Molecular Biology Commons^™

Methodology To Investigate Protein-Protein Interactions Of Jun During Neuronal Differentiation Of Pc12 Cells, Heather L. Leskinen

Theses and Dissertations

In humans and other mammals, injury to the central nervous system (CNS) can cause a permanent loss of neuronal function, leading to cognitive defects, limb paralysis, and other neurological disabilities. In contrast, studies have shown that some non-mammalian vertebrates like zebrafish, have the remarkable ability to functionally regenerate axons after CNS injury by reactivating and sustaining the expression of regeneration-associated genes (RAGs). Some RAGs encode transcriptional regulators that dimerize to control downstream gene expression necessary for functional axonal recovery. Our lab has previously identified Jun as an important transcriptional regulator of regeneration after optic nerve injury in zebrafish. After axon …

Complex Regulatory Pathways Connect Vfm Quorum-Sensing And Cyclic-Di-Gmp Signaling To Bacterial Virulence In The Phytopathogen Dickeya Dadantii 3937, Biswarup Banerjee

Theses and Dissertations

The bacterial second messenger Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates multiple cellular behaviors in most bacteria. Bacterial c-di-GMP signaling involves enzymes that synthesize and degrade c-di-GMP, c-di-GMP binding effectors, and targets that are acted upon by the effectors. So far, the c-di-GMP signaling pathways have been understudied. In this work, we explore the c-di-GMP signaling network further in the phytopathogen Dickeya dadantii 3937. In Chapter 2, we identified VfmE as a c-di-GMP binding transcriptional activator that represses pectate lyase production under a high c-di-GMP condition. VfmE was found to bind c-di-GMP in vitro via the RxxxR motif, similar to PilZ …

Molecular Evolution And Biogeography Of The New World Eptesicus Bats, Xueling Yi

Theses and Dissertations

Molecular evolution refers to a broad field of studies ranging from microevolution (e.g., population genetics) to macroevolution (e.g., phylogeny), including the bridging field of phylogeography. In natural populations, molecular studies are also combined with biogeography that links biological diversity with geographic distributions to provide a comprehensive understanding of evolutionary processes. The field of molecular evolution has been largely advanced from early exploratory descriptions to statistical tests on biological hypotheses and integrative analyses using sophisticated modeling. However, studies of molecular evolution still face some unresolved questions and challenges, especially in non-model systems. For example, the application of new technology has largely …

Roles Of Secreted Proteins And Iron Utilization Proteins In Virulence Of The Fish Pathogen Flavobacterium Columnare, Rachel Conrad

Theses and Dissertations

The Gram-negative bacterium Flavobacterium columnare is the causative agent of columnaris disease and is responsible for significant economic losses in aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Like many members of the phylum Bacteroidetes, F. columnare uses the type IX secretion system (T9SS) to secrete enzymes, adhesins, and proteins involved in gliding motility. When a core component of this system was deleted in the wild type strain, the resulting mutant was avirulent in zebrafish, rainbow trout, and channel catfish infection studies. This suggests that the individual secreted proteins may function …

Selection And Demography Drive Range-Wide Patterns Of Mhc Variation In Mule Deer (Odocoileus Hemionus), Rachel M. Cook

Theses and Dissertations

Variation at functional genes involved in immune response is of increasing concern as wildlife diseases continue to emerge and threaten populations. The amount of standing genetic variation in a population is directly associated with its potential for rapid adaptation to novel environments. For genes in the major histocompatibility complex (MHC), which are crucial in activating the immune response and which have extremely high levels of polymorphism, the genetic variation has been shown to be influenced by both parasite-mediated selection and historical population demography. To better understand the relative roles of parasite-mediated selection and demography on MHC evolution in large populations, …

Negative Regulation Of The Kinase Lin-45 By The E3/E4 Ubiquitin Ligase Ufd-2, Augustin Deniaud

Theses and Dissertations

The serine/threonine kinase BRAF is a key part of the Ras-Raf-MEK-ERK pathway, an inducer of cell growth, differentiation, and survival. In humans, activating mutations, most commonly BRAF(V600E), have been detected in several cancers, including melanoma and thyroid cancer. In the Caenorhabditis elegans ortholog LIN-45, the equivalent mutation LIN-45(V627E) results in elevated Raf-MEK-ERK signaling. We performed an unbiased genetic screen to identify negative regulators of LIN-45(V627E). Here, we report the identification of the E3/E4 ubiquitin ligase UFD-2, and show it is a negative regulator of LIN-45 protein activity and levels. Loss of UFD-2 leads to accumulation of wild-type LIN-45 protein as …

Structure & Function Of Enzymes In Two Uncharacterized Gene Clusters From Pseudomonas Brassicacearum & Streptomyces Griseofuscus, Lamia Tabassum Badhon

Theses and Dissertations

Pyridoxal 5’-phosphate (PLP)-dependent enzymes harness this versatile cofactor to catalyze a variety of reactions including transamination, decarboxylation, racemization and various elemination/subsitution reactions. Several years ago, a new class of PLP-dependent enzymes was discovered that uses PLP and molecular oxygen to catalyze the 4-electron oxidation of L-arginine to 4-hydroxy-2-ketoarginine. Work with the prototypical enzyme of this class, MppP from Streptomyces wadayamensis (SwMppP), showed that the dioxygen consumed during the reaction is reduced to hydrogen peroxide, and that the hydroxyl group installed in the product derives from water. Thus, SwMppP is an L-arginine oxidase, and not an oxygenase. This was surprising given …

Dissecting Molecular Mechanism Of Heat Stress On Anther Development In Fragaria Vesca, Shikha Malik

Theses and Dissertations

Strawberry is an important fruit crop in the Rosaceae family. Originally from the temperate region, strawberry is vulnerable to heat waves, which reduce fruit yield and quality. Previous studies have shown that heat stress impairs pollen development; however, the molecular mechanisms by which heat stress affects the development of anthers, where pollen develops, are unclear. Due to the genome complexity of cultivated strawberry (Fragaria × ananassa, 2n = 8x= 56), Fragaria vesca (2n = 2x = 14) was used, which emerges as a diploid model plant for Rosaceae, to study the effects of heat stress on anther development at morphological …

Understanding The Role Of Protein Kinases Kin1 And Kin2 In The Protein Folding Pathways In The Yeast Saccharomyces Cerevisiae, Chandrima Ghosh

Theses and Dissertations

Eukaryotic protein kinases catalyze the transfer of the -phosphate of an ATP to a serine/threonine/tyrosine residue present in a protein substrate. The phosphorylation of proteins has profound effects on their activity and protein-protein interactions, thus regulating a plethora of cellular processes, including cell growth, differentiation and protein homeostasis (or proteostasis). Our lab is the first to demonstrate that protein kinases Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae, orthologs of human microtubule affinity-regulating kinase (MARK), contribute to protein-folding homeostasis inside the endoplasmic reticulum (ER), in addition to their canonical roles in cellular exocytosis. The main aim of …

Photobiomodulation Modulates Bioenergetics And Oxidative Stress In An In Vitro Model Of Diabetic Retinopathy, Alexandria E. Hall

Theses and Dissertations

Diabetic Retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of blindness. The pathophysiology of DR is complicated, involving mitochondrial dysfunction, oxidative stress, inflammation and vascular degeneration. The most common therapeutic approach for DR includes the use anti-vascular endothelial growth factor (VEGF) drugs to reduce vascular proliferation. These treatments are invasive, frequently ineffective and have numerous adverse effects. A non-invasive more effective therapy is clearly needed. An alternative, non-invasive therapy using far-red light (photobiomodulation, PBM) may be an improvement over current therapy. PBM has documented efficacy in experimental and clinical studies of retinal disease including …

Gene Regulatory Mechanisms Driving Temporal Dynamics Of Lineage Specific Differentiation In The Cranial Neural Crest, Maria R. Replogle

Theses and Dissertations

Genetic and environmental perturbations impacting neural crest (NC) development can result in pleiotropic structural and functional birth defects, many of which are associated with pediatric syndromes. As developmental precursors, the NC has the unique capacity to give rise to a diverse array of ectodermal and mesoectodermal cell types, from neurons and glia of the peripheral nervous system to the cartilage and bone of the face. In order to transition from a multipotent progenitor to a specific cell type, NC cells must undergo a series of dynamic morphological and behavioral transformations that gradually unfold over time. However, the NC is rare …

A Tale Of Two Adaptors: The Role Of Two Adaptor Proteins In Pseudomonas Aeruginosa Chp Chemosensory System Signal Transduction And Implications For Chemosensory Array Formation, Zachary Hying

Theses and Dissertations

Bacteria use chemosensory systems to coordinate environmental signals to direct chemotaxis and make lifestyle decisions such as surface attachment and biofilm formation. Chemosensory systems form extended arrays with pseudo-hexagonal symmetry that are essential for efficient signal transduction. These arrays consist of three essential components: Methyl-Accepting Chemotaxis proteins (MCPs), which receive signals, a histidine kinase to coordinate cell responses through phosphorylation of response regulators, and an adaptor protein to transduce conformational change and facilitate array formation. Pseudomonas aeruginosa uses four chemosensory systems to control flagellar-based motility, type IV pili-mediated twitching motility and acute virulence, and biofilm formation. The Chp chemosensory system …

A Seascape Genetics Approach To Studying Genetic Differentiation In The Bull Kelp Nereocystis Luetkeana, Lily G. Gierke

Theses and Dissertations

The brown alga Nereocystis luetkeana is a foundation species found from Alaska to California. In the Salish Sea, N. luetkeana is declining, but little is known about its population structure. We explored N. luetkeana 1) allelic dissimilarity and richness using seven microsatellite markers, and 2) tested models of gene flow in the Salish Sea using a hydrodynamic transport model. Our results suggest that the N. luetkeana distribution is comprised of four genetic co-ancestry groups that are geographically coherent, apart from the separation of the Strait of Georgia/Puget Sound by the Strait of Juan de Fuca. Our model supported that environmental …

Development Of Indicators For Human Fecal Pollution Using Deep-Sequencing Of Microbial Communities, Shuchen Feng

Theses and Dissertations

The gut microbiome is shaped by both host physiology and environmental factors, which results in unique communities that contain certain members specific to a host. Microbial source tracking (MST) methods that rely on host-specific fecal microorganisms have been applied to detect human fecal pollution over the past two decades. The most common approach uses quantitative polymerase chain reaction (qPCR) to amplify certain sequences of these microorganisms, or human fecal markers. To date, most bacterial human fecal markers have focused on the HF183 cluster within the genus Bacteroides. However, cross-reactions with animals or variable Bacteroides abundance in humans have been found. …

Lorelei Localization And Ovule Ultrastructure In Arabidopsis Thaliana, Juleen May Dickson

Theses and Dissertations

Communication between the male and female gametophyte is vital to successful fertilization during sexual reproduction in plants. One of the proteins known to be important for communication between the male and female gametophyte is LORELEI (LRE). Several studies have shown that there are defects in pollen tube guidance and synergid degeneration, however this is the first study that shows that cell wall thickness in the female gametophyte may also be affected. Previous confocal studies have documented that LRE is present both in the filiform apparatus and found in puncta throughout the cytoplasm. This study confirmed this, but our studies suggest …

Flavobacterium Gliding Motility: From Protein Secretion To Cell Surface Adhesin Movements, Joseph Johnston

Theses and Dissertations

Flavobacterium johnsoniae exhibits rapid gliding motility over surfaces. At least twenty genes are involved in this process. Seven of these, gldK, gldL, gldM, gldN, sprA, sprE, and sprT encode proteins of the type IX protein secretion system (T9SS). The T9SS is required for surface localization of the motility adhesins SprB and RemA, and for secretion of the soluble chitinase ChiA. This thesis demonstrates that the gliding motility proteins GldA, GldB, GldD, GldF, GldH, GldI and GldJ are also essential for secretion. Cells with mutations in the genes encoding any of these seven proteins had normal levels of gldK mRNA but …

Mechanistic Insights Into Translational Silencing Of Hacl Mrna In Yeast S. Cerevisiae, Leena Sathe

Theses and Dissertations

Protein synthesis is a fundamental life process. Protein synthesis regulates cellular metabolism, cellular growth, the cell cycle, and cell morphogenesis. Technical advances in molecular biology, advanced high-throughput sequencing technologies and recent developments in crystallographic methodologies have aided in better understanding of the process of protein synthesis. Current knowledge of protein synthesis provides us with an overview of the initiation, elongation, and termination steps, general regulatory mechanisms, and molecular functions of the ribosomal rRNA and proteins. However, even with all this information we are far from understanding the detailed sequence of molecular interactions involved in the process of protein synthesis. We …

Dissection Of Floral Organ Development And Sterility In Sorghum Bicolor, Ashley R. Smith

Theses and Dissertations

Sorghum bicolor is a drought resistant cereal grain commonly grown for use in food, feed, fiber, and fuel production. Due to its versatility and modest sized genome it is poised to become an increasingly important research organism within the C4 plants. Sorghum produces spikelet pairs with one fertile sessile spikelet and one to two sterile pedicellate spikelets. One major area of interest in sorghum production is maximizing seed yield. While the importance of the grain is obvious there are several large gaps in the study of sorghum that prevent researchers and growers from maximum productivity. The first gap is that …

River Bank Inducement Influence On A Shallow Groundwater Microbial Community And Its Effects On Aquifer Reactivity, Natalie June Gayner

Theses and Dissertations

Placing groundwater wells next to riverbanks to draw in surface water, known as riverbank inducement (RBI), is common and proposed as a promising and sustainable practice for municipal and public water production across the globe. However, these systems require further investigation to determine risks associated with river infiltration especially with rivers containing wastewater treatment plant (WWTP) effluent. Since microbes drive biogeochemical transformations in groundwater and largely affect water quality, it is important to understand how the microbial communities in drinking water wells are affected by river infiltration. This study investigated if, and to what extent, the microbial community in a …

Role Of The Two Adaptor Proteins In The Chp Chemosensory System Of Pseudomonas Aeruginosa, Swati Sharma

Theses and Dissertations

The Chp chemosensory system in Pseudomonas aeruginosa controls two outputs: twitching motility (surface-mediated movement via Type IV pili) and intracellular adenosine 3’, 5’-cyclic monophosphate (cAMP) levels (by modulating the activity of major adenylate cyclase CyaB). This study was done to investigate the roles of the two adaptor proteins, PilI and ChpC in connecting one methyl-accepting protein (MCP) to one histidine kinase. We assayed -galactosidase activity as an indicator of the relative levels of intracellular cAMP and measured twitching motility. We also studied the interaction of the adaptor proteins within the Chp chemosensory system. Our bacterial adenylate cyclase two-hybrid analysis showed …

Sex Differences In Differential Fear Conditioning During The Acquisition And Consolidation Of Learned Safety, David Sylvan Reis

Theses and Dissertations

The ability to distinguish between threatening and non-threatening situations requires careful regulation of behavioral and physiological responses to stress and fear. Deficits in fear regulation are maladaptive and can lead to the development of anxiety disorders such as PTSD. Women are nearly twice as likely to develop PTSD as are men and laboratory animal studies have shown facilitated fear acquisition, resistance to fear extinction, deficits in extinction retention and impaired discrimination between danger and safety cues in females. Taken together this suggests a propensity for reduced inhibitory control over fear responding in females. Here we investigate the mechanisms underlying fear …

An Inducible Fluorescent Reporter System To Measure Lux Operon Promoter Activity, Nicole Thunes

Theses and Dissertations

Bioluminescence is the enzymatic production of light by a living organism. Many species of marine bacteria produce light with varying degrees of brightness. The lux operon is responsible for bioluminescence and is well studied, however it is currently unknown why different species of bacteria display different brightness levels. A dual-plasmid system designed to mimic the quorum-sensing induction of the lux operon was created and successfully implemented in E. coli. This was accomplished through the use of an arabinose-inducible plasmid containing a luxR gene from Vibrio harveyi, and then using the resulting LuxR protein to activate the lux promoter in a …

The Role Of Pilj And Its Structural Domains In The Localization And Function Of The Chp Chemosensory System In Pseudomonas Aeruginosa, Vibhuti Hemantkumar Jansari

Theses and Dissertations

Bacteria detect environmental signals using membrane-bound methyl-accepting chemotaxis proteins (MCPs), which are part of a larger complex of chemosensory proteins. Pseudomonas aeruginosa has four functionally distinct chemosensory protein complexes. The Chp chemosensory system regulates type IV pili mediated twitching motility and intracellular levels of cAMP by modulating the activity of an adenylate cyclase, CyaB. The Chp system is also proposed to be involved in type IV pili mediated directional twitching motility towards phosphatidylethanolamine (PE). PilJ is the only MCP predicted to be associated with the Chp system. In this study we investigated different domains of PilJ in order to decipher …

New Insights Into The Role Of Antimicrobials Of Xenorhabdus In Interspecies Competition, Kristin Jean Ciezki

Theses and Dissertations

Xenorhabdus spp. are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. The nematodes penetrate the insect midgut to enter the hemocoel where Xenorhabdus bacteria are released, transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the hemocoel. In addition, different species of nematodes carrying specific strains of Xenorhabdus can invade a single insect. Xenorhabdus spp thereby engage in competition with both related strains and nonrelated gut microbes. In complex media Xenorhabdus spp produce diverse antimicrobial compounds whose functions in biological systems remain poorly understood. R-type bacteriocins are contractile phage-tail-like structures that are bactericidal …

Microbial Communities And The Diverse Ecology Of Fecal Indicators At Lake Michigan Beaches, Danielle Cloutier

Theses and Dissertations

Fecal indicator bacteria (FIB) such as Escherichia coli (E. coli) and enterococci are used to assess microbiological water quality in recreational waters. The use of FIB follows the assumption that their presence correlates with that of fecal-associated pathogens in recreational waters. The beach ecosystem is complex however and multiple factors can influence the concentration of E. coli and enterococci in the beach environment. Microbial communities within beach sand play a key role in nutrient cycling and are important to the nearshore ecosystem function. E. coli and enterococci, two common indicators of fecal pollution, have been shown to persist in the …

Towards Autonomous Microcystin Detection: Investigating Methods For Automation, Maureen Anne Schneider

Theses and Dissertations

Due to increased anthropogenic activity, severe eutrophication is occurring in bodies of water around the world. Effects include decreased water quality, decreased value of surrounding land and recreational use (estimated loss in revenue of 0.67 and 3.96 U.S. billion dollars per year), and increased occurrence of toxin producing Harmful Algal Blooms (HABs). Microcystins are cyclic peptides made up of 7 amino acids and 800-1100 Daltons in size. They are one of the most predominantly produced of these toxins, and therefore was the focus of this study. Numerous structural variants of microcystin (referred to as congeners) exist, but microcystin-LR is one …

A Novel Link Between The Chemotaxis And Biofilm Dispersion Systems Of Pseudomonas Aeruginosa, Jesse Michael Reinhardt

Theses and Dissertations

Bacterial chemotaxis is the movement of a cell towards an attractant or away from a repellent. This controlled movement is possible due to the chemotaxis system, which is typically made up of several proteins that collectively sense the stimuli and transduce the signal within the cell to mediate a motility response. The chemotaxis proteins of Pseudomonas aeruginosa are encoded in two clusters, which are located at different regions of the chromosome: che I and che V. These gene clusters are known to control chemotaxis via swimming, or flagellar-based, motility. When expressed, these chemotaxis proteins associate with each other to form …

Characterization And Use Of Folate Receptor Isoforms For Targeting Of Epithelial And Myeloid Cells, Sreya Biswas

Theses and Dissertations

ABSTRACT

CHARACTERIZATION AND USE OF FOLATE RECEPTOR ISOFORMS FOR TARGETING OF EPITHELIAL AND MYELOID CELLS

by

Sreya Biswas

The University of Wisconsin-Milwaukee, 2016

Under the Supervision of Professor Douglas A. Steeber

Folate receptor (FR) is a GPI-anchored glycoprotein with high binding affinity for folic acid. FR has two membrane-associated isoforms, α and β, that are overexpressed on epithelial and myeloid tumors, respectively. Normal cells may also exhibit FR expression at very low levels but interestingly, FR-α on normal cells is restricted to the apical surface i.e., away from the blood stream. This differential expression and orientation of the FR-α isoform …

Molecular And Functional Analysis Of The Pixb Gene In Xenorhabdus Nematophila, John Lucas

Theses and Dissertations

Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae form a mutualistic relationship facilitating the infection, death and consumption of an insect host. The infective juvenile (IJ) form of S. carpocapsae invades the insect host through natural openings and proceeds to the hemocoel where exposure to hemolymph stimulates the release of X. nematophila from the anterior vesicle. Excreted X. nematophila releases immunosuppressive compounds and insect toxins into the insect hemolymph that facilitates death of the host. As X. nematophila reaches high cell density it secretes exoenzymes that degrade insect tissues and produces antibiotics that reduce microbial competition. S. carpocapsae utilizes the …

Discovery And Characterization Of A New Group Of Is10 Insertion Sequences, Rachel Marie Kinzelman

Theses and Dissertations

Insertion sequences (ISs) are small mobile genetic elements that can have significant impact on the genotype and phenotype of a host organism. Previous work in this laboratory revealed an insertion sequence that disrupted the luxA gene in Vibrio harveyi strain BCB451, knocking out light production. Phylogenetic analysis of this insertion sequence, dubbed IS451, reveals that it is in the IS10 family, but represents a novel variant that is only 79% identical to other known IS10 sequences. Twelve copies of IS451 were isolated from a genomic library and sequenced, and were found to be essentially identical, but located in dispersed chromosomal …