München, Techn. University, Diss., 2002.

Bielefeld, Universiẗat, Diss., 2004. / Dateiformat: tgz, Dateien im PDF-Format.

Made available in DSpace on 2015-12-06T23:47:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2007 / Objetivo: Investigar a possivel participacao de celulas B-1 na inducao de tolerancia oral em camundongos submetidos a um regime de alimentacao continua com OVA. Metodos: Camundongos femeos das linhagens BALB/c e BALB/xid, entre 6 e 8 semanas, foram utilizados na formacao de 9 grupos experimentais, composto respectivamente pelos grupos de animais tratados previamente com OVA por via oral, (B/c-T e Bx-T); grupo de animais que nao receberam tratamento oral com OVA mas foram imunizados, par via s.c. com OVA emulsificada (1:1) em Adjuvante completo de Freund (B/c-I e B/x-I); grupo de animais que foram somente desafiados com OVA agregada (B/c-N e B/x-N); grupos B/x-recN, B/x-recT e B/x-recl compostos por camundongos BALB/Xid reconstituidos por transferencia adotiva com celulas B-1 do sobrenadante de cultura de celulas aderentes peritoneais de camundongos BALB/c naive; tolerizados e imunes, respectivamente. Esses animais receberam transferencia adotiva de celulas B-1 tres dias antes da imunizacao. A resposta foi avaliada por: a) reacao inflamatoria local; b) linfoproliferacao pela incorporacao de 3+[H]Timidina e c) determinacao por ELISA de imunoglobulinas anti-¬OVA das subclasses (IgG total, IgG1 e IgG2a). Resultados: Em camundongos BALB/c 0 tratamento oral com OVA previamente a imunizacao foi capaz de suprimir reacao local de hipersensibilidade e resposta celular muito embora esse efeito nao tenha sido observado em camundongos BALB/Xid submetidos ao mesmo tipo de tratamento. No entanto, quando camundongos BALB/Xid foram transferidos adotivamente com celulas de sobrenadante de cultura celular de peritoneo de camundongos BALB/c tolerizados,que esses animais desenvolveram supressao da reacao local de hipersensibilidade e supressao da resposta celular. Conclusao: Celulas B-1 da cavidade peritoneal de camundongos BALB/c doadores, submetidos ao tratamento oral com OVA, foram eficazes em promover a supressao da reacao de hipersensibilidade local e da resposta celular linfoproliferativa, mas nao a de producao de anticorpos, quando adotivamente transferidas para camundongos BALB/Xid, refratarios a inducao de tolerancia oral / Purpose: To investigate a possible participation of B-1 cells in inducing oral tolerance in mice submitted to continuous feeding with OVA. Methods: Female mice of the BALB/c and BALB/Xid lineages, aged 6 to 8 weeks, were divided into 9 groups and submitted to the following conditions: previous oral treatment with OVA (B/c-T and Bx-T); without oral treatment with OVA, but with subcutaneous immunization with OVA emulsion (1:1) in complete Freund's adjuvant (B/c-I and B/x-I); challenge with aggregated OVA only (B/c-N and B/x-N); adoptive transfer (rebuilding), to BALB/Xid mice, of B-1 cells in culture supernatant with peritoneal adherent cells from naïve BALB/c mice (B/x-recN, B/x-recT, and B/x-recI); made tolerant and immune, respectively. These animals received adoptive transfer of B-1 cells three days prior to immunization. The immune responses were evaluated by means of: a) local inflammatory response; b) lymphoproliferation by incorporation of 3+[H]Thymidine and c) determination of anti-OVA immunoglobulin (subclasses total IgG, IgG1, and IgG2a) by ELISA. Results: Oral treatment with OVA prior to immunization suppressed the local hypersensitivity reaction and cellular response in BALB/c mice, although this effect was not observed in BALB/Xid mice in the same conditions. However, when BALB/Xid mice were reconstituted with cultured cells from tolerized peritoneal BALB/c mice, the suppression of local hypersensitivity and cellular response were observed. Conclusion: B-1 cells from BALB/c mice orally treated with OVA caused phenotypic and functional changes and suppression of cellular response local and linfoproliferative and local hypersensitivity reaction when adoptively transferred to BALB/Xid mice refractory to induction of oral tolerance. / BV UNIFESP: Teses e dissertações

[No abstract available] / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

L’arsenic est un métalloïde largement répandu dans l’environnement. Il se trouve essentiellement sous deux formes toxiques : l’arséniate [As(V)] et l’arsénite [As(III)]. De nombreux microorganismes sont capables de transformer l’arsenic (par oxydation, réduction et méthylation) et sont ainsi directement impliqués dans le cycle biogéochimique de ce métalloïde. Parmi les mécanismes de transformation de l’arsenic, le système ars semble largement répandu parmi les bactéries. Il consiste à transformer As(V) en As(III) (qui est la forme la plus toxique et la plus mobile de l’arsenic), puis à expulser ce dernier à l’extérieur de la cellule sous l’action d’un transporteur d’arsénite membranaire. Deux familles de transporteurs d’arsénite ont été décrites chez les procaryotes : la famille ArsB (la plus étudiée) et la famille Acr3p, elle même subdivisée en deux groupes. Dans le cadre de ce travail nous avons défini trois jeux d’amorces dégénérées permettant l’amplification des gènes arsB et ACR3. Ces amorces ont été utilisées pour la détection des gènes arsB/ACR3 de quarante et un isolats bactériens résistants à l’arsenic isolés à partir de deux sols lorrains présentant des concentrations différentes en arsenic. Les résultats PCR ont montré que 70,7 % des isolats contiennent un gène de transporteur d’arsénite avec une prévalence du génotype ACR3 par rapport à arsB. Ces résultats ont ainsi validé l’utilisation de nos amorces pour la détection des gènes de transporteurs d’arsénite. En plus de la validation des amorces, nous avons largement caractérisé la collection de quarante et un isolats bactériens résistants à l’arsenic : identification moléculaire, détermination des CMIs à As(III), As(V) et Sb(III) et recherche des activités arsénite oxydase et arséniate réductase. Parmi ces quarante et une souches, deux présentent une activité arsénite oxydase et trente-neuf une activité arséniate réductase. L’isolat A33 Microbacterium sp. s’est distingué par son niveau de résistance particulièrement élevé à As(III) et As(V). Nous avons identifié par une approche moléculaire, le système ars de cette souche. Notre analyse a révélé un système original composé de 6 gènes arsT, arsX, ACR3(1), arsRC2, arsC1 et arsC3 qui codent respectivement une thiorédoxine réductase, une thiorédoxine, un transporteur d’arsénite, une protéine de fusion "régulateur-arséniate réductase", une arséniate réductase et une autre arséniate réductase. Les cinq premiers gènes font partie d’un même opéron et arsC3 est transcrit dans une direction opposée. Bien que la thiorédoxine réductase et la thiorédoxine soient connues pour être indispensables au fonctionnement de certaines ArsC thiorédoxine dépendantes, les gènes arsT et arsX font rarement partie des opérons ars connus. De même ArsRC2 est une protéine atypique rarement trouvé dans les opérons ars connus. Une autre particularité de l’opéron ars de Microbacterium sp. est la protéine ArsC3 qui contient le motif "CX5R" spécifique des ArsC thiorédoxine dépendantes mais ne contient pas les deux résidus cystéines localisés du coté C-terminal de ces dernières et indispensables pour la réduction d’As(V) en As(III). Une stratégie est proposée pour analyser le rôle de chaque gène de l’opéron ars de Microbacterium sp. dans la résistance à As(III) et As(V). / Arsenic is an ubiquitous toxic metalloid. The two most common forms of arsenic in the environment are inorganic arsenate [As(V)] and arsenite [As(III)]. Many microorganisms are able to transform arsenic (by oxidation, reduction and methylation) and thus play an important role in the biogeochemical cycle of this metalloid. Among the mechanisms of arsenic transformation, the ars system seems to be widely distributed in bacteria. This detoxification system involves the reduction of As(V) in As(III) (the most toxic and most mobile form) which is then pumped out of the cell through an integral membrane transporter. Two unrelated families of arsenite transporters have been described in prokaryotes: the well-characterized ArsB family and the Acr3p family which is subdivided into two subgroups. In this study, we designed three sets of degenerate primers allowing the amplification of the arsB and ACR3 genes. These primers were used to screen a collection of forty-one arsenic-resistant strains isolated from two soil samples from Lorraine with contrasting levels of arsenic concentration. PCR results showed that 70.7 % of the isolates contained a gene related to arsB or ACR3 family. The ACR3 genotype was predominant over arsB. These results validated the use of our primers for the identification of arsenite transporter genes. In addition to validating the primers, we characterised a novel collection of forty-one arsenic-resistant strains: molecular identification, MIC determination for As(III), As(V) and Sb(III) and detection of arsenite oxidase and arsenate reductase activities. Among these forty-one strains, arsenite oxidase activity was detected in two strains and arsenate reductase activity in thirty-nine. The Microbacterium sp. A33 isolate showed high resistance level to both As(III) and As(V). We used a molecular approach to identify arsenic resistance (ars) genes of this strain. Our analysis revealed an original system composed of 6 genes : arsT, arsX, ACR3(1), arsRC2, arsC1 et arsC3 that encode a thioredoxin reductase, a thioredoxin, an arsenite transporter, a fusion protein "regulator-arsenate reductase", an arsenate reductase and another arsenate reductase respectively. The first five genes belong to the same operon and arsC3 gene is transcribed in the opposite direction. Thioredoxin and thioredoxin reductase are essential to thioredoxin-coupled arsenate reductase activity. However, the majority of ars operons don’t encode these proteins. The atypical protein ArsRC2 is also rarely present in ars operon. The ArsC3 arsenate reductase is another particularity of Microbacterium sp. ars system. ArsC3 contains the CX5R specific motif of thioredoxin-coupled arsenate reductases but don’t contains in the C-terminal extremity the two cysteine residues essential for reduction of As(III) to As(V). A strategy is proposed to analyze the role of each Microbacterium sp. ars gene in resistance to As (III) and As(V).

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No description available.

Tang Yuk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 130-144). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatitis B Virus (HBV) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- History of HBV --- p.2 / Chapter 1.1.3 --- Hepatitis B in Hong Kong --- p.3 / Chapter 1.2 --- Virology --- p.4 / Chapter 1.2.1 --- Hepadnavirus Family --- p.4 / Chapter 1.2.2 --- HBV Particles Types --- p.4 / Chapter 1.2.3 --- HBV Genome --- p.6 / Chapter 1.2.4 --- HBV Life Cycle --- p.8 / Chapter 1.2.5 --- Hepatitis B Surface Antigen --- p.14 / Chapter 1.3 --- HBV Immunobiology During Viral Infection --- p.17 / Chapter 1.3.1 --- Acute Hepatitis B Virus Infection --- p.19 / Chapter 1.3.2 --- Chronic Hepatitis B Virus Infection --- p.23 / Chapter 1.3.3 --- Cytokines in Suppression of HBV Infection --- p.23 / Chapter 1.3.4 --- The mechanism of HBV Persistence --- p.26 / Chapter 1.4 --- HBV Therapy --- p.28 / Chapter 1.4.1 --- Interferon alpha (IFN-α) --- p.29 / Chapter 1.4.2 --- Lamivudine --- p.31 / Chapter 1.5 --- Phyllanthus --- p.32 / Chapter 1.5.1 --- In vitro study --- p.32 / Chapter 1.5.2 --- In vivo study --- p.33 / Chapter 1.5.3 --- Clinical Trials --- p.34 / Chapter 1.5.4 --- Anti-tumor Effect of P. amarus --- p.35 / Chapter 1.5.5 --- Active component(s) of P. am arus --- p.35 / Chapter 1.6 --- Alexander cells --- p.37 / Chapter 1.7 --- Objectives --- p.38 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Phyllanthus urinaria --- p.39 / Chapter 2.2 --- In vitro study --- p.39 / Chapter 2.2.1 --- Materials --- p.39 / Chapter 2.2.1.1 --- Cell Lines --- p.39 / Chapter 2.2.2 --- Reagents and Buffers --- p.40 / Chapter 2.2.2.1 --- Phosphate Buffered Saline (PBS) --- p.40 / Chapter 2.2.2.2 --- Reagents and Buffers for mRNA Genes Expression Analyses --- p.40 / Chapter 2.2.2.3 --- Reagents and Buffers for Protein Analyses --- p.41 / Chapter 2.2.2.4 --- Reagents for Purification of Splenocytes --- p.43 / Chapter 2.2.3 --- Methods --- p.43 / Chapter 2.2.3.1 --- MTT Assay --- p.43 / Chapter 2.2.3.2 --- IMX Assay --- p.43 / Chapter 2.2.3.3 --- Semi-quantitative RT-PCR --- p.44 / Chapter 2.2.3.3.1 --- Extraction of RNA --- p.44 / Chapter 2.2.3.3.2 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.45 / Chapter 2.2.3.3.3 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.3.4 --- Protein Analysis --- p.48 / Chapter 2.2.3.4.1 --- Extraction of Protein --- p.48 / Chapter 2.2.3.4.2 --- Determination of Protein Concentrations --- p.48 / Chapter 2.2.3.4.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.49 / Chapter 2.2.3.4.4 --- Electroblotting of Protein --- p.50 / Chapter 2.2.3.4.5 --- Immunoblotting of Protein --- p.50 / Chapter 2.2.3.4.6 --- Enhanced Chemiluminescence (ECL) Assay --- p.51 / Chapter 2.2.3.5 --- Assay of preSl Promoter Activity --- p.51 / Chapter 2.2.3.5.1 --- Cloning of preSl Promoter from HBV Genome --- p.51 / Chapter 2.2.3.5.2 --- Transfection --- p.52 / Chapter 2.2.3.5.3 --- Luciferase Assay --- p.53 / Chapter 2.2.3.6 --- Pilot Experiments for the Investigation of Immunostimulatory Effect of (aq) P. urinaria --- p.53 / Chapter 2.2.3.6.1 --- Purification of Splenocytes --- p.53 / Chapter 2.2.3.6.2 --- Cytotoxicity Assay of Splenocytes --- p.54 / Chapter 2.2.3.6.3 --- [3Hl-thymidine Uptake Assay --- p.54 / Chapter 2.2.3.6.4 --- Purification of Macrophages --- p.55 / Chapter 2.3 --- In vivo Assay --- p.56 / Chapter 2.3.1 --- Materials --- p.56 / Chapter 2.3.1.1 --- Balb/c Mice --- p.56 / Chapter 2.3.1.2 --- Reagents for Cytokine Assay --- p.56 / Chapter 2.3.1.3 --- Reagents for Flow Cytometric Analysis of T Cell Subpopulations --- p.57 / Chapter 2.3.2 --- Methods --- p.57 / Chapter 2.3.2.1 --- Purification of Splenocytes --- p.57 / Chapter 2.3.2.2 --- Enzyme Assay --- p.57 / Chapter 2.3.2.3 --- Flow Cytometric Analysis of T Cell Subpopulations --- p.59 / Chapter 2.3.2.4 --- Cytokine Assays --- p.60 / Chapter 2.4 --- Statistical Analysis --- p.63 / Chapter Chapter 3 --- Results / Chapter 3.1 --- In vitro Assays --- p.64 / Chapter 3.1.1 --- Study of the Cytotoxicity of Crude Extract of (aq) P. urinaria on Alexander Cells by the MTT Assay --- p.64 / Chapter 3.1.2 --- Study of the Cytotoxicity of Crude Extract of (aq) P. urinaria on WRL 68 Cells by the MTT Assay --- p.66 / Chapter 3.1.3 --- Study of the Cytotoxicity of Crude Extract of (aq) P. urinaria on Primary Mouse Macrophages by the MTT Assay --- p.67 / Chapter 3.1.4 --- Effect on HBsAg Secretion by the IMX Assay --- p.69 / Chapter 3.1.5 --- Effect of Viral Gene Expression by Semi-quantitative RT-PCR --- p.73 / Chapter 3.1.6 --- Effect of Intracellular Viral Proteins Expression by Western Blotting Analyses --- p.75 / Chapter 3.1.7 --- preSl Promoter Activity in Alexander Cell Line and Hep3B Cell Line --- p.77 / Chapter 3.1.8 --- Effect of (aq) P. urinaria on preSl Promoter Activity in Hep3B Cell Line --- p.80 / Chapter 3.1.9 --- Pilot Experiments for the Investigation of Immunomodulatory Effect of (aq) P. urinaria --- p.82 / Chapter 3.1.10 --- Study of the Cytotoxicity of Crude Extract of (aq) P. urinaria on Primary Splenocytes by the MTT Assay --- p.85 / Chapter 3.2 --- In vivo Assay --- p.88 / Chapter 3.2.1 --- Study of the Immunomodulatory Effect of the Crude Extract of (aq) P. urinaria on Normal Balb/c Mice by [3H]-thymidine Uptake Assay --- p.88 / Chapter 3.2.2 --- Study of the Toxicity of Crude Extract of (aq) P. urinaria on Heart and Liver of Normal Balb/c Mice by Enzyme Assay --- p.91 / Chapter 3.2.3 --- Flow Cytometric Analysis of T Cell Subpopulations --- p.94 / Chapter 3.2.4 --- Analysis of Stimulatory Effect on Four Different Cytokines by ELISA --- p.100 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- In vitro Assay --- p.110 / Chapter 4.2 --- In vivo Assay --- p.116 / Chapter 4.3 --- Conclusions --- p.127 / Chapter 4.4 --- Future Prospects --- p.129 / References --- p.130

Thesis (MPH))University of Limpopo (Medunsa Campus), 2011. / Introduction: Hepatitis B virus (HBV) is a highly infectious virus responsible for considerable morbidity and mortality world wide. Chronic HBV carriers can transmit HBV parenterally in a hospital setting putting healthcare workers (HCWs) and their patients at risk of infection. Aim and objectives: This study aimed to investigate knowledge, attitudes and practices towards prevention and control of HBV amongst nurses, doctors and laboratory personnel. Objectives were to determine: (a) the knowledge; (b) the attitudes; (c) the practices of nurses, doctors and laboratory personnel; (d) if there are any associations between (1) knowledge and practice, and (2) attitudes and practice; (e) the predictors of HBV vaccination uptake. Materials and Methods: This was a cross-sectional descriptive study. Self-administered questionnaires were distributed to doctors, laboratory staff and nurses at Princess Marina Hospital. Results: Two hundred questionnaires were distributed and a total of 117 were returned, giving an overall response rate of 58.5%. More doctors had good knowledge (38.9% [7/18]), followed by 20% (4/20) of laboratory staff and 11.4% (9/79) of nurses. Most staff (100% [20/20] of laboratory staff; 97.5% [77/79] of nurses; 94.4% [17/18] of doctors) had positive attitudes. More laboratory staff (100 [20/20]) displayed good practices, followed by nurses (94.9% [75/79]); and lastly doctors (88.9% [16/18]). There were no significant associations between knowledge or attitudes and practices. Vaccination was inadequate, with 50.9% (59/116) of HCWs having received at least one dose, and of these only 61% (36/59) receiving all 3 doses. Needle stick injuries occurred in 31.6% (37/117), while 33.9% (39/115) reported blood or body fluid splashes. None of the HCWs accessed PEP after exposure. Being a laboratory worker (OR: 148.4) or doctor (OR: 125.7) were the only predictors of vaccination uptake. Conclusion: There is need to increase knowledge of HCWs, vaccination availability, vaccination uptake, PEP, and reduce the exposures of HCWs.

This thesis investigated how virologic and serologic kinetics of the hepatitis B virus (HBV) could influence the natural history and treatment of chronic hepatitis B (CHB). Virologic kinetics were described in the first five studies, with serologic kinetics being described in the next five. The first study delineated the HBV DNA profiles of 1,400 treatment-naive Asian CHB patients. Increasing viremia was noted with increasing age, highlighting the large therapeutic demand in Asian patients with hepatitis B e antigen (HBeAg)-negative CHB. The second study analyzed the association between viral load and liver histology in 319 patients, showing HBV DNA levels to have strong association with HBeAg-negative disease severity. The next three studies investigated the efficacy of baseline and on-treatment HBV DNA levels in predicting clinical outcomes in 117, 165 and 222 patients on telbivudine, lamivudine plus adefovir and entecavir respectively. Absolute on-treatment HBV DNA levels at week 12 or 24 predicted favorable outcome with telbivudine and lamivudine / adefovir therapy, while excellent viremic suppression with very low rate of resistance development was shown in the entecavir study. The following three studies examined the role of serum HBsAg measurements in different disease phases of CHB. First, histology specimens of 140 HBeAg-positive patients were analyzed together with HBsAg levels. High HBsAg titers (>25,000 IU/mL) were found to be predictive of insignificant fibrosis. In the next study involving 300 treatment-naive HBeAg-negative patients stratified by their viral loads, combination of low HBsAg and HBV DNA levels predicted significant HBsAg decline. This is followed by a study comparing HBsAg levels of 203 CHB patients achieving HBsAg seroclearance with 203 age- and sex-matched controls over a 3-year period. Serum HBsAg <200 IU/mL and a significant annual HBsAg reduction were found to be predictive of HBsAg seroclearance. The penultimate study investigated the usage of two novel HBV serologic markers, linearized HBsAg and hepatitis B core-related antigen, in 329 CHB patients achieving HBsAg seroclearance with a conventional HBsAg assay. More than 40% of patients had seropositivity to one or both serologic tests. Finally, the last study of this thesis investigated and compared the changes in serum HBsAg, intrahepatic HBV DNA and covalently closed circular DNA (cccDNA) after 1 year of nucleoside analogue therapy. Minimal changes in both serum HBsAg and intrahepatic cccDNA were noted after 1 year of therapy, but in patients with a significant decline in serum HBsAg levels, there was a corresponding significant reduction in cccDNA. This series of studies illustrated how the monitoring of serum HBV DNA and HBsAg levels could assist in optimizing management strategies for CHB. / published_or_final_version / Medicine / Master / Doctor of Medicine