Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.

Temperature is a critical environmental signal, which exerts powerful control over the development and virulence of diverse microbial pathogens. Fungi, along with other microbial species, exploit a diversity of mechanisms to sense and respond to temperature fluctuations that may be encountered in the host or under other conditions of temperature stress. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs, and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is strongly influenced by temperature, and most filament inducing cues depend on a concurrent increase of temperature to 37˚C before morphogenesis can occur. Further elevated temperature of 39˚C to 42˚C can serve as an independent filament-inducing cue, although the molecular mechanisms underpinning this temperature-dependent morphogenetic transition remain largely uncharacterized. Here, I provide the first comprehensive investigation of the molecular mechanisms mediating temperature-dependent morphogenesis in C. albicans. I establish that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, and that Hsp90 functions as a key temperature sensor, such that elevated temperature is required to relieve Hsp90-mediated repression of the morphogenetic program. Further, I demonstrate that Hsp90 controls morphogenesis via at least two distinct cellular signaling cascades. First, Hsp90 and its co-chaperone Sgt1 physically interact, and together regulate protein kinase A (PKA) signaling via an interaction with the adenylyl cyclase of the PKA cascade, Cyr1, such that genetic depletion of either Hsp90 or Sgt1 activates PKA signaling and induces filamentation. Second, Hsp90 controls temperature-dependent morphogenesis via previously uncharacterized cellular circuitry comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1, and the transcriptional regulator Hms1. Together, this research illuminates the central role of Hsp90 in coupling temperature sensing and morphogenesis in the human fungal pathogen C. albicans.

Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Richard D. Oberst / Viruses differ in their susceptibility and resistance to disinfectants and their ability to be inactivated based predominantly on structural and size differences. The virucidal methodology followed in this study was consistent with the U.S. Environmental Protection Agency guidelines for such efficacy testing. Various disinfectant chemistries were effective in rapid and complete inactivation of bovine coronavirus (BCV) including 25, 50, and 100% DF-200d, 10% bleach, 1% Virkon® S, and 70% ethanol following 10 min treatments. Efficacy was slightly reduced in the presence of organic challenge material (feces, soil). Treatment with 100% DF-200d or 10% bleach resulted in the degradation of BCV RNA. Treatment with 50 or 100% DF-200d, 10% bleach, or 1% Virkon® S resulted in the degradation of BCV proteins as detected by western blot analysis. Various disinfectants were effective in the complete inactivation of both mammalian and avian influenza test strains and included DF-200d, 10% bleach, 1% Virkon® S, and 70% ethanol following 10 min exposure but was reduced in the presence of organic challenge. Treatment with DF-200d or 10% bleach resulted in significant degradation of influenza RNA. Effective treatments against foot-and-mouth disease virus (FMDV) and bovine enterovirus-2 (BEV-2) included 10% bleach, 4% sodium carbonate, 2% sodium hydroxide, and Sandia DF-200. Treatment with 5% acetic acid was effective against FMDV, but not BEV-2. Treatment with 70% ethanol was not effective for inactivating FMDV or BEV. Additionally, 10 min treatment with 10% bleach, 2% sodium hydroxide, and Sandia DF-200 degraded FMDV and BEV-2 RNA. FMDV inoculated surfaces were exposed to 5% acetic acid, 10% bleach, 70% ethanol, 4% sodium carbonate, 2% sodium hydroxide, Sandia DF-200, 0.4% Oxy-Sept® 333, or 1% Virkon® S. All treatments except 70% ethanol were effective following 10 and 20 min contact on rubber and stainless steel. No treatments were shown to be effective for concrete, due to a low recovery of virus from this surface. Additionally, 10 and 20 min treatments with 10% bleach, 2% sodium hydroxide, and Sandia DF-200 degraded FMDV RNA on the surfaces evaluated.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / George C. Stewart / Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than the vegetative bacterial cells, are the source of anthrax infections. The spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ΔSterne that carries a deletion of the bclB gene. Immunofluorescence studies indicated that the mutant strain produced spores with increased amounts of the BclA glycoprotein expressed on their surface. Differences in exosporium composition between the mutant and wild-type spores were identified. The mutant was also found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. The resistance properties of the mutant spores were unchanged from that of the wild-type spores. The bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of a rigid and complete exosporium structure in B. anthracis. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

Master of Science / Department of Biology / Sherry D. Fleming / Helicobacter is a common intestinal pathogen of most laboratory mice from both commercial and academic sources worldwide. Not previously thought to have an effect, recent evidence indicates Helicobacter infection alters cytokine, chemokine, and gene expression in the stomach, intestine, and colon. Though the in vivo cell types responsible for these changes are currently unknown, in vitro results suggest macrophages are the likely source. In addition to detection and elimination of pathogens, intestinal macrophages play a role in maintaining homeostasis. By altering gene expression and cytokine production in the microenvironment, we hypothesized that Helicobacter infection altered the phenotype and inflammatory response of submucosal intestinal macrophages. To test this hypothesis, we examined macrophages within whole mounts of intestinal muscle as well as isolated macrophages from Helicobacter-infected or uninfected mouse intestine. Macrophages from the intestinal muscle of Helicobacter-infected mice showed increased expression of F4/80 and CD11b, altered gene expression, and increased phagocytosis when compared to macrophages from uninfected mice. Infection also altered the macrophage response to stimuli. Macrophages from infected mice produced significantly lower concentrations of cytokines, chemokines, and PGE[subscript]2 in response to stimulation with either IFN and LPS or IL-4 and IC. These data support our hypothesis demonstrating that the intestinal muscle macrophage phenotype, function, and response to stimulation are altered by Helicobacter infection both in vivo and in vitro.

Master of Science / Food Science Institute - Animal Science & Industry / Daniel Y.C. Fung / Proper microbiological examination of foods involves proper sample preparation in terms of mixing the solid or liquid food with a suitable sterile diluent (usually a 1:10 dilution) in a sterile bag and homogenizing them manually or by means of an instrument. This thesis addresses the effectiveness of Stomacher®, Pulsifier®, Bagmixer®, and Smasher® instruments in terms of: 1) Number of viable cell counts/g of ten food types, 2) Noise level of the four instruments ascertained by a) human and b) decibel meter at five feet (1.52 meters) from each instrument, 3) Ease in cleaning the instruments after use, and 4) Ergonomics. Following the ISO Method (7218:2007), 25 g each of alfalfa sprouts, spinach, peanuts, ground beef, fish meat, hot dogs, tofu, milk, chicken wing meat, and chicken drum stick meat were placed individually in a sterile sample bag containing 224.5 mL of 0.1% Peptone water plus 0.5 mL of E. coli inoculum diluent. Each food was homogenized for 60 seconds in each of the instruments. During each treatment four laboratory workers standing at five feet (1.52 meters) from the instrument assessed the noise level as: very quiet, quiet, nearly quiet, acceptable noise, and loud. Also the noise level was monitored instrumentally by the use of a decibel meter and recorded as Db. Ease of cleaning and ergonomics were determined with the aid of a subjective scale set with the Stomacher® as a reference point. The results indicate that all four instruments have similar performance in regards to viable cell counts. However, in regards to noise level, the Smasher® and the Bagmixer® are the quietest compared to the Stomacher® and then the Pulsifier®. The Smasher® is also the instrument with the highest ranking in ease of cleaning and ergonomics.

Master of Science / Department of Biology / Peter P. Wong / Contamination of water with various disease causing agents such as bacteria and viruses leads to 4 billion cases of diarrheal disease and 1.8 million deaths per year worldwide. The World Health Organization estimates that 94% of these cases can be prevented by increasing the availability of clean and safe water to those at risk. They also claim that 1.1 billion individuals worldwide do not have access to clean water sources, and suggest the best way to improve this situation is to increase household water treatment and safe storage (HWTS). HWTS can dramatically improve water quality, leading to a significant reduction in diarrheal disease. Being able to produce a small and inexpensive device that can be used in a household to improve water quality will significantly aid in preventing diarrheal disease. Water purification systems have been manufactured in the past in order to turn potentially disease causing water into safe drinking water. The metal iodine has been used in some of these systems. However, iodine is known to leach off and become part of the drinking water, which can lead to non-infectious diseases such as hyperthyroidism. This project shows how the development of new water purification systems utilizing silver and other heavy metal nanoparticles may be used to help purify water and have the potential to prevent diarrheal disease. Various heavy metals, including silver nitrate, magnesium oxide, brominated magnesium oxide and titanium-silicon-manganese dioxide nanoparticles, are used in conjunction with ion-exchange resins to develop a biocidal column to sanitize water. A model virus from each of the following families is used: Reoviridae (rotavirus), Picornaviridae (enterovirus), and Caliciviridae (calicivirus). This research shows not only that some of these metals can be used to purify water by inactivating viruses, but also shows the mechanism of viral inactivation. This includes protein denaturation and destruction of viral RNA.

Helicobacter pylori is a gram-negative bacterium that infects half the world population and is the etiological cause of numerous gastric pathologies. H. pylori possess numerous mechanisms to promote its survival and modulate host immunity. We propose that H. pylori can modulate intercellular communication by manipulating the host exosome pathway. Exosomes are secreted nanovesicles that contain different proteins and microRNAs that can be transferred between cells to alter cell signaling and gene expression. We demonstrate that H. pylori infection increases host exosome secretion. Furthermore, infection can alter exosome composition as VacA, a bacterial virulence factor, can be exported in exosomes and Argonaute 5, a miRNA effector protein, is upregulated in exosomes during infection. Lastly, we show preliminary evidence that infection-modulated exosomes can modulate immune-regulatory signaling in dendritic cells by activating STAT3. Together, these studies elucidate a novel mechanism by which H. pylori can modulate the host environment and promote its continued survival.