The objective of this study was to find a regression procedure that can better explain the relationship between patterns of antibiotic use and proportions of bacterial resistance. The sample for the study is comprised of 44 University Health System Consortium (UHC) member hospitals, and the data for antibiotic use and proportions of resistance are from the years 2002 to 2005. The hospitals are spread across the Northeast, South, Southwest, Midwest, and Northwest regions of the USA. Based on statistical analysis, MRSA continues to have the highest proportion of resistance among the bacteria examined and has increased significantly since 2002. The antibiotic use in the study was measured in indices called diversity indices. There were six such measures in the study. The study, first using ordinary least squares regression, did not find one single diversity index that adequately predicted the proportion of resistance. There were also concerns that the diversity indices could be measuring the same thing, and therefore all should not be used in the model. The correlations between the three general diversity indices were strong, positive, and linear. Likewise, the three Gram-negative indices were also positively correlated with one another. Multicollinearity diagnostics also showed that there were serious dependencies among general diversity indices. Given the multicollinearity results and the correlation coefficients for the indices, it can be concluded that all six indices should not be in the same model together. Logistic regression and weighted least squares regression using the logit transformation were also performed, and just like the ordinary least squares results, there was no one single diversity index or a combination of diversity indices that adequately predicted the proportion of resistance.

This thesis aims to give a reflection on the doctrine of the right of resistance in the Czech Republic, its liberal democratic system and legal order. The first part of the thesis presents a short description of the background and the development of the doctrine. It also deals with Article 23 of the Charter of Fundamental Rights and Basic Freedoms as an attempt of embedment the right of resistance in the Czech law. The subject matter of the second part are the transformation processes taking place in the current world that have an impact and threaten liberal democracies including the Czech Republic. This part also gives examples of the application of right of resistance. The first chapter contains a short historical and a modern definition of the right of resistance. It also defines the following related notions - the revolution, the coup d'état and civil disobedience, that this thesis applies. The second chapter focuses on the history of the right of resistance. It goes back to its roots in Ancient Israel, looks upon the Enlightment authors dealing with the theory of Social contract and concludes with the post-war development. Special focus is turned to the disputed aspects of the right of resistance. The third section deals with possible interpretations of Article 23 of the Charter of Fundamental...

Diabetes is a common chronic disease that affects almost all countries in the world and has continued to increase at an alarming rate in the last decades. It kills a person every seven seconds. Recent thinking treats both types of diabetes as inflammatory diseases. The aim of the thesis was to obtain a better understanding of the relationship between exercise and the management of diabetes by conducting surveys and experimental work. It investigates the effects of exercise on the physiology and metabolic control in Type 1 (T1D) and Type 2 diabetes (T2D), using non-diabetic (ND) people as a control. The management and treatment of T1D and T2D volunteers were first assessed in surveys and the novelty was second to expose both to exercise. In the latter, volunteers were compared biochemically including for inflammatory responses to their illness and to practical exercise. Four studies were undertaken in this thesis involving a mixed approach: questionnaire based studies (first and second surveys) and experimental based studies (first and second exercise studies). The first survey study was about insulin users with opinions gathered from both T1D and T2D (T2I) respondents (n=707). In this survey diabetic people were asked about the condition and coping strategies for the difficulties using insulin in daily life. The first survey does touch on exercise but only as part of the larger picture. The second survey study (n=240) evolved from the first one and was again about opinions but in this case oral anti-hyperglycaemics were included in the management of T2D respondents (T2T). This survey focused more strongly on the role of exercise. The surveys were conducted by post, email and online while detailed statistical analysis followed. Two exercise studies with the same volunteers (n=25; ND=7, T1D=7, T2T=7, T2I=4) were then carried out based on some findings of the surveys. These studies explored the effects of a combination of aerobic (AE) and resistance exercise (RE) components for a six week period on diabetes. The methodology of the first exercise study concentrated on the physiological variables, involving the use of exercise and measurement equipment to monitor for expired gases and anthropometric changes. Substrate oxidation, blood profiles for lipid, blood glucose (BG) and glycated haemoglobin (HbA1c) were also assessed. The second exercise study builds on this with specific inflammatory marker profiles such as tumour necrosis alpha (TNF-α), interleukin-6 (IL-6), leptin and resistin on ND, T1D and T2D volunteers over the same time period as in the first exercise study. The first survey study showed that many respondents (13-47%) lacked adequate professional information about the various separate aspects of their insulin-treated illness. For example, 38% of T1D and 28% of T2I reported that they did not have enough information regarding raised cholesterol levels. The results for diabetes complications revealed that T2I had greater complications compared to T1D (for example angina 18.5% for T2I compared to 4.6% for T1D), although the groups could not be matched for age, reasons for responding to the survey, duration of illness or severity of illness when starting insulin. The second survey revealed that insulin users often had an HbA1c that did not meet best practice expectations of 6.5% - 7.5% (48 -58 mmol/mol). It also showed that those who did exercise regularly were more likely to have acceptable HbA1c values (5-7% or 31-53mmol/mol), than those who did not. This is especially the case for the type 2 groups (eg for T2T 46% exercising compared to 31% non-exercising) who were less likely (19% respondents compared to 25%) to have HbA1c over 8% or 64 mmol/mol. It was of interest to know the risks, barriers and likely recommendations for the two groups. For example, fewer T2I people test BG frequently (12.5% compared to T1D 62%, testing four or more times daily), even when they are insulin basal bolus users, which could foster hypoglycaemic events during exercise. The findings of the first and second surveys showed that managing diabetes in the 21st century remains difficult for many people, despite the availability of diagnostic, monitoring and medication improvements. This leads to anxiety and illness over the short and long term. In the first exercise study, it was clear that for this combined exercise regimen, the chronic effects were notable. The most significant finding was that the effect of 6 weeks was the drop in HbA1c in all groups ND from 5.4-5.2% or 36-33mmol/mol (p ˂ 0.01), T1D 7.0 to 6.7% or 53-50mmol/mol (p ˂ 0.01), T2T 7.6 to 7.2% or 60-55mmol/mol (p ˂ 0.05), T2I 7.3 to 6.8 or 56-51mmol/mol (p ˂ 0.05). This is equivalent to raising insulin or other medication and while clearly very beneficial, especially as occurring as a result of moderate exercise over only 6 weeks. Lipid factors showed improvements, not all significantly but these were likely to be influenced by support medication such as statins. However, the heart rate (HR) and blood pressure (BP) reduced at rest for all groups over the six weeks. The respiratory exchange ratio (RER), a measure of substrates oxidation showed that the carbohydrate metabolism was steady. The muscular strength and the subjective assessment improved after the exercise period. The second exercise study showed the interleukin 6 levels fell with the chronic effects of combined exercise ND (3.97-2.7pg/ml), T1D (2.15-1.02 pg/ml), T2T (3.67-2.72pg/ml) and T2I (3.66-1.17pg/ml) as did TNFα and other cytokine levels which may thus be cardioprotective. This suggests that exercise could be part of the anti-inflammatory treatment of T1D and T2D. To conclude, the findings of the two survey studies showed that the management of diabetes is difficult for many diabetics. Furthermore, the exercise studies demonstrated that a regular combined (RE and AE) exercise trial at moderate intensity for six week could be physiologically beneficial for diabetics. The underlying mechanism for this could be improvements in glycaemic control, lipid profile, cardiovascular fitness level and strength, as well as the inflammatory features of both T1D and T2D.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / The use of antimicrobials in animal feed has come under increasing scrutiny from the public and regulatory agencies. Direct-fed microbials (DFM) are considered valuable alternatives to antimicrobials in food animal nutrition. DFM are products containing live (viable microorganisms). Studies in Europe have reported antimicrobial resistance (AMR) in organisms used in DFM. This is of serious concern because of the potential for transferring resistance to pathogenic bacteria in the gut. The aim of the present study is to characterize phenotypic and genotypic AMR profiles for 20 different antimicrobials in bacterial strains isolated from 10 commercially available DFM used in. Two antimicrobial susceptibility testing methods, disc diffusion and broth micro-dilution based assay were performed. Enterococcus faecium isolates showed resistance towards metronidazole (n=9/9) with a MIC of > 32 μg/mL, erythromycin (n=5/9) with a MIC of ≥ 8 μg/mL, ciprofloxacin (n=2/9) with a MIC ≥ 4 μg/mL, ceftriaxone (n=6/9) with a MIC ≥ 0.25 μg/mL, rifampin (n=8/9) with a MIC of > 4 μg/mL, trimethoprim/sulfamethoxazole (n=4/9) with a MIC ≥ 1 μg/mL and clindamycin (n=5/9) with a MIC of > 0.5 μg/mL. A Propionibacterium freudenreichii isolate showed resistance towards kanamycin with a MIC of > 64 μg/mL. The same strain also had a MIC of 16 μg/mL for levofloxacin. Two Lactobacillus acidophilus were resistant to vancomycin (n=2/6) with a MIC ≥ 32 μg/mL. All the Lactobacillus species including L. acidophilus (n=6), L. casei (n=4) and L. plantarum (n=2) were resistant to metronidazole, MIC > 32 μg/mL. Two strains of Bacillus subtilis showed resistance to clindamycin, with an MIC of 4 μg/mL and erythromycin with an MIC of > 8 μg/mL, and one strain had no zone of inhibition for metronidazole (MIC > 32 μg/mL). Microarray analysis revealed resistance genes in E. faecium strains of 3 different DFM, including aminoglycoside resistance genes, ant(4’)-Ia, erythromycin resistance genes, ere(A2) and ermB, tetracycline resistance genes, tet39, tet31, tetK and tetC, and beta-lactam resistance gene, pbp5. Conjugation with filter mating showed erythromycin resistance gene transfer, msrC gene, from donor strains to a recipient strain (E. faecium 45-24). These studies show that AMR is prevalent among bacterial strains used as DFM in the cattle industry in the U.S., justifying further characterization, detection and observation of transferable antibiotic resistance between the same genus. .

MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Introduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.

Faculty of Science School of Physiology 9808215t romainh@physiology.wits.ac.za / There is a high prevalence of insulin and leptin resistance and increased cortisol concentrations in sleep apnoea patients, independent of obesity. Chronic intermittent hypoxia is used an experimental animal model to simulate the hypoxia occurring in sleep apnoea patients. The aim of this study was to measure plasma insulin and leptin concentrations and hypothalamic-pituitary-axis activity in rats exposed to either intermittent hypoxia (CIH) or sham hypoxia (SH) for fourteen days. To induce CIH plexiglass cylinders were flushed with 100% nitrogen for nine seconds every 90 seconds, seven hours/day. The rats were weighed each day during the exposure period. Venous blood samples for insulin and leptin were collected on days one, three, five, eight and fifteen. Faecal samples were collected to measure glucocorticoid metabolites. There was no significant difference in the daily change in body weight between the rats exposed to CIH compared to the rats exposed to SH (unpaired t-test). Plasma insulin concentrations were not affected by CIH. In both groups of rats plasma leptin concentrations were significantly higher on day fifteen compared to day five (p=0.03, unpaired t-test). Glucocorticoid metabolites were significantly increased in the intermittent hypoxia group on day two (p=0.003 one-way ANOVA). In conclusion, exposing normal weight rats to CIH for fourteen days resulted in a transient iv increase in HPA axis activity on day two and an elevation in plasma leptin levels, in both groups of rats, at day fifteen.

Boston University. University Professors Program Senior theses. / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-02

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer which arises from the malignant transformation of B-cell or T-cell progenitors. Despite recent pioneering improvements in intensified combination chemotherapy, 20% of pediatric and 50% of adult ALL patients present with primary drug-resistant leukemia or develop relapse. Treatment of refractory and relapsed ALL has remained a significant clinical challenge with survival rates following relapse of only 40%, highlighting the need to understand the mechanisms which drive drug resistance and relapse of ALL. Through extensive sequencing analyses of matched diagnostic, remission and relapsed DNA samples from patients with B-precursor ALL (B-ALL) and T-cell ALL (T-ALL) we have identified recurrent relapse-specific gain-of-function mutations in the cytosolic 5'-nucleotidase II (NT5C2) gene in 25% of relapsed T-ALLs and 6% of relapsed B-ALLs. NT5C2 is a highly conserved, ubiquitously expressed enzyme which regulates intracellular purine nucleotide levels by dephosphorylating purine monophosphates. NT5C2 also dephosphorylates key metabolites in the activation of purine analog prodrugs such as 6-mercaptopurine and 6-thioguanine which are routinely used in the treatment of ALL, allowing purine analog nucleosides to be readily exported out of the cell. Here we show that mutant NT5C2 proteins have increased 5’-nucleotidase activity and confer resistance to 6-mercaptopurine and 6-thioguanine chemotherapy when expressed in leukemic cells. Consistently, NT5C2 mutations correlate with early relapse and relapse while under therapy. We present a novel T-ALL conditional inducible knock-in mouse model of the highly recurrent NT5C2 R367Q mutation and show that expression of one Nt5c2 R367Q allele from the endogenous locus in primary T-ALL lymphoblasts induces overt resistance and disease progression under therapy with 6-mercaptopurine in vivo, while surprisingly conferring reduced growth and decreased leukemia initiating activity in the absence of chemotherapy. Metabolically we show that the observed loss of fitness in Nt5c2 R367Q tumors can be explained by a severe depletion of endogenous purine monophosphate metabolites as a result of increased Nt5c2 5’-nucleotidase activity. Consistently, using ultra-sensitive mutation analyses we show that relapse-associated NT5C2 mutations are not detectable at initial disease presentation, indicating that NT5C2-mutant tumor cells are negatively selected by clonal competition in the early stages of disease development and only positively selected under prolonged 6-mercaptopruine chemotherapy which is the backbone treatment for ALL following remission. Our findings present the first known example of chemotherapy resistance and disease progression driven by a tumor clone with decreased leukemia initiating activity, highlighting the intense selective pressure of chemotherapy in the clonal evolution of tumors from diagnosis to relapse. Through extensive biochemical and structural characterizations of recombinant NT5C2 mutant proteins, we have grouped relapse-specific NT5C2 activating mutations into 3 different classes, each conferring unique enzymatic behavior in basal conditions and in response to allosteric activation, and each with unique structural features which mediate increased 5’-nucleotidase activity. Moreover, we identify a novel auto-regulatory switch-off mechanism of the NT5C2 enzyme involving movement of an unstructured flexible loop, and present the first crystal structure view of the NT5C2 C-terminal acidic tail, implicating it as an auto-inhibitory brake to the allosteric activation of the enzyme. The presence of multiple mutational mechanisms of activating such a highly conserved enzyme, especially in light of the inherent loss of fitness to the tumor cells, indicates a strong convergent evolution towards activating NT5C2. This is supported by our discovery that patients can harbor multiple leukemic clones with NT5C2 mutations at relapse. Overall our findings highlight NT5C2 as a major driver of drug resistance and relapse of ALL and pinpoint metabolic susceptibilities which could be exploited therapeutically to target NT5C2-mutant tumors in the future. Our in-depth structural and enzymatic knowledge of mutant NT5C2 proteins will serve as an essential tool in the rational drug development of novel NT5C2 inhibitors with increased specificity and selectivity for mutant NT5C2, while our novel Nt5c2 R367Q knock-in mouse model will serve as a platform for the pre-clinical testing of both NT5C2 inhibitors and alternative compounds selective for Nt5c2-mutant leukemias which can be used for prevention and treatment of relapsed ALL.

In Bihar state, India, the cure rate of antimonial compounds in the treatment of visceral leishmaniasis (VL) has declined from over 85% to less than 50%. This has been attributed to prolonged, widespread misuse of antimonials within the Indian private healthcare system. An alternative resistance hypothesis is that exposure to arsenic in drinking water in this region has resulted in antimony-resistant Leishmania parasites. Leishmania donovani were serially passaged in mice exposed to environmentally-relevant levels of arsenic in drinking water. Arsenic accumulation in organs of these mice was proportional to exposure. After five passages, isolated parasites were refractory to SbV in drug sensitivity assays. Treatment of infected mice with SbV confirmed that these parasites retained resistance in vivo, supporting this hypothesis. A retrospective field study on a cohort of antimony treated VL patients was performed in an arsenic contaminated area of Bihar to evaluate the presence of an increased risk of treatment failure and death in those exposed to arsenic. It demonstrated a significant increased risk of death from VL in arsenic exposed patients but did not indicate a significant relationship between arsenic exposure and antimonial treatment failure. Collectively these data suggest that it is biochemically possible that arsenic contamination may have contributed to the development of antimonial resistance in Bihar although issues of underpower and the retrospective nature of our epidemiological study made it difficult to conclusively demonstrate this. Further research in to the relationships between arsenic exposure and antimonial treatment failure and death in the leishmaniases is warranted.

Chemotherapeutika stellen nach wie vor eine der wichtigsten Behandlungs-optionen bei Krebs dar. Ihre akuten und chronischen Nebenwirkungen aber limitieren ihre Anwendung. Aktuelle klinische Studien deuten auf einen positiven Effekt von Kurzzeitfasten auf die Nebenwirkungen von Chemotherapeutika hin. Eine Erklärung hierfür könnte das unterschiedliche Ansprechen von normalen und malignen Zellen auf Chemotherapeutika in einer Mangelsituation sein, das als »differential stress resistance« (DSR) bezeichnet wird. Dieses Phänomen lässt nicht-maligne Zellen bei Restriktion von Glukose und Wachstumsfaktoren weniger sensitiv auf Chemotherapeutika reagieren als maligne Zellen. Das Ziel der vorliegenden Arbeit war zu untersuchen, ob ein Mangel an Glukose und Wachstumsfaktoren das Ansprechen nicht transformierten Zellen 5-FU abschwächen kann während das von kolorektalen Karzinomzellen (Colo741, LS174T, HCT116, HT29 und SW620) gleichbleibt. Optimale Kulturbedingungen für Tumorzellen in vitro stellen 11 mmol/l Glukose und 10 % FCS dar, während 3 mmol/l Glukose und 1 % FCS Mangelbedingun¬gen repräsentieren. Glukosewerte von 3 mmol/l werden auch mit Kurzzeitfasten erreicht. Da der Großteil der soliden Tumoren mit Sauerstoff unterversorgt ist, wurden Untersuchungen auch bei tumorphysiologischen Sauerstoffbedingungen von u. a. 5 % durchgeführt. Der antiproliferative Effekt von 5-FU wurde als halbmaximale inhibitorische Konzentration (IC50) für eine Kultur¬dauer von 72 Stunden bestimmt. Im Mangelmedium mit 3 mmol/l Glukose und 1 % FCS verstärkte sich der antiproliferative Effekt von 5-FU bei drei der fünf getesteten kolorektalen Karzinomzelllinien in Gegenwart von 5 % Sauerstoff im Vergleich zum Standard¬medium mit 11 mmol/l Glukose und 10 % FCS. Die Unterschiede in den IC50 Werten für 5-FU bei diesen drei Zelllinien (Colo741, HCT116, HT29) waren signifikant und bei den beiden anderen Zelllinien (LS174T, SW620) zeigten sich Tendenzen. Dagegen nahm bei Fibroblasten der antiproliferative Effekt von 5-FU im Mangelmedium ab, die Zellen waren somit besser vor dem Chemo¬therapeutikum geschützt. Eine Restriktion von Glukose und Wachstumsfaktoren verändert den antiproliferativen Effekt von 5-FU bei kolorektalen Karzinomzellen nicht und verringert den der Fibroblasten. Damit zeigen die Zellen das Phänomen der »differential stress resistance« (DSR), dass bei 5 % und 21 % Sauerstoff beobachtet wurde, nicht aber bei und 1 % Sauerstoff. Bei 5 % Sauerstoff wurde bei 3/5 Zelllinien sogar ein besseres Ansprechen auf 5 FU nachgewiesen. Der Einfluss von Sauerstoff auf die »differential stress resistance« ist bisher wenig untersucht und basiert vermutlich auf HIF-1-abhängige intrazelluläre Signal¬wege. Die Bedeutung von Sauerstoff und seinem Transkriptionsfaktor HIF-1 für DSR ist bisher nicht verstanden und sollte deshalb weiter untersucht werden. / The phenomenon of »differential stress resistance« in human colorectal carcinoma cell lines: augmentation of the antiproliferative effects of 5-fluoruracil under glucose restriction and low oxygen supply