full Length Pparα And Lxrα By Fatty Acids And Their Thioesters, Madhumitha Balanarasimha

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Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are known to play important roles in fatty acid metabolism, interact with each other, and function as heterodimeric partners. Although previous studies indicate that PPARα is activated by long chain fatty acyl-CoA thioesters (LCFA-CoA) and polyunsaturated fatty acids, little is known about the effects of these ligands on the function or interaction of PPARα and LXRα. In this study, hPPARα and hLXRα were shown to directly interact by circular dichroism, fluorescent binding assays, and co-immunoprecipitation. Further experiments suggested that although fatty acids resulted in small structural changes, they significantly altered binding …

Novel Roles For The Transcriptional Repressor Prdm1 In Human Natural Killer Cells And Identification Of An Inhibitor Of Its Interacting Methyltransferase G9a, Matthew Adams Smith

USF Tampa Graduate Theses and Dissertations

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation.

In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at …

The Role Of Brca1 Domains And Motifs In Tumor Suppression, Aneliya Velkova

USF Tampa Graduate Theses and Dissertations

Individuals that carry deleterious mutations in the breast and ovarian cancer susceptibility gene 1 (BRCA1) have much more elevated risk to develop breast and/or ovarian cancer than the individuals from the general population. The BRCA1 gene product has been implicated in several aspects of the DNA damage response, but its biochemical function in these processes has remained elusive. In order to probe BRCA1 functions we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. In this dissertation, we report the identification of the actin-binding protein Filamin A (FLNA) …

The Specific Role Of The Mll Cxxc Domain In Mll Fusion Protein Function, Laurie Ellen Risner

Dissertations

The MLL gene was first identified because it is involved in chromosome translocations which produce novel fusion proteins that cause leukemia. The CXXC domain of MLL is a cysteine rich DNA binding domain with specificity for binding unmethylated CpG-containing DNA. The CXXC domain is retained in oncogenic MLL fusions, and is absolutely required for the fusions to cause leukemia. This project explored the role of the CXXC domain by introducing structure-informed point mutations within the MLL CXXC domain that disrupt DNA binding, and by performing domain swap experiments in which different CXXC domains from other proteins, including DNMT1, CGBP and …

Wee1 Is A Biological Target Of The Mir-17-92 Cluster In Leukemia, Sonia Susan Olikara

Master's Theses

MicroRNAs are noncoding RNAs that bind to the 3' untranslated region of their mRNA targets, which causes downregulation of target gene expression. Previous studies have shown that the miR-17-92 cluster, which encodes six miRNAs, is overexpressed in leukemias arising from chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene. In the present study, prediction algorithms identified WEE1, a kinase that inhibits cell cycle progression, as a possible target of five of the six miRNAs. Through luciferase reporter assays, we found that miR-17, miR-20a, and miR-18a specifically target nucleotides 465 to 487 of the 3' UTR of WEE1, while miR-19a and …

Notch-1 Specifically Activates Erk1/2 In Multiple Breast Cancer Subtypes, Allison Schuyler Rogowski

Master's Theses

Notch-1 is a cell fate regulatory protein and a potent breast oncogene. Notch-1 and its ligand Jagged-1 are over-expressed in human breast cancers that are associated with poor overall survival (Reedijk, Odorcic et al. 2005). Deregulated Notch signaling may contribute to tumorigenesis by increasing proliferation, inhibiting differentiation, and preventing apoptosis (Miele, Golde et al. 2006). The mitogen-activated protein kinase (MAPK) pathway is a critical cell signaling pathway that has been implicated in the development and progression of cancer (Hanahan and Weinberg 2000). Four major MAPK pathways are involved in both cell growth and apoptosis. The regulation of these pathways is …

Microbial Ecology And Functional Genomics Of Deep-Water Coral-Associated Microbes, Julia Parker Galkiewicz

USF Tampa Graduate Theses and Dissertations

Lophelia pertusa is a cosmopolitan cold-water coral, often found in aphotic waters (>200m). Aggregations of L. pertusa (reefs) provide important habitat to many invertebrate and fish species and act as biodiversity hotspots in the deep sea. The health and diversity of these reefs is of vital importance to deep-sea ecosystems, and the microbial consortia associated with L. pertusa form the most basic ecological level. Deciphering the diversity and function of these microbes provides insight into the roles they play in maintaining reef health. This dissertation takes microbiological techniques that are used in shallow-water coral microbial research and applies them …

From Dna To Protein: A Study Of Genomic Instability Candidate Genes During Zebrafish Development, Kristine Griffett

USF Tampa Graduate Theses and Dissertations

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation …

Identification And Characterization Of The Human Herpesviruses 6a And 6b Genome Integration Into Telomeres Of Human Chromosomes During Latency, Jesse Herbert Arbuckle

USF Tampa Graduate Theses and Dissertations

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DR_R) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DR_L), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and …

Development Of An Elisa For Eastern Equine Encephalitis Virus That Can Differentiate Infected From Vaccinated Horses, Andrea Bingham

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) causes a fatal mosquito-borne virus that is vaccine preventable for horses. The conventional serological tests measure antibodies to the structural proteins of EEEV which are also found in the vaccine. This makes it difficult to differentiate infected and vaccinated animals (DIVA). Detection of antibodies to non-structural proteins (NSPs) is a theoretical strategy that would allow you to survey natural infections among vaccinated populations. This test would also allow for more accurate representations of the natural infection rate, vaccination rate, and help identify vaccine failures. The potential uses of the NSPs of Eastern Equine Encephalitis virus …

Inhibition Of P53 Dna Binding Function By The Mdm2 Acidic Domain, Brittany Lynne Cross

USF Tampa Graduate Theses and Dissertations

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild …

Analysis Of Ahr Expression And Stability In A Recombinant Yeast Model System, Sarah Elizabeth Cuccinello

USF Tampa Graduate Theses and Dissertations

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of …

Microbial Landscapes Of Corals And Ctenophores, Camille Arian Daniels

USF Tampa Graduate Theses and Dissertations

As technology and engineering allow mankind to survey nature at finer scales, the importance of bacteria has been elucidated in their metabolic diversity, ability to transfer genetic information, involvement in biogeochemical cycling, and sheer abundance. With an individual domain of life unto themselves, this diverse group of microorganisms plays an integral role in facilitating life on land and in the oceans, and is second only to viruses in abundance on Earth. They carve niches in a wide range of environments, including those inhospitable to other life forms, and reside in concert or to the detriment of other microbes and/or hosts …

Development Of A Non-Invasive Electrode For Intradermal Electrically Mediated Dna Vaccination, Amy Lynn Donate

USF Tampa Graduate Theses and Dissertations

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our …

Computational Network Analysis Of The Anatomical And Genetic Organizations In The Mouse Brain, Shuiwang Ji

Computer Science Faculty Publications

Motivation: The mammalian central nervous system (CNS) generates high-level behavior and cognitive functions. Elucidating the anatomical and genetic organizations in the CNS is a key step toward understanding the functional brain circuitry. The CNS contains an enormous number of cell types, each with unique gene expression patterns. Therefore, it is of central importance to capture the spatial expression patterns in the brain. Currently, genome-wide atlas of spatial expression patterns in the mouse brain has been made available, and the data are in the form of aligned 3D data arrays. The sheer volume and complexity of these data pose significant challenges …