The relentless evolution of antibiotic resistance in pathogens is one of the most pressing medical concerns of the 21st century. Antibiotic resistance and antibiotic drugs originated in environmental bacteria, where they have been integral to their evolution for millions of years. The application of antibiotics in medicine and agriculture has selected for mobilization and dissemination of resistance genes in pathogens. Understanding their evolution here will aid in combating their evolution in pathogens. This work expands the known mechanistic, functional, and genetic diversity of resistance (i.e. resistomes) in environmental bacteria. I systematically parse the extensively drug-resistant resistome of Paenibacillus sp. LC231, which was sampled from an underground ecosystem spatiotemporally isolated from the surface for over 4 Myr. Paenibacillus sp. LC231 was resistant to 26 of 40 drugs tested. Informatic annotation of resistance genes and functional genomes revealed 18 new resistance elements including five determinants without characterized homologs and three mechanisms not previously known to confer resistance. I investigated the resistome of Brevibacillus brevis VM4 to study the relationship between species diversity and resistance diversity in the Paenibacillaceae family, which includes Paenibacillus sp. LC231. I found that resistome diversity does not correlate with species diversity, consistent with horizontal transfer of resistance genes. In each of Paenibacillus sp. LC231 (MphI) and B. brevis VM4 (MphJ), I identified Mphs with unique substrate specifies. I identified the molecular determinants of substrate discrimination in MphI and in doing so, I developed a general strategy for understanding and predicting the functional evolution of resistance enzymes. Together, this work expands the known diversity of resistance that will enable better detection of resistance in pathogens. / Thesis / Doctor of Philosophy (PhD) / Infections caused by antibiotic resistant bacteria are a significant medical problem. Bacteria will always become resistant to antibiotic drugs. Understanding how resistance evolves is essential for increasing the effective lifetime of these drugs. Antibiotics have been naturally produced by bacteria for millions of years, which caused the spread of resistance in environmental bacteria. Medical and agricultural antibiotic use by humans caused resistance in environmental bacteria to transfer to pathogenic bacteria. My work expands the known causes of resistance in environmental bacteria so that we can better detect the causes of resistance in pathogens. In doing so, I demonstrate that multi-drug resistance is over 4 million years old and that environmental bacteria naturally transfer resistance genes. Furthermore, I develop a way to predict the evolution of new resistance functions by inferring their evolutionary histories.

In order to gain better insight into the possible mechanisms that influence resistance training-induced muscle hypertrophy, two groups of subjects were examined for changes in muscle protein synthesis, and protein, total RNA, and DNA content 4 (group A) and 24 (group B) hours following an isolated bout of unilateral elbow flexor resistance training. Subjects trained one arm by performing 3 different biceps exercises consisting of 4 sets of 6-12 repetitions to failure while the contralateral arm served as a control. Both groups received a primed-constant infusion of L-[1-¹³C] leucine (group A infused 0.68h post-exercise for 5.4h; group B infused 20.41h post:-exercise for 6.38h) and muscle protein synthesis was determined by the increment in L-[1-¹³C] leucine abundance in muscle biopsy samples relative to the mean plasma α-KIC enrichment at isotopic plateau. Protein, total RNA, and DNA were~ determined with standard methods and RNA capacity (total RNA (ug)/protein (ug)) and RNA activity (ug protein synthesized/hour/ug RNA) were calculated to assess changes in gene transcription and translation. Possible muscle damage was assessed by changes between pre and 15 minute post-exercise maximal voluntary elbow flexor torque (measured at 30°/S and 180°/S) and by 22 hour post-exercise changes in serum CK activity (assessed in group B only). Both groups had significantly elevated muscle protein synthetic rates (group A 43% ↑; group B 80% ↑) and RNA activities (group A 25% ↑ ; group B 89% ↑) in the exercised biceps compared to the control biceps. In addition, post-exercise torque declined by 22% at 30°/S and by 24% at 180°/S and mean serum CK activity increased by 35% in group B. It is concluded that an intense bout of resistance training stimulates increases in muscle protein synthesis 4 and 24 hours post-exercise. The elevations in muscle protein synthesis of the exercised arm are probably related to increases in translation with contractile protein damage being one possible signal for increasing protein synthetic rates. / Thesis / Master of Science (MSc)

The aim of this study was to investigate the mechanisms of action of and development of cellular resistance to various anticancer agents in human (HeLa) cells using a combined genetic and biochemical approach. The agents employed for this purpose included: the purine nucleoside analogues, toyocamycin, tubercidin, and 6-methyl-mercaptopurine riboside (6-MeMPR); the protein synthesis inhibitor, puromycin; and the microtubule stabilizer, taxol. To investigate the mechanisms of action and cellular resistance to the purine nucleoside analogues, stable first-step toyocamycin, tubercidin and 6-MeMPR resistant HeLa mutants were isolated. These mutants exhibited high degrees of resistance and cross-resistance to various adenosine kinase-activated nucleoside analogues, possessed <2% of the adenosine kinase activity of parental HeLa cell extracts and exhibited severely reduced cellular uptake and macromolecular incorporation of adenosine in vivo. These results indicate that in human cells the cytotoxic effects of toyocamycin, tubercidin and 6-MeMPR are dependent upon adenosine kinase-catalyzed phosphorylation of these drugs to their respective monophosphates and that resistance to these agents results from a deficiency in adenosine kinase activity in vivo. Further insight into the nature of the genetic and biochemical alteration(s) affecting adenosine kinase in these mutants was achieved using SDS-polyacryamide gel electrophoretic and immunoblot analysis. Immunoblots -revealed that each toyocamycin, tubercidin and 6-MeMPR resistant mutant contained similar amounts of cross-reacting material that had the same electrophoretic mobility as adenosine kinase in parental HeLa cells. Therefore, the lesion in these mutants must be a missense type of alteration in the structural gene for adenosine kinase. The utility of the 6-MeMPR resistant mutant selection system for quantitative mutagenesis studies was also investigated. Numerous favourable attributes appropriate to mutagenesis studies were found using this selection system. These included: the obtainment of highly resistant mutants which were stable in the absence of drug, the absence of cell density or cross-feeding effects in the selection system, maximum phenotypic expression required a relatively short time period and mutagen treatment increased the mutant frequency in a linear dose-dependent manner. Thus, selection for genetic alterations at the adenosine kinase locus appears to provide a valuable system for quantitative mutagenesis studies in human cells. The combined genetic and biochemical approach was also used to investigate the development of resistance to puromycin and taxol. Therefore, first-and second-step mutants resistant to each of these drugs were selected and characterized. Cross-resistance and uptake studies with the puromycin resistant mutants suggest that the most common -mechanism for the development of cellular resistance to puromycin in human (HeLa) cells involves an alteration in membrane permeability that reduces drug uptake/transport. Similar studies with the taxol resistant mutants suggest the existence of two possible mechanisms for the development of resistant to this agent in human (HeLa) cells. One mechanism involves a biochemical lesion that specifically affects a microtubule-related cellular component. The second mechanism, however, nonspecifically affects cellular membrane permeability and results in reduced drug uptake/transport. / Thesis / Master of Science (MS)

Eight men and 8 women trained 3 days/ week for 16 weeks by doing attempted ballistic unilateral ankle dorsiflexions against resistance which either rendered the resultant contraction isometric (one limb) or allowed a relatively high velocity (joint angular velocity of 5.23 rad.s- 1 ) isokinetic concentric contraction (other limb). Training sessions consisted of 5 sets of 10 contractions of each type. Pre and post-training tests of peak torque at 0, 0.26, 0.52, 1.04, 1.55, 3.02, 4.19, 5.23 rad.s-1 indicated a velocity specific training response (p<0.01), with increases of -5.9, 5.6, 8.6, 15.3, 13.9, 14.1, 19.3, and 27.4% respectively. In a separate test, maximal voluntary isometric peak torque (6 .1%) and maximum rates of torque development (20.4%) and relaxation (31.5%) increased after training (p<0.01). Electrically evoked isometric tetanic peak torque and rate of torque relaxation did not change but rate of torque development increased 12. 6% ( p<0. 01). Evoked peak twitch torque did not change but time to peak torque and 1/2 relaxation time decreased 6.2 and 11.9% respectively (p<0.01). Electromyographic (EMG) recordings of the agonist tibialis anterior (TA) during test contractions showed no change in integrated EMG, but there was an increase (14.6%, p<0.05) in antagonist soleus (S) EMG from mid-test to post-test. The velocity specific response to the isometric and isokinetic concentric training modes was the same, indicating that it was the intent to make a ballistic contraction, rather than the resultant velocity of contraction, that determined the velocity specific response. The data also suggest that both neural and muscular adaptations contributed to the velocity specific training response / Thesis / Master of Science (MS)

BACKGROUND: Antibiotics are essential medicines and their effectiveness is under threat due to antimicrobial resistance. Guidelines are one way to conserve antibiotic effectiveness given that they are intended to modify clinician prescribing. Guidelines that provide antibiotic recommendations should make explicit contextual considerations that influence antimicrobial resistance and their downstream effects on resistance emergence. METHODS: We conducted a systematic review of tuberculosis, gonorrhoea, and respiratory tract infection guidelines and recommendations to examine how and to what extent they are considering contextual factors that influence antimicrobial resistance. We also investigated whether there are guidelines and recommendations that can be adopted or adapted to local contexts. RESULTS: We found that within 74 included guidelines, two thirds of recommendations considered antimicrobial resistance. Of which only five guidelines considered all factors required to consider local aspects such as values, resource use, acceptability, feasibility, and equity. As such, these five guidelines can be either adopted or adapted to Canadian and other contexts. We also found that 39% of guidelines met credibility scores of 60% or greater in AGREE II domains: scope and purpose, rigor of development, and editorial independence. CLINCAL IMPLICATIONS: There are very few Infectious disease guidelines for highly prevalent diseases that do not consider all important contextual factors may influence antimicrobial resistance. Our findings can support societies and organizations, public health policy, and health care stakeholders to develop and implement guidelines that are applicable to local contexts efficiently and resourcefully. Our antimicrobial resistance recommendation framework, used in addition to GRADE Evidence to Decision frameworks, is a start to having this come to fruition. / Thesis / Master of Public Health (MPH)

With the aging of the baby boom population and an increased life expectancy, individuals aged 65 years and older are the fastest growing segment of our population. Aging brings about changes in skeletal muscle such as reduced muscle strength and mass, as well as cellular deficits such as increased production of reactive oxygen species (ROS), and mitochondrial DNA (MtDNA) deletions and mutations. Muscle mass declines at a rate of 1-2% each year after the age of 50, leading to muscle weakness, functional impairments, loss of independence, and an increase in falls. Additional declines in muscle mass and reduced muscle strength may result in a lower resting metabolic rate, reduced lipid oxidative capacity, increased adiposity, and insulin resistance. The rising number of individuals aged 65+ will increase demands on health care and health care costs, possibly leading to inadequate public resources and less care for the aged. This large societal impact, coupled with the aging of our population, suggests a clear need for methods that will improve the aging phenotype to enhance functionality, quality of life, and overall health for our aging population. This investigation aspires to delve into a relatively unexplored area of aging research and evaluate potential means that could help improve the aging phenotype. The associated mitochondrial impairments, mitochondrial mediated apoptosis, and mitochondrial DNA (MtDNA) deletions and mutations that accompany aging lead to a decline in physical fitness and oxidative capacity, and exercise has been shown to reverse or help prevent many of these disturbances. Resistance exercise training (RT) is currently the most effective known strategy to stimulate skeletal muscle hypertrophy and increase strength. Strength gains after RT lead to an improvement in activities of daily living and quality of life. There is some evidence suggesting that RT may lead to increased antioxidant enzyme capacity, decreased ROS production and increased electron transport chain (ETC) function in older individuals. The present study will lay a foundation for future research and further developments in the area of RT, mitochondrial function and aging. / Ph. D.

Terminal sire crossbreeding systems which improve growth performance while maintaining parasite resistance have the potential to enhance the profitability of hair sheep enterprises. Katahdin (KT, n = 4), Suffolk (SU, n = 3), and Texel (TX, n = 3) rams were randomly mated to KT ewes over two years (Y1, Y2) at the Virginia Tech Southwest Agricultural Research and Extension Center. Post-lambing until weaning (80 d), pairs were managed on fescue pasture. At weaning, lambs (n = 192) were moved to an ungrazed pasture and provided a concentrate pellet daily for a 90 d grazing trial. During this time, BW, strongylid egg count (FEC), FAMACHA score and packed cell volume (PCV) were collected every 14 d. FAMACHA score - 3 was utilized as the basis for anthelmintic treatment. Post-grazing, lambs were fed to approximately 50 kg BW. Lambs were harvested at the Virginia Tech Meat Center and carcass evaluation performed 1 d post-harvest. Statistical analyses were conducted using SAS (SAS Institute Inc., Cary, NC) with Proc MIXED for repeated measures analysis and Proc GLM with Tukey?s test for mean separation. No differences existed between sire breeds for adjusted number of lambs born or number of lambs weaned. Adjusted birth BW was greater for SU-sired lambs than KT-sired and TX-sired (P < 0.05) in Y2. Adjusted weaning BW was smallest for KT-sired lambs compared to SU- and TX-sired lambs (P < 0.05) in both years. During the grazing trials, BW, ADG, lnFEC, FAMACHA and PCV varied over time (P < 0.001) with lower FAMACHA scores for KT-sired lambs than SU- and TX-sired lambs in Y1 (P < 0.05). A greater proportion of SU-sired lambs tended to require deworming than KT-sired lambs (P = 0.08). Adjusted BW post weaning was greater for TX-sired lambs than KT-sired lambs (P < 0.05) in Y1. Post-grazing, BW and ADG varied over time (P < 0.01) with no sire breed differences for ADG. At harvest, SU-sired lambs were heavier than KT-sired lambs (P < 0.05). TX-sired lambs had greater LM area than KT-sired lambs (P = 0.05). KT-sired lambs had the smallest leg scores (P < 0.05). These results indicate the potential of terminal sires (SU- and TX-sires) to improve lamb growth and carcass merit. TX-sired lambs had more similar parasite resistance characteristics to KT-sired Iambs and may have potential as terminal sires in forage based hair sheep production systems. / Master of Science

Goosegrass is a problematic weed of golf courses, sports fields, and residential lawns that decreases playability and aesthetic quality of turf. With the recent banning of MSMA in sports fields and intensive restrictions in golf and sod production, turfgrass managers are seeking alternatives for postemergence goosegrass control and how to utilize currently labeled goosegrass control products more effectively. Studies were conducted to investigate a suspected-resistant (SR) goosegrass accession in Richmond, VA and characterize the resistance mechanism if present. The SR accession showed a hypersensitive response to oxadiazon treatment and reached maximum electrolyte leakage quicker than the susceptible (S) accession, but had significantly lower electrolyte leakage indicating less tissue damage and suggesting there is a physiological resistance mechanism within the SR accession. In absorption and translocation studies, percent oxadiazon absorption and translocation was not significantly affected by goosegrass biotype. Roots of both the S and resistant (R) biotypes contained over 95% of total detected oxadiazon, while the plant tissue above the treated foliage only contained small quantities. These results suggest that absorption or translocation is not the mechanism conferring oxadiazon resistance in the goosegrass biotype from Richmond, VA. Greenhouse and field trials were conducted to determine the lowest rate at which topramezone, with or without the addition of triclopyr, controls goosegrass while maintaining commercially-acceptable bermudagrass quality. In field trials, topramezone rate did not significantly affect goosegrass cover at 56 and 70 days after initial treatment (DAIT). All treatments reduced goosegrass cover below 3 and 7% with and without the addition of triclopyr, respectively at 70 DAIT. A significant herbicide effect on bermudagrass cultivar showed higher injury from topramezone within three weeks of application, but injury persisted longer from treatments containing triclopyr. Bermudagrass cultivars completely recovered by 4 weeks after treatment (WAT) from all treatments. Greenhouse trials were conducted to determine if goosegrass growth stage affects efficacy of nine postemergent herbicides or programs documented to have goosegrass activity. As goosegrass growth stage increased from four- to five-leaf to greater than eight-tiller stage, goosegrass control and biomass reduction decreased among all of the herbicides except topramezone and MSMA plus metribuzin at 4 and 8 WAT. These data suggest that one application of sulfentrazone is only effective for seedling stage (pre-tiller) goosegrass control; foramsulfuron, topramezone, and metribuzin suppress all growth stages of goosegrass; and diclofop, sulfentrazone plus metribuzin, fenoxaprop, and metamifop control up to three-tiller stage goosegrass. / Ph. D.

The incidence of mosquito-borne human diseases is increasing worldwide, with effective chemical control limited due to widespread insecticide resistance in the insect. Recent evidence also suggests that bacterial symbionts of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may play a significant role in facilitating mosquitocide resistance. Here, I examined the metabolic detoxification and toxicity of two mosquitocides, propoxur and naled, and the capacity of bacterial symbionts to modify the detoxification of the mosquitocides and, thus, alter their toxic action in the yellow fever mosquito, Aedes aegypti. The insecticide synergists piperonyl butoxide (PBO), triphenyl phosphate (TPP), and S,S,S-tributyl phosphorotrithioate (DEF) were used to examine the metabolic detoxification and toxic action of the two mosquitocides in mosquito larvae. A significant increase in the toxicity of propoxur was observed when applied in combination with PBO; however, there was no corresponding decrease in AChE activity. Naled applied in combination with PBO resulted in a decrease in anticholinesterase activity (higher residual AChE activity) and a subsequent decrease in toxicity of the insecticide. This suggests that esterases play a major role in the metabolic detoxification of both insecticides in mosquito larvae. The acute toxicities of naled and propoxur to Ae. aegypti larvae were also studied following a reduction of bacterial symbionts with the broad-spectrum antibiotics gentamycin, penicillin, and streptomycin. Antibiotic-treated mosquito larvae showed increased susceptibility and a reduction in cytochrome P450 monooxygenase and general esterase activities when treated with naled and propoxur. A reduction of bacteria in mosquito larvae treated with broad-spectrum antibiotics, therefore, appears to affect the metabolic detoxification of standard-use mosquitocides, such as propoxur and naled. The results also suggest that the bacteria themselves may contain metabolic detoxification enzymes that are functionally similar to those in the mosquito larvae. Additional experiments, however, are needed to fully elucidate the contribution of bacterial symbionts in Ae. aegypti larvae in the metabolic detoxification of mosquitocides. / Master of Science in Life Sciences

Escalating product recalls as a consequence of Salmonella-contaminated poultry products have resulted in detrimental economic impacts. One long-term alternative to Salmonella prevention, receiving increased attention, is selection to improve genetic resistance. This study evaluated the effects of an oral Salmonella Heidelberg (SH) challenge on bacterial colonization, and the ex vivo effects of SH on phagocytic and bactericidal leukocyte function in turkeys from six pedigree lines (A-F). Data were analyzed using JMP Pro (SAS) and differences were determined using Student’s t-test following ANOVA with significance reported at P ≤ 0.05. Interaction effects of treatment X gender X genetic line were significant on bacterial colonization in the ceca. Cumulatively, females exhibited higher phagocytosis potential than males. The main effect of genetic line was significant bactericidal activity of PBMCs. Microbial profiling of cecal DNA was performed to examine differences in colonization of Salmonella, E. coli, and Enterococcus species among the genetic lines. Results indicated line E having the highest Enterococcus but lowest Salmonella colonization than all other lines, while line A birds displayed the highest Salmonella colonization. These results suggest that gender and genetic line have a marked effect on susceptibility to Salmonella colonization, while genetic line X gender has a more eminent effect on Enterococcus cecal colonization. If able to determine genetic markers associated with these immune responses to Salmonella, genetic selection for increased resistance could be feasible in turkeys. / Master of Science