Dissertação (mestrado)-Universidade de Brasília, Programa de Pós-Graduação em Patologia Molecular, 2011. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2011-11-29T13:29:54Z No. of bitstreams: 1 2011_DeniseTakamatsu.pdf: 9119373 bytes, checksum: 141b36ca97f1837de6484ae5ef965e47 (MD5) / Approved for entry into archive by Elzi Bittencourt(elzi@bce.unb.br) on 2011-11-29T13:46:50Z (GMT) No. of bitstreams: 1 2011_DeniseTakamatsu.pdf: 9119373 bytes, checksum: 141b36ca97f1837de6484ae5ef965e47 (MD5) / Made available in DSpace on 2011-11-29T13:46:50Z (GMT). No. of bitstreams: 1 2011_DeniseTakamatsu.pdf: 9119373 bytes, checksum: 141b36ca97f1837de6484ae5ef965e47 (MD5) / A doença diarréica aguda, ou gastroenterite aguda, representa um grande problema de saúde pública no mundo, afetando mais de 1 bilhão de crianças e adultos a cada ano. A mortalidade associada a essa síndrome é estimada em 6 milhões de casos por ano. A etiologia das gastroenterites é bastante diversa, incluindo bactérias, parasitas e vírus. Os patógenos virais são os agentes mais comumente associados a essa patologia. Dentre os agentes virais, destacam-se os norovírus, considerados atualmente, os principais agentes causais de epidemias de gastroenterites virais no mundo. O gênero Norovirus é constituído por 5 genogrupos (GI, GII, GIII, GIV e GV), sendo o GII o que apresenta maior prevalência em humanos. Dentro deste genogrupo, destaca-se o genótipo 4 (GII/4), por ser o principal causador de gastroenterites em humanos. Recentes trabalhos indicam novas variantes de GII/4 disseminadas pelo mundo. Este trabalho teve como objetivo estudar a variação gênica de norovírus GII que ocorreu no Distrito Federal no período entre 2006-2008. Inicialmente, os isolados do DF foram detectados por meio de tiras imunocromatógráficas, utilizando um total de 182 amostras diarréicas cujos resultados para a presença de bactérias patogênicas, rotavírus e adenovírus foram negativos. A região N terminal do gene que codifica para a proteína do capsídeo foi amplificada por RT-PCR e seqüenciada. A análise filogenética dos 17 norovírus isolados juntamente com sequências referências mostrou que 16 isolados pertencem ao GII/4 e um ao GII/3. Uma amostra de GII/4 foi selecionada para sequenciamento de aproximadamente metade do genoma a partir da região 3’. A análise filogenética do gene que codifica para o capsídeo, de cerca de 1.5 kb, indicou que a estirpe brasileira pertence ao subtipo “2004” ou variante Hunter. Os dados mostraram a utilidade da tira imunocromatográfica para detecção dos norovírus e genótipo dos 17 isolados no Distrito Federal. Para subtipagem, mais estudos são necessários para elucidar a minuciosa circulação de NoV GII/4 no Brasil em relação com a disseminação global. ______________________________________________________________________________ ABSTRACT / Acute diarrhea or acute gastroenteritis is a major problem in public health worldwide, affecting more than 1 billion of children and adults per year. The mortality associated with this syndrome is estimated to be 6 million cases a year. The etiology of gastroenteritis is diverse including bacteria, parasites and virus. Viruses are the most common casual agents for this syndrome. Among viruses, nowadays, norovirus is considered the major causative pathogen of epidemics gastroenteritis in the world. A Norwalk virus (Norovirus), the monotipic species of genus Norovirus consisted of 5 genotypes (GI, GII, GIII, GIV and GV), and GII is the most prevalent genotype in humans. Within genogroup II, genotype 4 (GII/4) is the major causative agent in the world. Recent works showed that new variants of GII/4 disseminate worldwide. This study is aimed to moleculary-characterize the norovirus GII isolated in the Federal District during years from 2006 to 2008. At first, norovirus isolates were identified by immunochromatografic strips using a total of 182 diarrheic samples which were negative for bacteria, Rotavirus and Adenovirus. The capsid gene region of Nterminal capsid protein was amplified by RT-PCR and sequenced. Phylogenetic analysis of 17 norovirus isolates with reference sequences showed that 16 isolates belonged to GII/4 and one to GII/3. One GII/4 isolate was selected to be sequenced almost half of its genome at 3’ region. Phylogenetic analysis of whole capsid gene of 1.5 kb showed that this Brazilian isolate belonged to subtype “2004” or Hunter variant. Our results showed that the usefulness of immunochromatografic stripe for norovirus detection and genotype of 17 noroviruses isolated in the Federal District. For subtyping, futher study will be necessary to elucidate the detailed circulation of norovirus GII/4 in Brazil in relation to the global dissemination.

Orientador: Sandra C. B. Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-22T14:09:06Z (GMT). No. of bitstreams: 1 Nogueira_Eliana_M.pdf: 1773398 bytes, checksum: b10e465048807c44b7421ddd7e7eb4bb (MD5) Previous issue date: 1997 / Resumo: Os citomegalovírus (CMV) são uns dos principais agentes infecciosos que acometem pacientes imunocomprometidos. Antes do início da realização da triagem sorológica para lllV -1 e subseqüente tratamento para inativar viroses, por meio de aquecimento, indivíduos com deficiência nos fatores VIII ou IX apresentavam um alto risco de contrair infecção por estes vírus, mediante transfusão de produtos sangüíneos contaminados. Além deste problema em relação às infecções, as transfusões também estão associadas com alterações no sistema imunológico...Observação: O resumo, na integra, podera ser visualizado no texto completo da tese digital / Abstract: Cytomegalovirus (CMV) is of major concem in immunocompromised and immunosuppressed patients. Prior to the introduction of mv -1 antibody screening and subsequent heat-treatment to inactivate viruses, individuals with Factor VIll or Factor IX deficiency had a high risk of contracting mv -1 infection through infusion of contaminated blood products. In addition to such infections, blood transfusions are also frequently associated with alterations in immune function...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Farmacologia / Mestre em Ciências

Orientador: Joaquim Murray Bustorff-Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T00:31:46Z (GMT). No. of bitstreams: 1 Silva_MarciaAlessandraCavalaroPereirada_M.pdf: 4774627 bytes, checksum: acf23f8d62bddb933b1a7ee7fa63b21e (MD5) Previous issue date: 2004 / Resumo: Alterações histológicas precoces causadas por enterocolite necrosante já foram exaustivamente estudadas em modelos animais. No entanto, poucas informações sobre os efeitos tardios da doença sobre a motilidade intestinal podem ser encontradas na literatura atual. No presente estudo, trinta e dois ratos Wistar machos recém-desmamados pesando entre 58 e 103 gramas foram distribuídos ao acaso em quatro grupos: Controle (não operados), Sham (laparotomia), Isquemia por 30 minutos (laparotomia e isquemia da artéria mesentérica superior por 30 minutos) e Isquemia por 45 minutos (laparotomia e isquemia da artéria mesentérica superior por 45 minutos). A motilidade intestinal no período pós-operatório foi medida indiretamente através da medida do peso seco do total de fezes obtidas em 24 horas nos 3°, 7°, 14° e 210 dias após a cirurgia. Segmentos de duodeno, jejuno e íleo foram examinados à microscopia de luz para observação de alterações ocorridas nos plexos mioentéricos. Três semanas após a isquemia, os neurômos dos plexos mioentéricos apresentaram vacuolização citoplasmática, e seus núcleos apresentaram irregularidade no f:Ontomo e sinais degenerativos. Houve mudanças significativas na motilidade intestinal nos animais submetidos a 45 minutos de isquemia mesentérica, mas essas mudanças não provocaram alterações significativas no crescimento final dos animais. Os resultados sugerem que o processo de isquemia-reperfusão intestinal causa danos aos neurômos dos plexos mioentéricos. Nas condições referidas no presente estudo, essas mudanças foram suficientes para induzir alterações na motilidade, mas isso não afetou a função intestinal / Abstract: Background/Purpose: Early histological changes induced by necrotizing enterocolitis have been extensively studied using animal models. However, infonnation regarding late effects on intestinal motility are lacking. The aim of this study was to investigate the late effects of ischemia-reperfusion on myenteric pIexus histology and intestinal motility.Materiais and Methods: Thirty-two post-weaning male mice weighing between 58 and l03g were divided randomly into 4 groups: Control (unoperated), Sbam(celiotomy), 30 min Ischemia(celiotomy and superior mesenteric artery ischemia for 30 minutes) and 45 min Ischemia(celiotomy and superior mesenteric artery ischemia for 45 minutes). Postoperative intestinal motility was assessed by weighing total fecal output over 24 hours on the 3rd, 7th, 14th and 21 st day afier surgery. Segments of duodenum, jejunum and ileum were examined at light microscopy for changes in the myenteric plexus.Results: At three weeks afier ischemia, the myenteric neurons appeared, in light microscopy, spongy or foamy, containing many vacuoIes in their cytoplasm. The neuronal nucIeus became irregular, with degenerative signs. There were significantly changes in intestinal motility in the animals groups submitted to intestinal ischemia, but thest: changes did not affect the animal overall growth.Conclusions: The results suggest that intestinal ischemia-reperfusion causes late neuronal damage. Within that conditions of the present study, these changes were sufficient to induce alterations in intestinal motility, but this did not affect intestinal function / Mestrado / Cirurgia / Mestre em Cirurgia

Amphipods are the most ubiquitous animals, after nematodes, on earth. Although there are several terrestrial amphipod species, most are aquatic. They are familiar animals in the water table exposed in cave environments and boreholes. The food source on which the amphipods depend was not directly observable in the cave environments frequented by the amphipods. In order to establish the role cave-dwelling amphipods play in ecology, the primary purpose of this study was thus to determine what cave-dwelling amphipods feed on. Amphipod, water and sediment samples were collected from five different caves, in the northern part of South Africa, namely Koelenhof Cave, Sterkfontein Cave, Ficus Cave, Peppercorn’s Cave, and Irene Cave. Following collection and transportation, resulting in zero amphipod mortalities, all of the samples were transferred to rectangular fish tanks stored in an environmental room, set up in such a way as to mimic the conditions in the caves as closely as possible. Long term adaptability and survival proved to be a successful undertaking, resulting in the death of only two amphipods per tank. Scanning electron microscopy was used to observe the mouthparts of the amphipods in order to begin establishing their feeding behaviour. Standard microtechniques were carried out to establish the general histological orientation and histology of the gastrointestinal tract. A Histochemical Fluorescent staining method was employed, and a reddish-orange fluorescence was observed, thereby indicating the presence of mucous in the GIT. Several feeding experiments were carried out, and it was established that on average amphipods can survive without a food source for a maximum of sixty ABSTRACT xv days. Through a series of different feeding experiments, it was determined that amphipods ingest bat faeces, leaf litter, sediment and yeast, with leaf litter producing the highest rate of survival. It was also observed that amphipods, regardless of body size, are predators, scavengers, and cannibals, which may provide an explanation as to why amphipods display evasive behaviour. Microbiology plays a vital role in determining what amphipods feed on, and therefore water, soil, and digestive contents of amphipods were studied using a wide array of microbiological analyses: Heterotrophic Plate Counts; Total Coliforms; Faecal Coliforms; Faecal Streptococci; Confirmatory test for Escherichia coli; Detection of Clostridium, Pseudomonas, and Salmonella. According to the South African Bureau of Standards, the quality of the water contained within all four of the caves in this study may not be used for human consumption prior to undergoing various purification processes. Once the role that cave-dwelling amphipods play in ecology has been firmly established it may then be possible to make use of amphipods as biological indicators, because since they inhabit cave streams and groundwater and are sensitive to pollutants, declines in their populations could indicate a decline in the water quality in their streams and surrounding groundwater supply. / Dr. J.F. Durand

Background: Malnutrition and gastrointestinal (GI) involvement are common in patients with systemic sclerosis (SSc). Despite malnutrition being common, little is known about its associations and predictors. Although patients are frequently screened and assessed for malnutrition, different clinically applicable assessment modalities in SSc have not been compared. An understanding of the relationship between dietary intake and energy expenditure is important for nutritional assessment and management. However, studies have not compared these. For many years, home parenteral nutrition (HPN) has been used in patients with intestinal failure, but little outcome data exists to support its role in SSc. GI involvement results in dysmotility, the underlying mechanism for the development of which is unknown. However, autonomic dysfunction has been proposed. Aims: To explore aspects of the nutritional assessment and management of patients with SSc. To seek associations and predictors of nutritional decline. To investigate for a link between GI dysmotility and autonomic dysfunction. Methods: Study 1: A retrospective review of the survival and outcome data of patients commenced on HPN over 22 years. Study 2: An assessment of 168 patients recruited over 12 months and restudied after approximately 1 year. Assessment included demographics, clinical data, GI and functional questionnaires, nutrition screening tool, oral aperture, mid-upper arm and 4-site anthropometry, bioelectrical impedance and biochemical testing. Re-study included weight change. Study 3: A 3 day assessment of dietary intake and energy expenditure using food record charts and SenseWear® Armband involving 36 patients recruited to Study 2. Study 4: Patients and matched controls completed GI and autonomic questionnaires, an autonomic battery, a gastric emptying study and postprandial cardiovascular measures and GI sensations and symptoms scores. Results: Study 1: The cumulative probabilities of surviving on HPN at 2, 5 and 10 years were 75%, 37% and 23%. HPN-associated complication rates were low. Study 2: Nutritional screening failed to identify all patients who lost weight. Mid-arm circumference correlated with body mass index (BMI) and weight change. Four-site anthropometry correlated with BMI more strongly (r=0.65 vs. r=0.49) than bioelectrical impedance analysis. Small intestinal, but not oesophageal, involvement correlated with baseline nutritional status. No clear predictors of nutritional decline were identified. Study 3: Predicted energy intakes correlated with measured expenditures, but absolute values differed. Energy intakes did not correlate with expenditures. Study 4: Autonomic measures did not correlate with gastric emptying. However, autonomic results were hindered by patient-related and technical limitations. Conclusion: Nutritional screening tools cannot be relied upon to detect all at risk patients. MAC and 4-site anthropometry may have a role in nutritional assessment. When an accurate appreciation of energy requirements is needed, kinematic monitors should be used rather than predictive equations. For those patients who progress to intestinal failure, HPN is safe and effective. Autonomic studies were inconclusive. However, the autonomic apparatus has been refined for utilisation in more definitive studies in younger patients.

Gel filtration coupled with radioimmunoassay of fractions has demonstrated the existence of an 8000 dalton immunoreactive form of GIP (glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide), which may be a precursor in the biosynthetic pathway. A monoclonal antibody to GIP has been shown to have highly suitable characteristics for affinity purification of different species of IR-GIP. An enzyme-linked immunosorbent assay (ELISA) was developed for GIP, employing the monoclonal antibody and was used for screening fractions for peptides with the same antigenic determinant i.e. IR-foras of GIP. Classical strategy used in peptide purification may result in loss of related peptides if they are sensitive to the pH or temperature conditions used. Tissue from hog duodenal and jejunal mucosa was boiled and extracted into acetic acid. Peptides were then adsorbed to alginic acid, eluted with 200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted with methanol. The methanol insoluble fraction demonstrated the highest content of IR-GIP₈₀₀₀⋅ The overall acidic charge on the larger IR-GIP oUUU moiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP₅₀₀₀ was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A µBondapak C₁₈ column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

A study on the digestive tract of a harpacticoid copepod, 2i3lio£Ss californicus, was carried out using techniques of light and electron microscopy. It was found that a curved, cuticulized esophagus extends from the ventral mouth to the midgut. Its musculature and shape allows fairly large food particles to enter the gut. The noncuticulized portion of the digestive tract consists of; 1. A single, anterior, spherical midgut caecum, 2. An anterior midgut extending from the midgut caecum to the joint at the beginning of the urosome, 3. A posterior midgut extending almost the length of the urosome. The cuticulized hindgut can be divided, structurally, into anterior and posterior regions. It is suggested that the anterior hindgut functions in ion and water regulation as well as begins the formation of a faecal pellet. The posterior hindgut compacts the faecal pellet and retains it until defaecation. At the light and electron microscope levels four cell types could be distinguished. By studying the cell's position in the gut, electron density, amount of lipid, amount and type of vesiculation and the abundance and position of the cell*s organelles, functions for these cells were determined: 1. cell type one is an embryonic cell which will replace cells worn away or lost in secretion. 2. Cell type two functions mainly in the synthesis and secretion of proteins and also plays a role in lipid absorption. 3. Cell type three appears to function mainly in lipid absorption. 4. Cell type four also functions in lipid absorption but this cell is only found in the anterior midgut and the type of vesicles found in this cell suggest a different type of absorption is occurring than in cell type three. From the abundance of each cell type, the length of the microvilli, the development of the basal lamina and luminal projections, the following conclusions were made: 1. The midgut caecum functions mainly for absorption of digested nutrients. 2. The anterior midgut also functions for nutrient absorption but plays a more important role in merocrine and exocrine secretion. The presence of concretions in cell types two and three of the anterior midgut suggest a role in excretion, water or ion regulation. 3. The posterior midgut functions mainly in absorption, though some holocrine secretion is evident. / Science, Faculty of / Botany, Department of / Graduate

Les biofilms bactériens sont des communautés répandues, dans lesquelles la densité cellulaire élevée, la diffusion réduite et la structure hétérogène favorisent les contacts physiques et métaboliques entre les bactéries et induisent de nouveaux comportements par rapport aux microorganismes individuels. [Si] la plupart des connaissances actuelles sur la formation du biofilm et le métabolisme découlent des études réalisées dans les modèles bactériens aérobies pathogènes et environnementaux, […] il existe peu d'informations sur la formation de biofilm dans des anaérobies strictes en général et des anaérobies commensaux associés à l'hôte de la gastro-microbiota intestinal en particulier. […] Nous avons utilisé des tests de biofilm in vitro statiques et dynamiques combinés avec des approches génétiques et transcriptomiques pour étudier les mécanismes moléculaires de la formation de biofilm et du métabolisme dans un prothèses intestinales Bacteroides thetaiotaomicron. Notre écran initial sur 35 souches de B.thetaiotaomicron […] a révélé une capacité généralisée de cette espèce à former un biofilm. Cependant, la production de biofilm semblait réprimée […] dans le VPI 5482 de type sauvage de référence et cela nous a poussé à développer les travaux de thèse selon deux axes principaux : l'identification de conditions induisant la formation de biofilm in vitro dans VPI 5482 et l'identification et la caractérisation de mutants transposons avec augmentation de la capacité du biofilm par rapport à WT VPI 5482. Après avoir testé divers composés qui sont pertinents pour l'environnement intestinal, nous avons montré que les sels biliaires […], induisent la formation de biofilm chez VPI 5482 et nous avons utilisé le RNAseq pour identifier les fonctions impliquées dans la formation de biofilm induite par le sel biliaire. Les résultats de RNAseq montrent que les bactéries dans le biofilm augmentent l'expression des gènes impliqués dans la glycosylation de la surface cellulaire et présentent un modèle d'expression spécifique des opéron de synthèse capsulaire, suggérant que les polysaccharides de surface cellulaire pourraient jouer un rôle clé lors de la formation du biofilm dans B. thetaiotaomicron. Nous avons ensuite utilisé la mutagenèse du transposon pour révéler les facteurs d'adhérence impliqués dans la formation du biofilm en présence de sels biliaires, ce qui a conduit à l'identification d'un opéron non caractéristique (BT3560-2) impliqué putativement dans le transport de micronutriments dépendant de TonB. Même si les cibles des systèmes de transport identifiés par mutagenèse et RNAseq sont […] inconnues, leur caractérisation supplémentaire peut conduire à l'identification des voies réglementaires qui régissent la formation du biofilm. En utilisant la mutagenèse du transposon pour débloquer l'adhérence dans le pauvre biofilm B. thetaiotaomicron VPI 5482, nous avons identifié deux loci - un putatif V Mfa1 pili operon BT3148-7 et un opérat de synthèse de capsule 4 (CPS4) impliqué dans la formation de biofilm indépendamment des sels biliaires. Sur la base de l'homologie structurale avec les pilins mfa1, BT3148 et BT3147 constituent vraisemblablement une structure pilus qui requiert une troncature C-terminale de la pilule BT3147 de la tige prédite pour l'allongement et l'augmentation conséquente de la formation du biofilm. La microscopie électronique continue combinée à la détection d'immunoglole du pilus putatif devrait élucider complètement le mécanisme de formation de biofilm médié par BT3148-7. Le deuxième facteur de biofilm identifié indépendant de la bile est la capsule 4 (CPS4). Comme la suppression du CPS4 à l'état sauvage a conduit à une forte production de biofilm, nous avons émis l'hypothèse que le CPS4 pourrait masquer une adhésine putative. / Bacterial biofilms are widespread communities, in which high cell density, reduced diffusion and heterogeneous structure favor physical and metabolic contacts between bacteria and induce novel behaviors as compared to individual microorganisms. Most of the current knowledge about biofilm formation and metabolism is derived from the studies performed in pathogenic and environmental aerobic bacterial models, by contrast, there is little information on biofilm formation in strict anaerobes in general, and host-associated commensal anaerobes of the gastro-intestinal microbiota in particular. In this project we used static and dynamic in vitro biofilm assays combined with genetic and transcriptomic approaches to study molecular mechanisms of biofilm formation and metabolism in a prominent gut commensal Bacteroides thetaiotaomicron. Our initial screen on 35 B. thetaiotaomicron strains collected from various academic and clinical collections revealed a widespread ability of this species to form biofilm. However, biofilm production seemed repressed or masked in the reference wild type VPI 5482 and this prompted us to develop the thesis works along two main axis: the identification of conditions inducing in vitro biofilm formation in VPI 5482 and the identification and characterization of transposon mutants with increased biofilm capacity compared to WT VPI 5482. Having tested various compounds that are relevant to gut environment, we showed that bile salts – an important environmental clue for gut microbiota, induce biofilm formation in VPI 5482 and we used RNAseq to identify functions involved in bile salt-induced biofilm formation. RNAseq results show that bacteria in biofilm increase the expression of genes involved in glycosylation of cell surface and exhibit a specific expression pattern of capsular synthesis operons, suggesting that cell surface polysaccharides could be playing a key role during biofilm formation in B. thetaiotaomicron. We then used transposon mutagenesis to reveal adhesion factors involved in biofilm formation in presence of bile salts, which led to identification of an uncharacterized operon (BT3560-2) putatively involved in TonB-dependent transport of micronutrients. Even though the targets of the transport systems identified via mutagenesis and RNAseq are yet unknown, their further characterization may lead to identification of regulatory pathways that govern biofilm formation. Using transposon mutagenesis to unlock adhesion in the poor biofilm-former B. thetaiotaomicron VPI 5482, we identified two loci – a putative type V Mfa1 pili operon BT3148-7 and capsule synthesis operon 4 (CPS4) involved in biofilm formation independently of bile salts. Based on structural homology with mfa1 pilins, BT3148 and BT3147 likely constitute a pilus structure that requires C-terminal truncation of the predicted stalk pilin BT3147 for elongation and consequent increase in biofilm formation. Ongoing electron microscopy combined with immunogold detection of the putative pilus should fully elucidate the BT3148-7-mediated biofilm formation mechanism.

Equine piroplasmosis is a tick-transmitted disease caused by Theileria equi or Babesia caballi leading to haemolytic anaemia. Imidocarb is an effective treatment of piroplasmosis, but adverse clinical signs, including colic and diarrhoea, from cholinesterase inhibition are associated with its use. Atropine is advocated for the treatment of cholinesterase inhibiting compounds. Atropine is known to have a prolonged inhibitory effect on gastrointestinal motility. Glycopyrrolate is an anticholinergic drug that has similar effects to atropine on gastrointestinal motility, but with decreased penetration of blood-brain and blood-aqueous barrier. This study was performed to assess the adverse clinical effects of a therapeutic dose of imidocarb, the effect of this dose on gastrointestinal motility, and on cholinesterase activity. The ability of atropine or glycopyrrolate to ameliorate imidocarb’s adverse clinical signs, and the effect of the combination of atropine and imidocarb or glycopyrrolate and imidocarb on gastrointestinal motility was evaluated. A blinded crossover trial was performed in 8 horses. All horses were administered saline (CON), imidocarb 2.4 mg/kg im and saline iv (IMI), imidocarb 2.4mg/kg im and atropine 0.02 mg/kg iv (IMATROP) and imidocarb 2.4mg/kg im and glycopyrrolate 2.5 µg/kg iv (IMGLYCO), with a one week wash-out period between treatments. Butrylcholinesterase activity was measured in the CON and IMI group. Clinical signs, gastrointestinal motility and faecal production were assessed. Gastrointestinal motility was measured by abdominal auscultation and frequency of contractions in the duodenum, caecum and right dorsal colon visualized with transcutaneous abdominal ultrasound. Total faecal production, faecal dry matter, wet matter, faecal water percentage, frequency of defaecation and time to first defaecation was assessed. Abdominal pain and diarrhoea were observed in the IMI group. Borborygmi and frequency of intestinal contractions were not different in the IMI group compared to CON. Percentage water content, faecal production, faecal dry matter and frequency of defaecation were significantly increased in the IMI group. Butrylcholinesterase activity was not significantly decreased in the IMI group compared to CON. In the IMATROP group colic signs were observed, heart rate was significantly elevated and mydriasis was evident. Borborygmi and frequency of contractions in the right dorsal colon was significantly reduced in the IMATROP group. In the IMGLYCO group the incidence and severity of colic induced by imidocarb was reduced. Heart rate was significantly increased and borborygmi significantly decreased compared to CON. The effect of IMGLYCO on heart rate and borborygmi was significantly less than the effect of IMATROP. In the IMGLYCO group the frequency of ultrasound visualised intestinal contractions and faecal variables were not different from CON. Therapeutic doses of imidocarb are associated with clinical signs of muscarinic stimulation including colic and diarrhoea, and enhanced faecal production. Clinical signs of cholinesterase inhibition can be present without significant depression in plasma cholinesterase activity. Atropine prevents diarrhoea and normalises faecal water percentage but is not effective in decreasing incidence of abdominal pain, and causes a prolonged inhibition of gastrointestinal motility, which might make this drug undesirable to use as a pre-treatment to imidocarb in clinically affected horses. Glycopyrrolate only partially reduces gastrointestinal motility and decreases adverse signs and thus its use as a pre-treatment to imidocarb is preferred. / Dissertation (MMedVet)--University of Pretoria, 2006. / Companion Animal Clinical Studies / unrestricted