A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfillment of the requirements for the degree of Masters of Science in Medicine in Emergency Medicine. Johannesburg, 2013 / To determine the types of uropathogens encountered in patients presenting to a busy private emergency department in Johannesburg and compare sensitivity patterns of the bacteria identified with current antimicrobial prescribing patterns.

Sexual transmission of the Human Immunodeficiency Virus Type 1 (HIV-1) accounts for the majority of newly acquired infections. Vaccination efforts have induced only modest protection in HIV clinical trials. HIV-1 induces a robust local immune response in genital mucosa of exposed individuals. Understanding the function of naturally occurring antibodies against HIV-1 in genital mucosa, the primary site of transmission, might be instrumental to improving vaccines and antibody-based microbicides. This study focused on HIV-specific antibody responses in the male genital tract (MGT), which is underexplored. We characterized antibody subclasses and specificities in genital tract secretions (seminal plasma, urethral secretions) and blood from a cohort of HIV-1-infected men to determine the origin and distinct nature of antibodies in the MGT. We detected similar HIV-1 IgG titers and specificities in all three body fluids, indicating that MGT IgG likely originates from blood. In contrast, gp41-specific IgA was restricted to genital secretions suggesting a local niche of IgA antibody production. Genital secretions from a subset of individuals neutralized cell-free HIV-1 and blocked cell-to-cell HIV-1 transmission. Statistically, these functions correlated positively with gp41 IgA titers. HIV-specific IgA monoclonal antibodies were also effective in these assays. To explore cell-dependent activities of HIV-specific antibodies in genital mucosa we surveyed Fc receptor expression in mucosal epithelial tissue. The IgG-engaging neonatal Fc receptor (FcRn), the IgA receptor FcαRI and the high-affinity intracellular Fc receptor TRIM21 were detected by immunohistochemistry and western blots. In stratified squamous epithelia (foreskin, vagina) FcRn+ epithelial cells were detected primarily in the basal layer, FcαRI+ epithelial cells in suprabasal layers and TRIM21 throughout. Deposits of immunoglobulins in the stratified squamous epithelium colocalized with FcRn, FcαRI and TRIM21. Our findings indicate that the MGT is capable of expressing a local anti-HIV IgA response to achieve antiviral defense through antibody neutralization and cell-dependent functions involving classical immune effector cells and epithelial cells.

In adult mammals, the gastrointestinal (GI) epithelium exhibits the highest turnover rate among the endodermal tissues. The harsh luminal environment of the GI tract necessitates replenishment of epithelial cells to maintain organ structure and function during routine turnover and injury repair. This delicate balance between gain and loss of cells is called tissue homeostasis, and multipotent tissue specific adult stem cells serve as the continuous source of self-renewal. Due to their important contribution to homeostatic maintenance the proliferative capacity of the stem cells needs to be tightly controlled, as an imbalance can result in diverse pathologies such as cancer or insufficient injury repair. Despite the crucial role for regulatory processes the molecular mechanisms and the genes governing these processes remain poorly understood. Rnf43 and its paralogue Znrf3 (RZ) act as tumour suppressors in the intestine, but their role in the gastric epithelium has not been previously investigated. Using a novel unpublished stomach specific CreERT2 expressing mouse line I found that simultaneous knockout of RZ (RZ DKO) result in gastric hyperplasia of the corpus epithelium. Gastric RZ DKO organoids show independence from the essential growth factor Rspondin-1 but require exogenous Wnt. A similar exogenous Wnt dependence was identified in a human gastric cancer cell line harbouring homozygous Rnf43 inactivating mutations. Thus, Wnt secretion inhibition might provide a new treatment paradigm for a subset of patients carrying Rnf43 mutations. The prominent role of the E3s Rnf43 and Znrf3 in the intestinal and gastric epithelial led to the question of whether other E3s either closely related to RZ or specifically expressed in stem or niche cells could play a role in homeostatic regulation, specifically in the small intestine. Using a retroviral overexpression screen I identified Rnf24 and Rnf122, two E3s that rendered intestinal organoids insensitive to withdrawal of the BMP inhibitor Noggin. Moreover, potential substrate candidates located at the cell surface membrane were identified and the generation of in vivo models initiated to provide a basis for further studies investigating the role of these E3s. In trying to address the function of the abovementioned genes using in vitro functional genetics I identified gaps in the current technology for organoid genetic engineering. I therefore developed two gene editing methods; a gRNA concatemer system allowing simultaneous knockout of multiple genes and CRISPR-FLIP enabling generation of conditional gene knockouts In summary, this thesis describes the first stomach specific knockout of Rnf43 and Znrf3 in the gastric epithelium, showing that it results in gastric hyperplasia located to the corpus epithelium. The dependence of the Rnf43 and Znrf3 knockout epithelium on exogenous Wnt signalling provides a potential treatment strategy for a subset of patients harbouring Rnf43 mutations. Next, it identifies Rnf24 and Rnf122 as E3 ubiquitin ligases involved in intestinal stem cell regulation and provide preliminary data and a basis for future studies. Finally, it describes the establishment of two advanced genetic engineering approaches which can be applied to various in vitro culture systems such as 3D organoids, mouse embryonic stem cells and conventional cell lines. Collectively this work and the developed methods will contribute to our understanding of the mechanisms regulating adult stem cell homeostasis.

Resumo: A triagem de crianças para bacteriúria assintomática objetivando prevenir pielonefrite e danos renais é amplamente recomendada. Amostras de urina, colhidas sem contaminação, de 500 pré-escolares com idade entre 2 a 7 anos foram submetidas ao teste com cloridrato de trifeniltetrazólio (TTC) e a urocultura. Culturas quantitativas foram realizadas usando dois diferentes meios de cultura: ágar CLED e ágar MacConkey. As colônias foram contadas, após 18-24 horas de incubação à 35-37ºC. O achado de 105 ou mais UFC/mL do mesmo microrganismo foi considerado como positivo. Para realizar o teste com TTC, 4 mL da urina foram misturados com 1 mL da solução aquosa de TTC estéril à 1% e incubados à 35-37ºC por 4 horas. Uma segunda urocultura foi realizada para as crianças que apresentaram resultado positivo. A sensibilidade aos antimicrobianos foi determinada. Uma comparação entre a urocultura e o teste com TTC foi feita, para avaliação do teste. Um questionário foi aplicado para avaliar fatores predisponentes comportamentais e funcionais. A triagem para bacteriúria assintomática, em pré-escolares em Araraquara-SP-Brasil mostrou uma prevalência de 1,4%. Escherichia coli foi o microrganismo mais isolado e a resistência a tetraciclina foi significante. Os resultados mostram que o teste com TTC possui 91,3% de sensibilidade; 64,3% de especificidade; 15,5% de valor preditivo positivo e 99,0% de valor preditivo negativo. Esses valores mostram que este teste pode ser usado como metodologia de triagem. O fato de já ter desenvolvido ITU anteriormente; usar o papel de trás para frente na higienização anal; beber menos de 1L de água por dia; e usar roupa íntima apertada foram considerados possíveis fatores de risco para o desenvolvimento de bacteriúria assintomática. / Abstract: Urinary tract infection (UTI) is the most commom of bacterial infections. Screening children for asymptomatic bacteriuria to prevent pyelonephritis and renal scarring is widely recommended. Urine samples, revealed without contamination, from 500 pre-school children aged 2 to 7 years were submited to the tryphenyl tetrazolium chloride (TTC) test and urine culture. Quantitative urine cultures was performed using two different agar types: CLED and MacConkey. Colonies were count after 18-24 hours of incubation at 35-37ºC. The finding of 105 or more CFU/mL of the same microorganism constituted a positive culture. To perform the TTC test, 4 mL of the urine were mixed with 1 mL of the TTC 1% aqueous sterile solution and incubated at 35-37ºC for 4 hours. We performed a second urine culture for all children with a positive result. Antimicrobial susceptibility was determined. A comparison between the quantitative culture and the TTC test were made, for the evaluation of the test. A questionnaire were used to assess predisposing behavioral and functional abnormalities. The screening survey for asymptomatic bacteriuria in pre-school children in Araraquara-SP-Brazil showed a prevalence of 1.4%. Escherichia coli was the commonest organism isolated and resistence to tetracycline was significant. The results show that the TTC test has sensitivity 91.3%, specificity 64.3%, positive predictive value 15.5% and negative predictive value 99.0%. This test can be use as a screening test. History of the urinary tract infection, inadequate hygiene, poor fluid intake and use of tigh-fitting underwear appear to be risk factors for asymptomatic bacteriuria. / Orientador: Maria Stella Gonçalves Raddi / Coorientador: Antonio Carlos Pizzolitto / Coorientador: Elisabeth Loshchagin Pizzolitto / Banca: Maria Jacira Silva Simões / Banca: Isabel Cristina Affonso Scaletsky / Mestre

The microflora inhabiting the human gastrointestinal tract can be considered an essential 'metabolic organ', in a symbiotic relationship with its host. Due to the low cultivability and inappropriate sampling methodology the microflora is poorly explored and ill-defined. Preliminary, molecular-based research at the University of Waikato revealed the presence of 16S rRNA gene sequences originating from novel Mollicute-like species inhabiting the human GI tract. A ~830bp 'consensus' sequence representing these novel Mollicute-like sequences was classified within the Mollicute Genus Anaeroplasma the type species of which is Anaeroplasma abactoclasticum. It also displayed near exact matches with 16S rRNA sequences obtained from the human GI tract and matches of high similarity to those from the mouse GI tract in the NCBI database. This thesis describes an attempt to design and create primers that would amplify and characterize full-length versions of these Mollicute-like sequences from samples obtained from the mucosal surface of the human gastrointestinal tract. Primers sets targeted extended 5' and 3' versions of these novel 'known' sequences and were designed from sequence matches found in the preliminary work and other related sequences from the NCBI database. The attempt to amplify a full-length version of these novel Mollicute-like sequences was proven to be unsuccessful. No sequences were classified within the Genus Anaeroplasma, although 81% of amplicons from the 5' extending primer sets were classified within the same division as the Mollicutes, the Firmicutes, only 6% of the sequenced amplicons from the 3' extending primer set belonged to this division. Phylograms containing these 'relevant' sequences and the 'consensus' sequence grouped the 'consensus' sequence separately, indicating a lower relatedness than would have been seen if any of the amplicons contained the 'consensus' sequence.

SYNOPSIS Included in this work, is a general historical overview of the development of astronomical knowledge in the West from the realms of Greek scholarship in classical times through to the Renaissance and the threshold of modern physics. The subject matter of both the Irish Tract and this review extends beyond the strict confines of astronomy, encompassing the physical sciences in general. The extent of astronomical knowledge in medieval Ireland is given specific attention with a review of scholarly works in Latin since the seventh century. This includes a number of specialist studies on astronomical topics and related cosmographical fields. Also included are numerous incidental references to astronomical matters from both Irish and Latin literature during the Middle Ages. Attention is devoted to the surviving manuscript copies of the Tract and the question of its sources, origin and purpose. A possible Dominican context for the compilation and dissemination of the Tract is considered. A detailed commentary of the technical content of each chapter is presented, together with reference to contemporary developments in the West and to the occasional clues as to the institutional, geographical and chronological origins of the Tract. A study of the technical terminology used by the Irish compiler is presented in detail. Reference is made both to earlier Irish terminology where appropriate, as well as to the limitations imposed by the fact that many of the scientific concepts were yet to attain clarity that came with the advent of Newtonian physics, Copernican astronomy and post-Colombian geography. The data entries on ms Stowe B are evaluated and compared with computer generated data of astronomical movements in the 14th and 15th centuries with a view to ascertaining the time of compilation of the Tract and its working life. A A revised English translation of the Tract is included in the appendices together with Maxwell Close's unpublished commentary to relevant portions. An Irish edition, closely following the ITS edition of 1914 is also included. Corruptions to the text are footnoted together with the likely run of the original text.

<p><strong>Introduction</strong> Every tenth patient in Sweden is affected by nosocomial infections. Among these, urinary tract infection (UTI) is the most frequently occurring within the hospital environment. Nosocomial infections lead to increasing costs for care, more suffering among the patients, increasing use of antibiotics, and longer treatment spells. The Academic hospital have engaged all wards in the so called VRISS-project (“nosocomial infections must be stopped”) in order to reduce the number of nosocomial infections.</p><p><strong>Aim</strong> In connection to the VRISS-project new routines for removal of uretrahl catheters are introduced at ward 70E2 at the Academic hospital. The new routines imply that the catheter should be removed during the first post-op day. According to the old routines the catheter was to be removed no later than on the third post-op day. The authors of this study choose to investigate whether the new routines for uretrahl catheter removal have any effect on the occurrence of nosocomial UTI’s.</p><p><strong>Methods </strong>The study is a restrospective record study with quantitative, descriptive design. 411 journals from 2007 (188) and 2009 (223) have been reviewed. All patients that underwent knee or hip operations during January, February, March, and April 2007 and 2009 are included in the sample.</p><p><strong>Results</strong> The study shows that the new routines at ward 70E2 had some, but not full, influence on the share of patients with nosocomial UTI. Among women under 71 years of age and men older than 70 years, the number of days with uretrahl catheter and the share of patients with nosocomial UTI have decreased. However, among the younger men (<71 years) and the older women (>70 years) no effects of the new routines are detected.</p><p><strong>Conclusion</strong> To summarize it can be concluded that the routines for how to use uretrahl catheters at ward 70E2 work fairly well. However, the results also show that the number of days with uretrahl catheter and the share of patients with nosocomial UTI among risk patients, above all among the older women, are the same in spite of the new routines for removal of uretrahl catheters. Thus, strategies for detecting risk patients in an early stage can be improved upon. Such measures can reduce the risk for nosocomial UTI among these patients.</p>

Introduction Every tenth patient in Sweden is affected by nosocomial infections. Among these, urinary tract infection (UTI) is the most frequently occurring within the hospital environment. Nosocomial infections lead to increasing costs for care, more suffering among the patients, increasing use of antibiotics, and longer treatment spells. The Academic hospital have engaged all wards in the so called VRISS-project (“nosocomial infections must be stopped”) in order to reduce the number of nosocomial infections. Aim In connection to the VRISS-project new routines for removal of uretrahl catheters are introduced at ward 70E2 at the Academic hospital. The new routines imply that the catheter should be removed during the first post-op day. According to the old routines the catheter was to be removed no later than on the third post-op day. The authors of this study choose to investigate whether the new routines for uretrahl catheter removal have any effect on the occurrence of nosocomial UTI’s. Methods The study is a restrospective record study with quantitative, descriptive design. 411 journals from 2007 (188) and 2009 (223) have been reviewed. All patients that underwent knee or hip operations during January, February, March, and April 2007 and 2009 are included in the sample. Results The study shows that the new routines at ward 70E2 had some, but not full, influence on the share of patients with nosocomial UTI. Among women under 71 years of age and men older than 70 years, the number of days with uretrahl catheter and the share of patients with nosocomial UTI have decreased. However, among the younger men (&lt;71 years) and the older women (&gt;70 years) no effects of the new routines are detected. Conclusion To summarize it can be concluded that the routines for how to use uretrahl catheters at ward 70E2 work fairly well. However, the results also show that the number of days with uretrahl catheter and the share of patients with nosocomial UTI among risk patients, above all among the older women, are the same in spite of the new routines for removal of uretrahl catheters. Thus, strategies for detecting risk patients in an early stage can be improved upon. Such measures can reduce the risk for nosocomial UTI among these patients.

The current study involved two experiments which were conducted at the Texas A&M Agricultural Research and Extension Center in Uvalde (semi-arid environment) from 2002 to 2004. In experiment one, Bonsmara bulls (n = 19; 20-24 mo of age) were joined with multiparous, crossbred females (n =586) for 90 d in 2003 and 2004. Bulls were allotted by selected physical traits, seminal traits, social rank, and serving capacity to one of two bull to female ratio (BFR) treatments: Conventional (1:21-1:29; n = 6 pastures) or Low (1:47-1:52; n = 2 pastures) BFR. Pregnancy rate (P = 0.33), calving rate (P = 0.26), and calving date (P = 0.22) did not differ between Conventional and Low BFR treatments. Post-breeding evaluation of bulls in 2002 (n = 16) indicated that social rank, but not seminal traits, was significantly correlated with pre-breeding values (P < 0.05). The current study demonstrates that Low BFR can be utilized in single- and multisire, 90-d breeding pastures of up to 2,090 ha without adversely affecting reproductive performance. In experiment two, yearling, one-half or three-quarter Bonsmara heifers (n = 106; 11-14 mo of age) were palpated per rectum and assigned a reproductive tract score (RTS) immediately prior to the beginning of the breeding season. Reproductive performance was measured in their two subsequent breeding years in order to estimate the value of the RTS system in extensively-managed, natural mating, 90-d breeding season programs. RTS was positively correlated (p < 0.01) with frame score (r = 0.25), age (r = 0.31), weaning weight (r = 0.47), and the weight of the heifer on the day of RTS exam (r = 0.56). The RTS means by dam parity also differed (P < 0.03). A lower (P < 0.01) percentage of females conceived during each of their first two breeding seasons for heifers of RTS 1 and 2 (65.2%) than for heifers of RTS 3, 4, and 5 (91.2%). Females with a RTS of 1 had a lower pregnancy rate over each of their first two breeding seasons, conceived later during their first breeding season, weaned lighter first calves, and remained lighter each year for fall body weight and body condition score than did heifers with RTS of 2 to 5 (P < 0.05). Collectively, the results of the current study indicate that heifers with a RTS of 1 immediately prior to a 90-d breeding season should be culled. Consideration should also be given to eliminating RTS 2 heifers, but further studies will be needed to confirm the potential economic advantage of this practice.

Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions.