The Heat Shock Protein 90 (Hsp90) is an essential and highly conserved chaperone that facilitates the maturation of a wide array of client proteins, including many kinases. These clients in turn regulate a wide array of cellular processes, such as signal transduction, and transcriptional reprogramming. As a result, the activity of Hsp90 has the potential to influence physiology, which in turn may influence the ability to adapt to new environments. Previous studies using a deep mutational scanning approach, (EMPIRIC) identified multiple substitutions within a 9 amino acid substrate-binding loop of yeast Hsp90 that provides a growth advantage for yeast under elevated salinity conditions and costs of adaptation under alternate environments. These results demonstrate that genetic alterations to a small region of Hsp90 can contribute to evolutionary change and promote adaptation to specific environments. However, because Hsp90 is a large, highly dynamic and multi-functional protein the adaptive potential and evolutionary constraints of Hsp90 across diverse environments requires further investigation. In this dissertation I used a modified version of EMPIRIC to examine the impact of environmental stress on the adaptive potential, costs and evolutionary constraints for a 118 amino acid functional region of the middle domain of yeast Hsp90 under endogenous expression levels and the entire Hsp90 protein sequence under low expression levels. Endogenous Hsp90 expression levels were used to observe how environment may affect Hsp90 mutant fitness effects in nature, while low expression levels were used as a sensitive readout of Hsp90 function and fitness. In general, I found that mutations within the middle domain of Hsp90 have similar fitness effects across many environments, whereas, under low Hsp90 expression I found that the fitness effects of Hsp90 mutants differed between environments. Under individual conditions multiple variants provided a growth advantage, however these variants exhibited growth defects in other environments, indicating costs of adaptation. When comparing experimental results to 261 extant eukaryotic sequences I find that natural variants of Hsp90 support growth in all environments. I identified protein regions that are enriched in beneficial, deleterious and costly mutations that coincides with residues involved in co-chaperone-client-binding interactions, stabilization of Hsp90 client-binding interfaces, stabilization of Hsp90 interdomains and ATPase chaperone activity. In summary, this thesis uncovers the adaptive potential, costs of adaptation and evolutionary constraints of Hsp90 mutations across several environments. These results complement and extend known structural and functional information, highlighting potential adaptive mechanisms. Furthermore, this work elucidates the impact environment can have on shaping Hsp90 evolution and suggests that fluctuating environments may have played a role in the long-term evolution of Hsp90.

<p>In&nbsp; this&nbsp; study,&nbsp; tolerance&nbsp; of&nbsp; soil&nbsp; bacteria&nbsp; to&nbsp; hexavalent&nbsp; chromium&nbsp; (Cr(VI))&nbsp; was&nbsp; investigated.&nbsp; First,&nbsp; influence&nbsp; of&nbsp; high chromium levels of anthropogenic and geogenic origin on the&nbsp; soil cultivable&nbsp; bacterial community was examined. Next, a number&nbsp; of&nbsp; bacterial&nbsp; strains&nbsp; with&nbsp; high&nbsp; Cr(VI)&nbsp; tolerance&nbsp; were&nbsp; isolated&nbsp; from&nbsp; diverse&nbsp;&nbsp; environmental&nbsp; samples&nbsp; such&nbsp; as&nbsp; soil, sediment, water and waste material.&nbsp; Strains were&nbsp; identified&nbsp; and&nbsp; tested for&nbsp; the&nbsp; level of&nbsp; Cr(VI) tolerance&nbsp; and&nbsp; the&nbsp; ability to<br />reduce toxic Cr(VI) to more innocuous Cr(III). Selected&nbsp; <em>Bacillus cereus</em>&nbsp; group strains&nbsp; were further characterized&nbsp; -&nbsp; their morphological&nbsp; and&nbsp; biochemical&nbsp; characteristics,&nbsp; 16S&nbsp; rRNA&nbsp; and&nbsp; pycA&nbsp; gene&nbsp; sequences,&nbsp; biofilm&nbsp; formation&nbsp; potential&nbsp; and resistance to other heavy metals were determined. Also, more detailed study of their tolerance level and&nbsp; Cr(VI) reduction was&nbsp; conducted.&nbsp; Strain&nbsp; with&nbsp; the highest&nbsp; resistance&nbsp; together&nbsp; with the&nbsp; control&nbsp; chromate&nbsp; sensitive&nbsp; strain&nbsp; were&nbsp; analyzed&nbsp; by STEM EDS for their cellular and endospore Cr content under different conditions. Results indicate Cr(VI) tolerant bacteria are&nbsp; present&nbsp; both&nbsp; in&nbsp; low&nbsp; and&nbsp; high&nbsp; Cr&nbsp; environments.&nbsp; Majority&nbsp; of&nbsp; isolates&nbsp; belonged&nbsp; to&nbsp; the&nbsp;<em> B.&nbsp; cereus&nbsp;</em> group&nbsp; indicating&nbsp; its overall high tolerance to&nbsp; Cr(VI). Certain strains exhibited high&nbsp; tolerance and reduction&nbsp; ability,&nbsp; indicating their possible<br />usefulness&nbsp; in practical&nbsp; bioremediation&nbsp; application.&nbsp; STEM&nbsp; EDS&nbsp; analysis&nbsp; of&nbsp; Cr(VI)-sensitive&nbsp;<em> B.&nbsp; subtilis&nbsp;</em> PY79&nbsp; strain&nbsp; and Cr(VI)-resistant&nbsp; <em>B. cereus&nbsp;</em> group strain&nbsp; NCr1a revealed&nbsp; significant differences in their response to Cr(VI)&nbsp; and in&nbsp; their&nbsp; Cr cellular and endospore content.</p> / <p>U ovom radu ispitana je tolerantnost&nbsp; zemlji&scaron;nih&nbsp; bakterija na &scaron;estovalentni hrom (Cr(VI)). Prvo, ispitan je uticaj visokog nivoa&nbsp; hroma&nbsp; antropogenog&nbsp; i&nbsp; geogenog&nbsp; porekla&nbsp; na&nbsp; kultivabilnu&nbsp; bakterijsku&nbsp; zajednicu&nbsp; zemlji&scaron;ta.&nbsp; Dalje,&nbsp; izolovani&nbsp; su bakterijski sojevi sa visokom tolerancijom na Cr(VI) iz različitih sredinskih uzoraka &nbsp; kao &scaron;to su zemlji&scaron;te, sediment, voda i otpadni materijal. Sojevi su identifikovani i određen je nivo njihove Cr(VI) tolerancije i sposobnost redukcije toksičnog Cr(VI)&nbsp; u&nbsp; manje&nbsp; toksični&nbsp; Cr(III).&nbsp; Odabrani&nbsp; sojevi&nbsp; <em>Bacillus&nbsp; cereus&nbsp;</em> grupe&nbsp; su&nbsp; dalje&nbsp; karakterisani&nbsp; &ndash;&nbsp; određene&nbsp; su&nbsp; njihove morfolo&scaron;ke i biohemijske karakteristike, 16S rDNK i&nbsp; pycA&nbsp; sekvence, potencijal formiranja biofilma i otpornost na druge te&scaron;ke&nbsp; metale.&nbsp; Takođe,&nbsp; sprovedeno&nbsp; je&nbsp; detaljnije&nbsp; ispitivanje&nbsp; njihove&nbsp; tolerancije&nbsp; i&nbsp; redukcije&nbsp; Cr(VI).&nbsp; Soj&nbsp; sa&nbsp; najvi&scaron;om otporno&scaron;ću&nbsp; je&nbsp; uporedo&nbsp; sa&nbsp; kontrolnim&nbsp; osetljivim&nbsp; sojem&nbsp; analiziran&nbsp; pomoću&nbsp; STEM&nbsp; EDS&nbsp; na&nbsp; sadržaj&nbsp; hroma&nbsp; u&nbsp; ćelijama&nbsp; I endosporama u različitim uslovima. Rezultati ukazuju da su bakterije tolerantne na Cr(VI) prisutne i u sredinama sa niskim i&nbsp; sa&nbsp; visokim&nbsp; koncentracijama&nbsp; hroma.&nbsp; Većina&nbsp; izolata&nbsp; pripadala&nbsp; je&nbsp; B.&nbsp; cereus&nbsp; grupi&nbsp; &scaron;to&nbsp; ukazuje&nbsp; na njenu&nbsp; uop&scaron;teno&nbsp; visoku otpornost na Cr(VI). Pojedini sojevi su pokazali visoku otpornost i sposobnost&nbsp; redukcije Cr(VI), &scaron;to ukazuje na mogućnost njihove praktične primene u bioremedijaciji. STEM EDS analiza osetljivog<em> B. subtilis</em> PY79 soja i Cr(VI)- rezistentnog soja <em>B.&nbsp; cereus</em>&nbsp; grupe&nbsp; NCr1a&nbsp; otkrila&nbsp; je&nbsp; značajne&nbsp; razlike&nbsp; u&nbsp; njihovom&nbsp; odgovoru na&nbsp; Cr(VI)&nbsp; i&nbsp; sadržaju&nbsp; Cr&nbsp; u njihovim&nbsp; ćelijama&nbsp; i endosporama.</p>

Scotts Creek Marsh (SCM) is a small coastal wetland ecosystem in Davenport, CA. The vegetation of SCM is dominated by three halophytic zones comprised of saltgrass, salt rush, cattails. The objectives of the study were (i) to investigate the variables that influence the zoning of the three dominant halophyte communities in SCM and (ii) to the test the effectiveness of Indicator of Reduction in Soil (IRIS) tubes to indicate the reduction of S. The study examined the following parameters from April 6 to July 21, 2013: (i) the HGM of Scotts Creek Marsh, (ii) soil oxidation and reduction (redox) conditions, (iii) salinity, and (iii) the effectiveness of Adobe Photoshop CS 5.1 (AP5) to analyze IRIS images. All three halophytes were well suited for anoxic, redox, and saline conditions by utilizing morphological adaptations (arenchyma, adventitious roots) to their root systems. The study concluded that the spatial zoning of the three dominate halophyte species within SCM was most likely due to slight differences in the water levels and salinity. The halophytes within SCM were zoned with saltgrass occupying the areas with the lowest water table and highest EC (26.98 dS/m). The cattails dominated the low average saline areas (9.60 dS/m) near the marsh channels with the highest water level. The salt rush zones had a mild EC level of 15.24 dS/m and intermediate water level. The IRIS tubes that were installed as indicators of both sulfur and iron reduction were effective. The tubes that were withdrawn after the closure of Scott’s Creek all had more than 30% reduction of the Fe3+ paint. The results from the IRIS study indicate that they are effective at recording the reduction of sulfur. The use of AP5 seemed to be an effective tool for analyzing IRIS images. The analyzed data from the study suggests that changes to the HGM of SCM could potentially alter the ecology of the marsh.

No description available.

Death and decomposition are natural processes that are generally well-understood. However, large events of death, such as mass mortality events (MMEs) are increasing in frequency and their impacts on the ecosystem are largely unknown. These events may have both bottom-up effects from increased nutrient input as well as top-down effects from loss of an ecological functional group by the affected population. Different functional MMEs may result in different top-down effects, creating cascading effects. In Chapter 1, I test the hypothesis that scavenger and herbivore simulated MMEs generate novel bottom-up and top-down effects. Results indicate that MMEs have a significant effect on communities, including on soil chemistry, plant tissue, soil microbes, and soil arthropods. Carrion effects on the community were both a result of biomass (MMEs vs. single carcasses) as well as functional group exclusion (herbivores, scavengers). Further, MMEs may also generate long-lasting community effects due to the size and nature of the disturbance. In Chapter 3, I evaluated the potential long-term of effects of MMEs by sampling an experimental MME that was conducted four years earlier. I found that MMEs generated long-term asymmetrical effects on ecosystems, with some noticeable changes in increased soil nutrients as well as an unexpected effect of biomass on aboveground arthropod communities, with very little effect on belowground soil arthropods. However, studies of long-term decomposition from mass carcasses may expand beyond studying MMEs. Composting of carrion is a continuous disturbance event, with numerous carcasses being deposited in the same location over a longer period of time. In Chapter 2, I analyzed potential effects on the surrounding community at a unique instance of concentrated carcass disposal (5 years old). Significant differences were revealed between samples taken near the compost pit (0 m, 5 m) compared to further distances (10 m, 25 m, 50 m) with calcium being increased away from the pit, different soil microbial communities at the pit than farther distances and increased aboveground arthropod abundance at the pit. These experiments provide us with a greater, holistic understanding of previously understudied events of mass death on community structure and ecosystem function.

No description available.

No description available.

A rising problem in agriculture is the increase of antibiotic-resistant Salmonella cases associated with chicken eggs, which transmit infection to humans. To counter this, new approaches to combat Salmonella in chickens and desiccated on eggshells are vital in the prevention of human foodborne illness. Disrupting signaling pathways with naturally occurring compounds provides a potential novel avenue for prevention of Salmonella infections, as this would disrupt sensing of these environments and inhibit subsequent optimal gene expression. Starting with signals identified in previous studies, salicylic acid was found to inhibit Salmonella desiccation survival on both eggshells and plastic. To expand upon this, a desiccation inhibition screen of 285 signals resulted in 9 additional potential desiccation inhibitors, including deoxyribose and guanine. By using natural signals to disrupt bacterial communication pathways, novel therapeutics that serve as viable antibacterial alternatives could be developed to prevent Salmonella contamination at a major source.

Matthew R. Borglin This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection Agency (EPA) to create a new category of sanitizer specifically designed for treatment of mature biofilms. The efficacy of sanitizers in this new regulatory category is determined by the EPA protocols MB-19 and MB-20. The EPA’s protocols outline methods for cultivating, treating, and measuring effects on Pseudomonas aeruginosa biofilms in a continuous flow stir bar bioreactor. Biofilm modification by OSA was verified by the presence of octenyl esters on OSA treated biofilms with single point Raman spectrophotometry. OSA modified biofilm’s antimicrobial properties were first investigated with crystal violet staining in 96-well microtiter plates with inconclusive results. However, effective antimicrobial properties where apparent when using the CDC Biofilm Reactor. OSA treatments consistently returned a 6-log CFU/coupon reduction in biomass compared to controls. Inhibition of planktonic and/or biofilm regrowth was demonstrated using the 96-well plate methodology. This thesis demonstrated the effectiveness of OSA chemical esterification reaction as a biofilm treatment. In doing so, this work suggests a new approach for biofilm remediation by chemically modifying the structural components of biofilm.

<p>Streptomycetes biology and genetics encompasses a variety of interesting features including multicellular growth, rich secondary metabolite production, and extensive environmental sensory and response systems. The characteristically large genomes of streptomycetes makes studying the diverse external stimuli intricate and internal regulation of these gene systems a challenge. Currently, there does not exist an efficient, cost-effective method of high throughput gene expression analysis in streptomycetes. Luciferase reporters have been used successfully in <em>Streptomyces coelicolor</em> to measure select promoter activity, however, they have demonstrated limited success in other strains and are not favourable to gene expression studies on a larger scale. Here, I present pLHR, a novel luciferase-based reporter tool designed specifically for high throughput gene expression studies in streptomycetes as well as the preliminary results which support the further development of this tool for gene expression profiling in <em>S. coelicolor</em>. Once developed, pLHR may be used to generate libraries of <em>Streptomyces</em> reporter stains to measure promoter activity repeatedly under variable conditions for the duration of the organism’s complex lifecycle.</p> / Master of Science (MSc)