Life Sciences Commons^™

The Key Question In Symbiotic Nitrogen Fixation: How Does Host Maintain A Bacterial Symbiont?, Onur Oztas

Doctoral Dissertations

The fact that plants cannot use nitrogen in the gaseous form makes them dependent on the levels of usable nitrogen forms in the soil. Legumes overcome nitrogen limitation by entering a symbiotic association with rhizobia, soil bacteria that convert atmospheric nitrogen into usable ammonia. In root nodules, bacteria are internalized by host plant cells inside an intracellular compartment called the symbiosome where they morphologically differentiate into nitrogen-fixing forms by symbiosome-secreted host proteins. In this project, I explained the host proteins required to maintain bacterial symbionts and described their delivery to the symbiosome. I showed that the SYNTAXIN 132 (SYP132) gene …

Adaptors At Work: Regulation Of Bacterial Proteolysis By Adaptor Hierarchies, Kamal Joshi

Doctoral Dissertations

Regulated protein degradation is essential for all life. Bacteria use energy-dependent proteases to regulate protein degradation. Recognition of a substrate is enabled by the inherent specificity of the protease and by the use of adaptor proteins that widen the spectrum of recognized substrates. In Caulobacter crescentus, the timed destruction of many regulators including CtrA by the ClpXP protease drives cell cycle progression. Although, in a test tube, ClpXP can degrade CtrA by itself and does not need any helping factors, additional factors such as CpdR, RcdA and PopA are required in vivo. Understanding how these factors modulate protease …

Modifications Of Myofilament Structure And Function During Global Myocardial Ischemia, Mike K. Woodward

Masters Theses

Cardiac arrest is a prevalent condition with a poor prognosis, attributable in part to persistent myocardial dysfunction following resuscitation. The molecular basis of this dysfunction remains unclear. We induced cardiac arrest in a porcine model of acute sudden death and assessed the impact of ischemia and reperfusion on the molecular function of isolated cardiac contractile proteins. Cardiac arrest was electrically induced, left untreated for 12 min, and followed by a resuscitation protocol. With successful resuscitations, the heart was reperfused for 2 h (IR2) and the muscle harvested. In failed resuscitations, tissue samples were taken following the failed efforts (IDNR). Actin …

Characterizing The Inhibition Of Katanin Using Tubulin Carboxy-Terminal Tail Constructs, Corey E. Reed

Masters Theses

Understanding how the cellular cytoskeleton is maintained and regulated is important to elucidate the functions of many structures such as the mitotic spindle, cilia and flagella. Katanin p60, microtubule-severing enzymes from the ATPase associated with cellular activities (AAA+) family, has previously been shown in our lab to be inhibited by free tubulin as well as α- and β-tubulin carboxy-terminal tail (CTT) constructs. Here we investigate the inhibition ability of several different tubulin CTT sequences. We quantify the effect of the addition of these constructs on the severing and binding activity of katanin. We find that some constructs inhibit katanin better …

Investigating The Balance Between Estrogen Receptor Mediated Cell Proliferation And Genomic Surveillance, Margarita Brown

Masters Theses

Breast cancer is a leading cause of cancer in women and the second leading cause of cancer death. Lifetime exposure to estrogen contributes to this risk but high dose estrogen has been used to induce apoptosis as treatment for breast cancer. These opposing tumorigenic and anti-tumorigenic effects of estrogen may be regulated differently by the two Estrogen Receptors (ER), Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ). Although the receptors share a 96% homology in their DNA binding domain, they are unique in the ligand-binding domain with 53% amino acid homology. Previous studies have shown that ERα drives cell …

Control Of Proteolysis During The Caulobacter Cell Cycle, Joanne Lau

Doctoral Dissertations

Intracellular protein destruction is a carefully coordinated and timed regulatory mechanism that cells utilize to modulate growth, adaptation to environmental cues, and survival. In Caulobacter crescentus, a bacterium known for studies of bacterial cell division cycle, the response regulator CpdR couples phosphorylation events with the AAA+ protease ClpXP to provide punctuated degradation of crucial substrates involved in cell cycle regulation. CpdR functions like an adaptor to alter substrate choice by ClpXP, however it remains unclear how CpdR influences its multiple targets. In this thesis, we show that, unlike canonical ClpXP adaptors, CpdR alone does not strongly bind its substrate. …

Novel Advancements For Improving Sprout Safety, Kyle S. Landry

Doctoral Dissertations

All varieties of bean sprouts (mung bean, alfalfa, broccoli, and radish) are classified as a “super-food” and are common staples for health conscious consumers. Along with the proposed health benefits, there is also an inherent risk of foodborne illness. When sprouts are cooked, there is little risk of illness. The purpose of this dissertation was to explore novel techniques to minimize or prevent the incidence of foodborne illness associated with the consumption of sprouts. Three areas were investigated: 1) the use of a biocontrol organism, 2) the use of a novel spontaneous carvacrol nanoemulsion, and 3) the influence of the …

Photolysis Of Triazenylbenzoic Acids For Click Chemistry, Adam Gann

Doctoral Dissertations

Copper catalyzed cycloaddition of terminal alkynes and azides has revolutionized the field of bioconjugate chemistry. Unfortunately, typical copper catalysts are known to disrupt relevant biological systems, so it has become necessary to develop new, copper-free methods that are less cytotoxic. particular interest are "click" probes which can be activated with an outside light source, giving the user spatial and temporal control over the system being investigated. We have developed a method in which an aryl diazonium salt is rapidly generated using photolysis of the triazene functional group, and subsequently coupled with an electron rich aromatic nucleophile to yield an azobenzene. …

Exploring The Impact Of The E. Coli Proteostasis Network On The Folding Fate Of Proteins With Different Intrinsic Biophysical Properties, Kristine Faye R. Pobre

Doctoral Dissertations

The three-dimensional (3D) native structure of most proteins is crucial for their functions. Despite the complex cellular environment and the variety of challenges that proteins experience as they fold, proteins can still fold to their native states with high fidelity. The reason for this is the presence of the cellular proteostasis network (PN), consisting of molecular chaperones and degradation enzymes, that collaborates to maintain proteostasis, in which the necessary levels of functional proteins are optimized. Although extensive research has been carried out on the mechanisms of individual components of the proteostasis network, little is known about how these components contribute …

Promoting Extracellular Matrix Crosslinking In Synthetic Hydrogels, Marcos M. Manganare

Masters Theses

The extracellular matrix (ECM) provides mechanical and biochemical support to tissues and cells. It is crucial for cell attachment, differentiation, and migration, as well as for ailment-associated processes such as angiogenesis, metastases and cancer development. An approach to study these phenomena is through emulation of the ECM by synthetic gels constructed of natural polymers, such as collagen and fibronectin, or simple but tunable materials such as poly(ethylene glycol) (PEG) crosslinked with short peptide sequences susceptible to digestion by metalloproteases and cell-binding domains. Our lab uses PEG gels to study cell behavior in three dimensions (3D). Although this system fosters cell …

Rnai Validation Of Resistance Genes And Their Interactions In The Highly Ddt-Resistant 91-R Strain Of Drosophila Melanogaster, Kyle Gellatly

Masters Theses

4,4’-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as …

The Evolution Of Thermotolerance A Characterization Of A Directionally Evolved Cyanobacterium, Nathen Emil Bopp

Masters Theses

Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their …

New Insights Into The Role Of The Udp-Glucose: Glycoprotein Glucosyltransferase 1 In The Endoplasmic Reticulum Quality Control, Abla Tannous

Doctoral Dissertations

The UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control factor in the Endoplasmic Reticulum (ER). It surveys the folding status of proteins in the ER and redirects them, via its reglucosylation activity, to bind to the ER carbohydrate binding (lectin) chaperones calreticulin (CRT) and calnexin (CNX). However, the cellular mechanism of UGT1 is not completely understood. Using a cell based reglucosylation assay, we found that UGT1 reglucosylated proteins that eventually fold. This modification was transient and resulted in delay of protein trafficking in the secretory pathway and prolonged binding to lectin chaperones in the ER. In addition, terminally misfolded …

Molecular Mechanisms Underlying The Contralateral Repeated Bout Effect (Crbe) In Human Skeletal Muscle, Ling Xin

Doctoral Dissertations

Eccentric (muscle lengthening) exercise induces temporary muscle damage that can lead to long-term muscle adaptation, a process known as the repeated bout effect where subsequent exercise results in less damage. The existence of a contralateral repeated bout effect (CRBE) has been controversial. The primary goals of this study were to: 1) validate the existence of the CRBE; and 2) define the underlying molecular mechanisms. Thirty-six young men performed 100 maximal eccentric actions of the knee extensors using one leg (bout 1) and repeated the exercise with the contralateral leg five weeks later (bout 2). Vastus lateralis muscle biopsies were …

Thermocycle-Regulated Wall Regulator Interacting Bhlh Encodes A Protein That Interacts With Secondary-Cell-Wall-Associated Transcription Factors, Ian P. Whitney

Masters Theses

Lignocellulosic biomass is one of the most abundant raw materials on earth that can be utilized to created carbon-neutral biofuels as a replacement for conventional fossil fuels. In order to create ideal energy crops, the regulation and deposition of cell wall polysaccharides must first be fully understood. Improved understanding of cell wall regulation will enable selection of traits that can optimize biofuel feedstocks. Herein, I utilize the grass model system Brachypodium distachyon in order to understand the transcriptional regulation of secondary cell wall deposition. Gene expression profiling was used to elucidate transcription factors that regulate secondary cell wall biosynthesis. Through …

The Estradiol-Induced Transcriptome Of The Female Mouse Anteroventral Periventricular Nucleus: More Than Just A Kiss, Leah K. Aggison

Doctoral Dissertations

Estradiol (E₂) is critical in the reproductive mechanisms of mammals. In female rodents E₂ acts through the neurons of the anteroventral periventricular nucleus (AVPV) to exert neuroendocrine control over ovulation, via synaptic activation of the gonadotropin releasing hormone (GnRH) neurons. The neurocircuitry of the AVPV is complex, receiving input from the suprachiasmatic nucleus and ventral premammillary nucleus and the as well as projecting to organum vasculosum of lamina terminalis and the arcuate. This suggests a broader role for the AVPV as a center of multisignal-integration in regards to ovulation. I used full genome expression microarrays to assess …

Expression And Purification Of Human Lysosomal Β-Galactosidase From Pichia Pastoris, Sarah E. Tarullo

Masters Theses

Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into …

Sequence Analysis Of Maize Yellow Stripe3 Candidate Genes, Dennis B. Depaolo

Masters Theses

The work presented here focuses on the molecular mechanism of phytosiderophore secretion in graminaceous plants. In maize, yellow stripe3 (ys3) is a mutant that is deficient in its ability to secrete iron-chelating compounds of the mugineic acid family known as phytosiderophores. Phytosiderophores are specific to grasses and are used for the acquisition of iron. Genetic linkage mapping of the ys3 locus lead to a region of interest on chromosome 3 defined by marker UMC1773. The sequence of eleven candidate genes (GRMZM2G390345, GRMZM2G390374, GRMZM2G342821, GRMZM5G800764, GRMZM2G502560, GRMZM5G849435, GRMZM2G105766, GRMZM5G876835, GRMZM2G036976, GRMZM2G502563, miR167g) revealed several small deletions …

Designing A Pore-Forming Toxin Cytolysin A (Clya) Specific To Target Cancer Cells, Alzira Rocheteau Avelino

Masters Theses

Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can …

Novel Strategies To Modulate Synaptic Communication And Investigate The Role Of Hdac6 In Alzheimer’S Disease, Kathryne A. Medeiros

Doctoral Dissertations

Neuronal communication is mediated by chemical signaling at the synapse. The underlying molecular mechanisms of learning and memory are poorly understood. Very few tools are available to study how memories are formed in the mammalian brain. This dissertation focuses on developing novel strategies to study neural activity. Here we develop and use a chemical-genetic approach to enable target-specific photocontrol of inhibitory synaptic neurotransmission of GABA_A receptor subtypes. The tools developed here selectively photocontrolled GABA_A receptor subtypes. This enabled the investigation of the functional role these receptor subtypes have in inhibitory synaptic neurotransmission. This dissertation also focuses on identifying …

Engineering Probes To Detect Cholesterol Accessibility On Membranes Using Perfringolysin O, Benjamin B. Johnson

Doctoral Dissertations

Cholesterol is an essential component of mammalian cell membranes and it is important to regulate the structure and function of lipid bilayers. Changes in cholesterol levels are involved in many physiological and pathological events such as the formation of arterial plaques, viral entry into cells, sperm capacitation, and receptor organization. Determination of cholesterol trafficking and distribution is essential for understanding how cells regulate cholesterol. A key factor in the regulation of cholesterol is cholesterol accessibility. Through it interactions in the membrane, cholesterol is sequestered below the surface of the membrane. Based on the composition of the membrane, a certain amount …

Inhibition Of Lung Carcinogenesis By Polymethoxyflavones, Noppawat Charoensinphon

Open Access Dissertations

Lung cancer is the leading cause of cancer-related death worldwide. Exclusively found in citrus peels, the inhibitory effects of polymethoxyflavones (PMFs) on 3 human non-small cell lung cancer cells have been investigated. Results showed that monodemethylated PMFs at 5-position potently inhibited lung cancer cells than those of their permethoxylated counterparts. The inhibition of cancer cells caused by monodemethylated PMFs was associated with both extensive cell cycle arrest and apoptosis as a result of modulation of key oncogenic signaling proteins. Treatment with different bioactive compounds in combination may enhance inhibitory effects on lung cancer due to their synergistic interaction among these …

The Role Of Ykl-40 In The Progression Of Glioblastoma, Ralph Anthony Francescone

Open Access Dissertations

Glioblastoma Multiforme (GBM) is the most common brain cancer and one of the most fatal forms of cancer overall. The average survival time is 10-14 months, and less than 10% of patients survive more than 5 years after diagnosis. It is characterized by extreme vasculature, chemo/radioresistance, and invasiveness into the normal brain. The current standard of care, which includes surgical removal of tumor, radiation, and the chemotherapeutic agent temozolomide, initially stunt tumor growth. Nevertheless, the tumor invariably rebounds and the patient succumbs to the disease. Therefore, there is an urgent need to develop new therapies for this devastating disease.

YKL-40 …

Molecular Characterization Of The Pathophysiology Of The Digital Laminae In Acute Carbohydrate-Induced Equine Laminitis, Erica Ann Pawlak

Open Access Dissertations

Equine laminitis is a devastating condition that results in the failure of the tissue responsible for suspending the skeleton within the hoof capsule. The digital laminae is composed of two interdigitated layers, the dermal lamellae surrounding the distal pedal bone, and the epidermal lamellae, which interfaces with the hoof wall. During laminitis, these layers separate, allowing for rotation and sinking of the pedal bone. While there are multiple diseases and physiological conditions associated with the development of laminitis, including sepsis, metabolic syndrome, and unequal weight bearing, the exact cause remains elusive. Prior work by our research group identified the metalloprotease …

Effects Of Phytochemicals From Rhodiola Crenulata On Highly Invasive Breast Cancer Cell Lines And Embryonic Models Of Migration, Adaris Rodriguez-Cortes

Open Access Dissertations

The root of the Tibetan plant Rhodiola crenulata is part of eastern traditional medicine. Studies have suggested that members of the Rhodiola genus display anticancer properties. In this study we examine the effect of R. crenulata in a cellular model of invasive breast cancer, this disease being the second cause of cancer death among women in the US. Deregulation of the Wnt/β-catenin pathway has been frequently observed in breast cancers and appears to have a key role in the transformation of benign cells to a malignant form. Although mutations of the Wnt growth factor are rarely observed in cancer, the …

The Unavoidable Threat Of Aggregation: Implications For Folding And Function Of A Β-Rich Protein, Mylene Hazelle Anne Ferrolino

Open Access Dissertations

Protein aggregation has been implicated in several catastrophic diseases (neurodegeneration, diabetes, ALS) and its complexity has also become a major obstacle in large-scale production of protein-based therapeutics. Despite the generic behavior of proteins to aggregate, only a few globular proteins have known aggregation mechanisms. At present, there have been no clear connections between a protein folding, function and aggregation. We have tackled the challenge of understanding the links between a protein's natural tendency to fold and function with its propensity to misfold and aggregate. Using a predominantly beta-sheet protein whose in vitro folding has been explored in detail: cellular retinoic …

Regulation And Action Of Skp2 And Rhoa In Cell And Tumor Models: Investigation Into The Molecular Mechanisms Responsible For The Aggressive Phenotype Of Triplenegative Breast Cancer, Katrina D. Fagan-Solis

Open Access Dissertations

Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue.

In this study, we use the Tamoxifen-selected, MCF-7 derivative, TMX2-28 breast cancer cell line. TMX2-28 cells are triple-negative in that …

Identification And Functional Characterization Of The Zebrafish Gene Quetschkommode (Que), Timo Friedrich

Open Access Dissertations

Locomotion in vertebrates depends on proper formation and maintenance of neuronal networks in the hind-brain and spinal cord. Malformation or loss of factors required for proper maintenance of these networks can lead to severe neurodegenerative diseases limiting or preventing locomotion. A powerful tool to investigate the genetic and cellular requirements for development and/or maintenance of these networks is a collection of zebrafish mutants with defects in motility. The zebrafish mutant quetschkommode (que) harbors a previously unknown gene defect leading to abnormal locomotor behavior. Here I show that the que mutants display a seizure-like behavior starting around four days post fertilization …

The Role Of Bacteriocins In Mediating Interactions Of Bacterial Isolates From Cystic Fibrosis Patients, Emine Suphan Bakkal

Open Access Dissertations

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa(Pa) and …

Xenopus Adam13 And Adam19 Are Important For Proper Convergence And Extension Of The Notochord, Russell David Neuner

Open Access Dissertations

Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal …