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Studies On The Small Ubiquitin-Like Modifier (Sumo) E2 Conjugases Of The Sumoylation System In Chlamydomonas Reinhardtii And Their Role In Stress Physiology, Amy R. Knobbe

Department of Biochemistry: Dissertations, Theses, and Student Research

The eukaryotic protein post-translational modification by SUMOylation is involved in a diverse array of cellular processes, including various stress responses. A fully functional SUMOylation system is present in the unicellular green alga Chlamydomonas reinhardtii, and SUMOylation of multiple high molecular weight proteins is induced in response to abiotic stress in this organism. We report here the characterization of a SUMO E2 conjugase deletion mutant in C. reinhardtii, mut5. SUMO E2 conjugase enzymes are responsible for the conjugation of the protein SUMO to a lysine residue within a target protein. C. reinhardtii mutants in which the SUMO E2 …

Developing A High Throughput Protocol For Using Soil Molecular Biology As Trace Evidence, Sabreena A. Larson

Department of Biochemistry: Dissertations, Theses, and Student Research

The use of soil as trace evidence has changed significantly with the addition of new techniques. These techniques include using the biochemical molecules from soil microbial communities to make a fingerprint of the specific soil. This research examines the changes to the microbial community profile that take place during storage of a soil sample. To observe such changes both the DNA and fatty acid profiles will be examined.

The DNA profiles were made with capillary electrophoresis-single stranded conformation polymorphism (CE-SSCP). After statistical analysis using Bray-Curtis distances and ANOSIM (analysis of similarity) it was shown that storage of soil does not …

Development Of An Assay To Screen For Novel Proteins From Bacillus Thuringiensis With Antibacterial Activity, Auriole T. Tamegnon

Theses and Dissertations - UTB/UTPA

Bacillus thuringiensis (Bt) produces the non specific Cyt protein and the specific Cry protein. Past research has shown these proteins are toxic to insects, red blood cells, cancer cells, and some bacteria. While many antibacterial substances are known to exist, efforts to find new ones still continue. In this project we used Bt 6-2-56 (containing a novel filament associated parasporal body which appears non-toxic to insects and protozoan) and Bt 4Q13 (which has been shown to have activity against E. coli and S. aureus) to develop a simple, easy, scalable assay in a 96 well Microtitre plate to look for …

Activation Of The Carbon Concentrating Mechanism By Co2 Deprivation Coincides With Massive Transcriptional Restructuring In Chlamydomonas Reinhardtii, Andrew J. Brueggeman, Dayananda S. Gangadharaiah, Matyas F. Cserhati, David Casero, Donald P. Weeks, Istvan Ladunga

Department of Biochemistry: Faculty Publications

A CO₂-concentrating mechanism (CCM) is essential for the growth of most eukaryotic algae under ambient (392 ppm) and very low (ppm) CO₂ concentrations. In this study, we used replicated deep mRNA sequencing and regulatory network reconstruction to capture a remarkable scope of changes in gene expression that occurs when Chlamydomonas reinhardtii cells are shifted from high to very low levels of CO₂ (£100 ppm). CCM induction 30 to 180 min post-CO₂ deprivation coincides with statistically significant changes in the expression of an astonishing 38% (5884) of the 15,501 nonoverlapping C. reinhardtii genes. Of these genes, …

Development And Evaluation Of A Mouse Neocortical Cells Neurodevelopment Assay To Assess The Effects Of Pyrethroids, Stephanie I. Savino

Pell Scholars and Senior Theses

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the global insecticide market with household, agricultural and public health applications. With increasing use of pyrethroids over recent years, human exposure to these chemicals is likely. The acute toxicity of pyrethroids to mammalian adults has been documented as moderately toxic by the U.S. Environmental Protection Agency. However, little evidence is available regarding the potential toxicological effects of pyrethroids on mammalian neurodevelopment. In this study, a neurodevelopment assay was developed for future investigation of potential pyrethroid effects on the neuronal development of Mus musculus neocortical cells.

Syntaxin 6- And Microtubule- Mediated Intracellular Trafficking Contributes To Golgi And Nuclear Translocation Of Egfr, Yi Du

Dissertations and Theses (Open Access)

Receptor-mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER), and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completed understood. Here we report a mechanism of Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 …

Determining The Genotype-Phenotype Connection In Synthetic Inducible Gene Expression Systems, Rhys M. Adams

Dissertations and Theses (Open Access)

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise.

Methods I created probabilistic …

Increased Geranylgeranylated K-Ras Contributes To Antineoplastic Effects Of Farnesyltransferase Inhibitors., Mandy A. Hall

Dissertations and Theses (Open Access)

The Ras family of small GTPases (N-, H-, and K-Ras) is a group of important signaling mediators. Ras is frequently activated in some cancers, while others maintain low level activity to achieve optimal cell growth. In cells with endogenously low levels of active Ras, increasing Ras signaling through the ERK and p38 MAPK pathways can cause growth arrest or cell death. Ras requires prenylation – the addition of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) group – to keep the protein anchored into membranes for effective signaling. N- and K-Ras can be alternatively geranylgeranylated (GG’d) if farnesylation is inhibited but are …

Regulation Of Protein Degradation In The Heart By Amp-Activated Protein Kinase, Kedryn K. Baskin

Dissertations and Theses (Open Access)

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of …

Chemosensitization Of Hepatocellular Carcinoma To Gemcitabine By Non-Invasive Radiofrequency Field-Induced Hyperthermia, Mustafa Raoof

Dissertations and Theses (Open Access)

Gemcitabine is a potent nucleoside analogue against solid tumors however drug resistance rapidly emerges. Removal of gemcitabine incorporated in the DNA by repair mechanisms could potentially contribute to resistance in chemo-refractory solid tumors. In this study, we evaluated homologous recombination repair of gemcitabine-stalled replication forks as a potential mechanism contributing to resistance. We also studied the effect of hyperthermia on homologous recombination pathway to explain the previously reported synergy between gemcitabine and hyperthermia. We found that hyperthermia degrades and inhibits localization of Mre11 to gemcitabine-stalled replication forks. Furthermore, gemcitabine-treated cells that were also treated with hyperthermia demonstrate a prolonged passage …

Regulation Of Protein Degradation In The Heart By Amp-Activated Protein Kinase, Kedryn K. Baskin, Kedryn K. Baskin

Dissertations and Theses (Open Access)

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of …

A Fast Response Highly Selective Probe For The Detection Of Glutathione In Human Blood Plasma, Yixing Guo, Xiaofeng Yang, Lovemore Hakuna, Aabha Barve, Jorge O. Escobedo, Mark Lowry, Robert M. Strongin

Chemistry Faculty Publications and Presentations

A fluorescent probe for glutathione (GSH) detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys) and homocysteine (Hcy). The proposed method is precise with a relative standard deviation (R.S.D) lower than 6% (n = 3) and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.

Genome-Wide Identification Of Conditionally Essential Genes In Salmonella Typhimurium Using Tn-Seq Method, Anita Khatiwara

Graduate Theses and Dissertations

ABSTRACT

As more whole genome sequences become available, there is an increasing need for high-throughput methods that link genes to phenotypes and facilitate discovery of new gene functions. The objective of this study was to develop a high-throughput method to study gene functions in bacteria and use this method to study gene functions of S. enterica serotype Typhimurium (S. Typhimurium) under various environmental conditions encountered during its life cycle. Chapter I of this dissertation reviews the history and evolution of functional genomics in bacteria with focus on Salmonella, along with the recent techniques available. Chapter II, deals with the development …

Stromal-Epithelial Interactions Modulate Cross Talk Between Prolactin Receptor And Her2/Neu In Breast Cancer, Cong Xu

All Dissertations

The tumor microenvironment is a crucial factor in breast tumorigenesis. Tumor epithelial cells maintain 3D structure in tumor stroma and they interact with soluble factors secreted by stromal cells such as cancer associated fibroblasts (CAFs) or directly with the extracellular matrix (ECM). Recent studies have shown that the hormone prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been demonstrated not only to be able to directly inhibit PRLR activation but also …

An Investigation Of Trypanosoma Brucei Hexokinases: Localization, Oligomerization, And Inhibition, April Joice

All Dissertations

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and nagana in livestock. The parasite inhabits multiple environmental niches including the bloodstream of the mammalian host and the mid-gut of the tsetse fly vector. While in the bloodstream of its mammalian host, the organism depends solely on glycolysis for production of ATP. My studies focus on the first enzyme in glycolysis, hexokinase.
T. brucei has two hexokinases, TbHK1 and TbHK2 that are 98.5% identical at the nucleotide level. The hexokinases are expressed in the glycosomes of both procyclic form and bloodstream form parasites. Glycosomes are peroxisome-like organelles …

Development Stage-Specific Proteomic Profiling Uncovers Small, Lineage Specific Proteins Most Abundant In The Aspergillus Fumigatus Conidial Proteome, Moo-Jin Suh, Natalie D. Fedorova, Steven E. Cagas, Susan Hastings, Robert D. Fleischmann, Scott N. Peterson, David S. Perlin, William C. Nierman, Rembert Pieper, Michelle Momany

Biochemistry and Molecular Medicine Faculty Publications

Background

The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process.

Results

To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae …

Activation Of An Nlrp3 Inflammasome Restricts Mycobacterium Kansasii Infection, Chang-Chieh Chen, Sheng-Hui Tsai, Chia-Chen Lu, Shiau-Ting Hu, Ting-Shu Wu, Tsung-Teng Huang, Najwane Saïd-Sadier, David M. Ojcius, Hsin-Chih Lai

All Dugoni School of Dentistry Faculty Articles

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, …

Heterotopic Ossification: Cellular Basis, Symptoms, And Treatment, Brian Wolfe

Senior Honors Theses

Heterotopic ossification (HO) is the process by which calcified bone develops in soft tissues. Because of the abnormal calcification, complications such as bone deformation, loss of range of motion, and joint immobility adversely affect patients. There are many genetic types of heterotopic ossification, namely fibrodysplasia ossificans progressiva, progressive osseous heteroplasia, and Albright hereditary osteodystrophy. However, this condition can also arise from surgery, burns, or traumatic injuries, so it is seen as an important area for research in the future. There are various treatments available such as non-steroidal anti-inflammatory drugs and radiation therapy, as well as combinations of the two. The …

31p Nmr Of Backbone Conformation And Dynamics In Dna At Cre Binding Site In Terms Of Sequence Context, Kelly A. Garton

Scripps Senior Theses

The Cre sequence (ACGT) is a site responsible for the binding of specific transcription factors that determine the activation of genes. Due to its major role in gene transcription, it has become a subject of immense research. The binding of transcription factors to the Cre binding site has been determined to be dependent on DNA conformation. In this study, the effects of flanking sequence around the Cre binding site on the conformation and the dynamics of DNA were investigated. The Cre binding site was studied in its native form with differing flanking sequences to determine the BI/BII profile (conformation) and …

The Role Of Thd2 In Deposition-Related Deactylation And Chromatin Maturation, T. Alexandria Dumas

Scripps Senior Theses

During S phase of the cell cycle, newly synthesized histones are acetylated in the cytoplasm in patterns specific to DNA replication. Once incorporated into nucleosomes, these histones are rapidly deacetylated by enzymes known as histone deacetylases. Though common in all organisms, the significance of this molecular mechanism is not fully understood. Homologous to HDAC6 in humans and HDA1 in budding yeast, Tetrahymena histone deacetylase 2 (Thd2) has been identified as the only known histone deacetylase that performs this task in the unicellular eukaryote Tetrahymena thermophila. Localizing to the transcriptionally inactive germline nucleus, the micronucleus, Thd2 has been found to deacetylate …

Analysis Of The Effects And Current Treatments Of Laminin Deficiency, Joshua Mark Reynolds

Senior Honors Theses

Laminin (LM) is a network of proteins that functions as a connective framework of most cells in the body. It is composed of multiple different subunits and therefore has many different variations. It is a trimeric protein, meaning that it is composed primarily of ⍺, β, and γ chains. The differentiation of these subunits is what gives the different variants their functions. In addition, although LM is the primary molecule in scope, the network of other connective proteins involved in LM-associated diseases will also be covered in lesser detail because molecules like dystrophin, dystroglycan, collagen, and integrin are vital to …

A Plant Virus Substrate Induces Early Upregulation Of Bmp2 For Rapid Bone Formation, Pongkwan Sitasuwan, L. Andrew Lee, Peng Bo, Erin N. Davis, Yuan Lin, Qian Wang

Faculty Publications

Many nanoscale materials have been developed to investigate the effects on stem cell differentiations via topographical and chemical cues for applications in tissue engineering and regenerative medicine. The use of plant viruses as cell supporting substrates has been of particular interest due to the rapid induction of bone marrow derived mesenchymal stem cells (BMSCs) towards osteogenic cells. In this study, the role of Tobacco mosaic virus (TMV) and its early effects on osteoinduction with particular emphasis on the regulation of bone morphogenetic protein-2 (BMP2) was examined. We observed that the cells on the virus substrate immediately aggregated and formed bone-like …

The Role Of Hsp70 In Cancer: A Study Of The Hsp70 / Akt Relationship, John Koren

USF Tampa Graduate Theses and Dissertations

The Hsp70 family of molecular chaperones is essential for

protein folding, re-folding misfolded client proteins, clearance

of aberrant client proteins, and can also inhibit programmed

cell death. There are two major cytosolic members of this

family: the constitutive Hsc70, and the inducible Hsp72. Under

stress conditions the Hsp70 family protects the cell from

protein related damage by the induction of Hsp72. Hsc70 and

Hsp72 are highly homologous with minor differences in

substrate binding. In cancers, Hsp72 is commonly induced and

this induction is thought to aid in cancer cell survival. In these

studies we demonstrate the differential regulation of the …

Paramyxovirus Fusion And Entry: Multiple Paths To A Common End, Andres Chang, Rebecca Ellis Dutch

Molecular and Cellular Biochemistry Faculty Publications

The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins …

2-Allylphenyl Glycosides As Complementary Building Blocks For Oligosaccharide And Glycoconjugate Synthesis, Hemali Premathilake, Alexei Demchenko

Chemistry & Biochemistry Faculty Works

The O-allylphenyl (AP) anomeric moiety was investigated as a new leaving group that can be activated for chemical glycosylation under a variety of conditions, through both direct and remote pathways. Differentiation between the two activation pathways was achieved in a mechanistic study. The orthogonal-type activation of the AP moiety along with common thioglycosides allows for the execution of efficient oligosaccharide assembly.

Isolated Amyloid-Β(1−42) Protofibrils, But Not Isolated Fibrils, Are Robust Stimulators Of Microglia, Geeta Paranjape, Lisa Gouwens, David Osborn, Michael Nichols

Chemistry & Biochemistry Faculty Works

Senile plaques composed of amyloid-β protein (Aβ) are an unshakable feature of the Alzheimer’s disease (AD) brain. Although there is significant debate on the role of the plaques in AD progression, there is little disagreement on their role in stimulating a robust inflammatory response within the context of the disease. Significant inflammatory markers such as activated microglia and cytokines are observed almost exclusively surrounding the plaques. However, recent evidence suggests that the plaque exterior may contain a measurable level of soluble Aβ aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aβ deposits led us to …

Using Bioinformatics To Efficiently Organize And Analyze Significant Immunogenic Epitope Sequences In Various Stages Of Trypanosoma Cruzi, Karla Singh^, Andrea Wurm, Clemente Aguilar, Alexandre Marques, M-Y Leung*, Igor Almeida*

COURI Symposium Abstracts, Spring 2012

No abstract provided.

Prostate Cancer-Specific And Potent Antitumor Effect Of A Dd3-Controlled Oncolytic Virus Harboring The Pten Gene, Miao Ding, Xin Cao, Hai-Neng Xu, Jun-Kai Fan, Hong-Ling Huang, Dong-Qin Yang, Yu-Hua Li, Jian Wang, Runsheng Li, Xin-Yuan Liu

Molecular and Cellular Biochemistry Faculty Publications

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN …

Differentiation Of Capsicum Species Via Protein Extraction And Sds-Page Analysis: A Two-Day Experiment For Undergraduate Biochemistry Laboratories, Trey Polvadore, Brittney P. Cooper, Michele R. Harris

Undergraduate Research Conference

The Capsicum genus is comprised of more than 200 varieties of pepper plant that collectively represent only five cultivated spices. As a result of extensive cross-breeding, an 88% similarity in protein coding DNA has developed. While this has made species determination based solely on physical appearance all but impossible, the 12% difference in protein coding DNA has led to subtle differences in translated protein. SDS-PAGE analysis facilitates the generation of protein banding patterns that, after careful analysis, was used to differentiate one variety of pepper from another. This project was used to distinguish between pepper plant species and was carried …

Geometric Surface Function Of A Giant Marine Virus Cafeteria Roenbergensis Virus, Tiffany H. Blankenship^, Rishabh Jain, Chuan Xiao*

COURI Symposium Abstracts, Spring 2012

No abstract provided.