As toxinas LT são expressas por linhagens de Escherichia coli enterotoxigênicas e possuem marcantes efeitos adjuvantes. No entanto, não se conhece se a exposição prévia à toxina afeta sua atividade adjuvante durante imunizações subsequentes. Assim, o presente estudo avaliou o impacto da imunidade pré-existente nas propriedades inflamatórias e adjuvantes da LT quando inoculada pela via subcutânea. Como antígeno modelo, empregou-se a proteína NS1 do vírus dengue e duas abordagens experimentais distintas: (i) incubação in vitro de LT com anticorpos anti-LT e com o receptor gangliosídio (GM1) antes da administração em camundongos não imunizados em conjunto com a proteína NS1 do vírus dengue; (ii) imunização com NS1 coadministrada à LT em camundongos previamente expostos à LT. Foram avaliados os efeitos inflamatórios locais e os efeitos adjuvantes por meio da resposta imunológica humoral anti-NS1. Nossos resultados indicam que a imunidade prévia contra a LT não afeta seu potencial inflamatório e atividade adjuvante. Além disto, a exposição ao receptor GM1 reduziu as reações inflamatórias locais sem, no entanto, reduzir os efeitos adjuvantes de LT. / LT toxins are expressed by enterotoxigenic Escherichia coli strains and display strong adjuvant effects. Nonetheless, the impact of preexisting immunity on the on LT adjuvant activities is still unknown. Thus, the present study evaluated the impact of pre-existing immunity in the inflammatory and adjuvant properties of LT after subcutaneous administration. the NS1 of dengue virus was employed as a model antigen and two experimental approaches were evaluated: (i) in vitro incubation of LT with LT-specific antibodies and the ganglyoside receptor (GM1) before administration to naïve mice in combination with NS1; (ii) immunization with NS1 co-administered with LT in mice previously exposed to LT. The local inflammatory effects induced by LT were evaluated as wel as the adjuvant effects by means of NS1-specific humoral response. Our results indicate that the LT pre-existing immunity does not affect the inflammatory and adjuvant activities of the toxin. In addition, exposure to GM1 reduced the local inflammatory reactions without affecting the toxin adjuvant effects.

C. perfringens é uma bactéria anaeróbia presente no intestino delgado do homem e animais em equilíbrio e, sob a ação de alguns fatores predisponentes como mudança brusca de alimentação ou super alimentação, stress no manejo ou alto parasitismo intestinal, há a proliferação do microrganismo com a consequente produção de potentes toxinas que provocam a morte do animal. Dentre as toxinas principais destaca-se a toxina alfa, importante fator de virulência, produzida por todos os tipos de C. perfringens, sendo os pertencentes ao tipo A os maiores produtores. A fim de caracterizar o microrganismo em suspeitas de enterotoxemia em ruminantes, trabalhamos com 61 amostras de intestino delgado de bovinos e 12 de ovinos como grupo estudo e no grupo controle composto de animais hígidos levados ao abate, 73 amostras de intestino delgado de bovinos e 24 de ovinos. Foram realizados procedimentos de isolamento e tipagem molecular de C. perfringens e quantificação celular, detecção molecular da toxina &beta;2, além de avaliações moleculares qualitativa (PCR convencional) e quantitativa (PCR em tempo real) do gene da toxina alfa dos diferentes isolados. Em 29 amostras do grupo estudo bovino (47,54%) e em 4 (33,33%) do grupo estudo ovino isolou-se o microrganismo, em contrapartida no grupo controle bovino não houve isolamento do bacilo e 5 amostras do grupo controle ovino (20,83%) foram positivas. Houve diferença estatisticamente significante somente entre os grupos de bovinos (p<0,05). Todos os isolados (100%) foram classificados como tipo A, e os resultados das quantificações celulares de C. perfringens revelaram que todos os bovinos controle apresentaram <10 UFC/g de conteúdo enquanto que o grupo estudo apresentou mediana de 104 UFC/g com variações de <10 UFC/g até 108 UFC/g. Nos ovinos, a mediana no grupo controle foi 101 UFC/g assim como no grupo estudo, entretanto com clara separação de valores entre os grupos. Tanto na PCR convencional quanto na PCR em tempo real para detecção do RNAm da toxina alfa foi observado limiar de detecção de 102 cópias de cDNA por reação, porém provavelmente devido aos valores das amostras estarem próximos ao limite da sensibilidade analítica da reação, não foi observada boa reprodutibilidade da última. Já na reação molecular convencional, observou-se a presença de detecção de RNAm da toxina alfa em 60,52% dos isolados o que revela alguma diferença da presença do transcrito entre as culturas, já que nas cepas restantes não foi detectada a presença do RNAm em questão. A pesquisa do gene da toxina &beta;2 revelou sua presença em 54,55% dos isolados de C. perfringens corroborando com a afirmativa de que o gene está amplamente distribuído entre os ruminantes. A metodologia aplicada para avaliação da expressão do gene da toxina alfa nos isolados mostrou que há diferenças dos níveis de transcrição porém não permitiu quantificar esses valores. A tipagem molecular corrobora com outros estudos quanto à importância epidemiológica do tipo A nos quadros de enterotoxemia em ruminantes, e os dados da quantificação celular permite-nos concluir que animais sadios possuem um nível basal de C. perfringens <10 UFC/g de conteúdo que não possibilita o seu isolamento. / C. perfringens is an anaerobe present in small intestine of man and animals in equilibrium, and under some predisposing factors such as sudden feeding change or super feeding, rough management or high intestinal parasitism, the microorganism multiplies with the consequent production of potent toxins that can cause animal death. Amongst the main toxins, alpha toxin is an important virulence factor, that is produced by all C. perfringens types, and those belonging to type A are its higher producer. Aiming to characterize the microorganism in ruminants suspect of enterotoxaemia, we evaluated 61 bovine small intestinal samples and 12 sheep small intestines as the study group, and for the control group composed by higid animals led to slaughterhousing, 73 bovine small intestines and 24 ovine samples. We performed microbiological culture and molecular typing of C. perfringens isolates, cellular quantification, molecular detection of 2 toxin, and qualitative and quantitative molecular evaluations of alpha toxin from different isolates by means of conventional PCR and real time PCR, respectively. In 29 samples from the bovine study group (47.54%) and in 4 (33.33%) from ovine study group the microorganism was isolated, however in the bovine control group there was no isolation success and 5 samples from sheep control group (20.83%) were positive. There was statistically significant difference only between bovine groups (p<0,05). All isolates (100%) were classified as type A, and C. perfringens cellular quantification results showed that every control bovine presented <10 CFU/g of intestinal contents while the study group presented a median of 104 CFU/g with results ranging from <10 CFU/g to 108 CFU/g. In sheep, the median value in the control group was 101 CFU/g as in the study group, but with a clear division of values between the groups. We observed the threshold detection of 102 cDNA copies per reaction in both conventional and real time PCR reactions for alpha toxin mRNA detection, however since the samples quantification values were close to the analytical sensitivity of the test, we could not observe the reproducibility in the last technique. In the conventional PCR reaction, alpha toxin mRNA was detected in 60.52% of the isolates. This result reveals some difference in the transcript presence among the cultures, since we could not detect the presence of the described mRNA in the other isolates. Beta2 toxin gene was detected in 54.55% of C. perfringens isolates corroborating with the affirmative that this gene is widely distributed among ruminants. The methodology presented herein for the evaluation of alpha toxin gene expression showed that there are differences in the transcription levels, however it didnt allow to quantify these values. Molecular typing results agree with other studies regarding the epidemiological importance of type A in the enterotoxaemia processes in ruminants, and the cellular quantification data allow us to conclude that healthy animals show a basal level of C. perfringens <10 CFU/g of intestinal content that doesnt allow its isolation.

Os objetivos deste estudo foram: caracterizar, comparar e correlacionar, em indivíduos com distonia laríngea, antes e após 30 e 120 dias da aplicação da toxina botulínica tipo A no músculo tireoaritenoideo unilateralmente, os parâmetros vocais perceptivo-auditivos e visuais e o impacto vocal na qualidade de vida. Participaram do estudo 16 indivíduos com diagnóstico de distonia laríngea, sendo 11 de gênero feminino e cinco de gênero masculino, com idade média de 57 anos e dois meses, sendo que 68,75% deles apresentaram tremor vocal associado. Todos foram submetidos à aplicação da toxina botulínica tipo A (Dysport®), no músculo tireoaritenoideo unilateralmente, guiado por eletromiografia, gravação do sinal vocal e preenchimento do protocolo de qualidade de vida, Índice de Desvantagem Vocal, antes da injeção da toxina botulínica, no primeiro retorno de seguimento, em média, após 36 dias, e no segundo retorno, coincidente com o dia da nova aplicação de toxina botulínica, que foi em média, 137 dias. Os resultados para análise perceptivo-auditiva e visual de sinais e sintomas vocais, realizada por dois juízes especialistas em voz, revelou, na comparação entre a pré-aplicação e o primeiro retorno, diminuição da severidade da oscilação de intensidade, quebras de voz (sonoridade) e tremor vocal (p=0,002), portanto sem alterações no segundo retorno. No grau de severidade da qualidade da voz, voz tensa-estrangulada, rugosidade, soprosidade e astenia não foram observadas alterações nos três tempos. Para esta análise, notou-se concordância excelente e muito boa (0,71 a 0,99) intrajuízes. Na comparação entre a pré-aplicação e o primeiro retorno, para o Índice de desvantagem vocal, verificou-se diminuição dos valores para o escore total (p=0,039) e para o domínio emocional (p=0,035). Quanto à comparação entre a pré-aplicação e segundo o retorno, houve redução significativa para o domínio funcional (p=0,03) unicamente. De modo que, entre a análise perceptivo-auditiva e visual, do grau de severidade da qualidade vocal e o escore total do índice de desvantagem vocal, evidenciou-se correlação moderada na pré-aplicação e segundo retorno e fraca no primeiro retorno. Portanto, concluiu-se que o tratamento com toxina botulínica para os indivíduos com distonia laríngea mostrou-se eficiente na diminuição de sinais e sintomas vocais, bem como no impacto vocal na qualidade de vida, gerando mudanças na função fonatória e facilitando a comunicação após 30 dias da aplicação, com retorno dos sintomas vocais para a maioria dos indivíduos após 120 dias. / The purpose of this study was to characterize, compare, and correlate the laryngeal dystonia in individuals before, after 30, and 120 days after the application of botulinum toxin type A in the unilateral thyroarytenoid muscle the perceptual vocal and visual parameters and the vocal impact in quality of life. 16 individuals with diagnosed laryngeal dystonia participated in the study, 11 being of the female gender and five of the male gender, with an average age of 57 years and two months, being that 68.75% of individuals having presented associated vocal tremor. All individuals were subjected to: the application of botulinum toxin type A (Dysport®) via electromyography in the unilateral thyroarytenoid muscle, a recording of the vocal sound waves, filling out a survey about his/her quality of life and an Voice Handicap Index (VHI) before the botulinum toxin injection, in the first follow-up on average 36 days after, and in the second follow-up concurrent with the new botulinum toxin application visit, which was on average after 137 days. The results for the perceptual auditory and visual analysis of vocal sound waves and symptoms, conducted by two adjudicating voice specialists, revealed in the comparison between the pre-application and the first follow-up a decrease in the severity of the oscillation intensity, voice breaks (sonority), and vocal tremor (p=0.002), however, revealed no changes in the second follow-up. During the three visits, no changes were observed in grading the severity of voice quality in tense-strangled voice, roughness, breathiness and asthenia. For this analysis, a very good and excellent agreement between the adjudicators was noted (0.71 to 0.99). Comparing the pre-application and the first follow-up, for the index of vocal disability, a decrease in the values of total score (p=0.039) and of emotional mastery (p=0.035) was observed, whereas comparing the pre-application and the second follow-up, there was a significant decrease only for functional mastery (p=0.03). In this way, between perceptual auditory and visual analysis, grading the severity of vocal quality and the total score of the Voice Handicap Index, a moderate correlation was evident in the pre-application and second follow-up and a faint correlation was evident in the first follow-up. Therefore, it can be concluded that the treatment with botulinum toxin for individuals with laryngeal distonia who participated in this study proved effective in the decrease of symptoms, as well as in a vocal impact of quality of life which created changes in the phonatory function and facilitated communication after 30 days of the application, with a return of vocal symptoms for the majority of individuals after 120 days.

EPEC atípicas (EPECa) são isoladas de surtos diarréicos em crianças, adultos e animais em países industrializados e em desenvolvimento. Hemolisinas são toxinas que atuam sobre a membrana de eritrócitos e de diversos tipos celulares. Tal grupo de toxinas é reconhecido como importante fator de virulência envolvido na patogênese bacteriana, por isso o objetivo foi avaliar a expressão de enterohemolisina em isolados de EPECa e analisar características fenotípicas como interferência do meio de cultivo e adesão a componentes de matriz extracelular. Observou-se prevalência de 5,4% de isolados enterohemolíticos, e variações na expressão da toxina foram associadas à concentrações de lactose e caseína nos meios de cultivo. Além disso, verificou-se forte associação entre produção de enterohemolisina e ligação à fibronectina celular. / Atypical EPEC (aEPEC) are isolated from diarrheal outbreaks in children, adults and animals in industrialized and developing countries. Hemolysins are toxins that act on the red cell membrane and various cell types. This group of toxins is recognized as an important virulence factor involved in the bacterial pathogenesis. The aim of this study was the evaluation of the enterohemolysin expression in aEPEC isolates as interference of the culture media and adhesion to components of extracellular matrix. The prevalence of enterohemolytic isolates was 5.4%, and variations in toxin expression were associated with lactose and casein concentrations in culture media. Moreover, it was found association between production of enterohemolysin and adhesion to cellular fibronectin.

INTRODUÇÃO: O blefaroespasmo essencial é uma distonia focal caracterizada por contrações involuntárias, espasmódicas e bilaterais dos músculos protratores das pálpebras. O glaucoma é a principal causa de cegueira irreversível em adultos de países desenvolvidos e a elevação da pressão intra-ocular é o maior fator de risco para o desenvolvimento da doença. A contração voluntária forçada das pálpebras pode causar aumentos da pressão intra-ocular de até 90 mmHg. Uma vez que pacientes com blefaroespasmo essencial apresentam contrações palpebrais freqüentes e de forte intensidade, eles poderiam compor um grupo de risco para o desenvolvimento do glaucoma. MÉTODOS: Vinte e oito pacientes com blefaroespasmo essencial e 28 pacientes de grupo controle, formado por indivíduos com doenças palpebrais ou no seu pós-operatório, foram submetidos a um exame oftalmológico completo. No grupo de pacientes com blefaroespasmo, a avaliação foi realizada em 8 a 11 dias após o tratamento com toxina botulínica. Efetuou-se exame de refração e medida de melhor acuidade visual corrigida com tabela de Snellen, biomicroscopia em lâmpada de fenda, tonometria de aplanação, campo visual computadorizado, teste de sobrecarga hídrica e biomicroscopia de fundo para avaliação da papila óptica. RESULTADOS: 1) A prevalência de glaucoma nos pacientes com blefaroespasmo foi significativamente maior do que nos indivíduos do grupo controle, sendo diagnosticada em 14,3% e 3,6% dos casos, respectivamente (p=0,008). 2) A pressão intra-ocular foi significativamente maior nos pacientes com blefaroespasmo (15,80 ± 3,80 mmHg) do que nos indivíduos do grupo controle (13,90 ± 2,75 mmHg) (p= 0,004). 3) O pico da pressão intra-ocular na prova de sobrecarga hídrica foi significativamente maior nos pacientes com blefaroespasmo essencial (18,82 ± 4,47 mmHg) do que nos indivíduos do grupo controle (16,27 ± 2,69 mmHg) (p=0,0421). DISCUSSÃO: Diversos estudos descreveram a influência da compressão palpebral sobre a pressão intra-ocular. A prova de sobrecarga hídrica tem sido considerada uma ferramenta indireta para se avaliar a capacidade do fluxo de drenagem do trabeculado, além de exibir correlação com os picos de pressão intra-ocular que muitas vezes não são detectados em exames de rotina. Os pacientes com blefaroespasmo essencial apresentaram pressões intra -oculares e picos de pressão intra-ocular na prova de sobrecarga hídrica mais elevados do que os pacientes do grupo controle, o que poderia indicar um baixo fluxo de drenagem. Estas altíssimas variações de pressão intra -ocular a que estes indivíduos são submetidos constantemente, em pacientes com menor fluxo de drenagem poderiam causar um aumento crônico da pressão intra -ocular e o desenvolvimento do glaucoma. CONCLUSÕES: Os resultados observados sugerem que estes pacientes compõem um grupo de risco para o desenvolvimento de glaucoma e esta doença deve ser pesquisada de maneira sistemática na avaliação inicial e durante o seguimento desses casos / INTRODUCTION: Essential blepharospasm is a focal distonia characterised by involuntary, spasmodic, bilateral contractions of eyelid protractors. Glaucoma is the most important cause of irreversible blindness in adults in developed countries and high intraocular pressure is the major risk factor for development of the disease. Voluntary forced eyelid closure can produce an intraocular pressure increase of 90 mmHg. As patients with essential blepharospasm present frequent and strong eyelid contractions, they could be at risk for glaucoma development. METHODS: Twenty eight patients with essential blepharospasm and 28 patients of a control group, with eyelid diseases or in the post-operative period, were submitted to a complete ophthalmic examination. In the group of patiens with blepharospasm, the evaluation was done between 8 to 11 days after botulinum toxin treatment. We performed refractometry and best corrected visual acuity with Snellen chart, slitlamp biomicroscopy, applanation tonometry, automated perimetry, water drinking test and dilated funduscopy to evaluate optic discs. RESULTS: 1) Prevalence of glaucoma in patients with blepharospasm (14,3%) was higher than in the individuals from control group (3,6%) (p=0,008). 2) Intraocular pressure was higher in patients with blepharospasm (15,80 ± 3,80 mmHg) than in the individuals from control group (13,90 ± 2,75 mmHg) (p=0,004). 3) Intraocular pressure peaks in the water drinking test were higher in patients with essential blepharospasm (18,82 ± 4,47 mmHg) than in the individuals from control group (16,27 ± 2,69 mmHg) (p=0,0421). DISCUSSION: Several studies reported the influence of eyelid compression over intraocular pressure. The water drinking test has been considered as an indirect tool to measure outflow facility and it has good correlation with intraocular pressure peaks which are frequently missed in routine examinations. Patients with essential blepharospasm had higher mean intraocular pressures and intraocular pressure peaks in the water drinking test than patients from control group, which could indicate low outflow facility. These high intraocular pressure variations in patients with low outflow facility could increase the intraocular pressure chronically and lead to development of glaucoma. CONCLUSION: Our results suggest that patients with essential blepharospasm be at higher risks for development of glaucoma and this disease should be always investigated at presentation and during follow-up of these cases

Shiga toxin-producing E. coli (STEC) are globally relevant bacterial pathogens responsible for epidemic outbreaks of hemorrhagic diarrhea with variable progression to potentially fatal systemic Shiga toxicosis. Predictive clinical biomarkers and targeted therapeutic interventions for systemic Shiga toxicosis in diagnosed STEC patients are not available, and the impact of Shiga toxin production on STEC colonization and survival remain unclear. Improved murine models of STEC infection are needed to address knowledge gaps surrounding the gastrointestinal effects of Shiga toxins, as previously published models utilize ablation of host defense responses or microbiota depletion to facilitate colonization and are poorly suited for study of the effects of Shiga toxins on host responses. Dextran sulfate sodium (DSS) colitis in rodents has been associated with outgrowths of commensal E. coli in the literature, suggesting that DSS colitis could open a gastrointestinal niche usable by pathogenic STEC. This DSS colitis-based approach successfully induced susceptibility to robust colonization by two clinical isolate STEC strains in standard C57BL/6 mice. Studies using a Shiga-like toxin 2 (STX2)-producing clinical isolate STEC strain and its paired isogenic STX2 deletion strain (STEC(ΔSTX2)) revealed that STX2 was associated with delayed gastrointestinal clearance of STEC and concurrent reduction in colonic interleukin 23 (IL-23) axis transcripts known to be critical for pathogen clearance in other gastrointestinal pathogen models. In vivo reductions in IL-23 axis transcripts in the DSS+STEC model were supported by decreased IL-23 protein secretion by human macrophage-like cells during Shiga intoxication in vitro. Increased morbidity during STX2-producing STEC infection was associated with renal injury consistent with murine systemic Shiga toxicosis characterized by elevations in renal transcripts of molecular injury markers and histologically apparent renal tubular injury in a subset of mice. The dissertation research establishes a novel model of DSS colitis-facilitated murine STEC infection that recapitulates progression to systemic Shiga toxicosis in a subset of infected mice and demonstrates a clear STEC survival benefit associated with STX2 production. Shiga toxin-induced suppression of IL-23 axis signaling is a novel finding facilitated by the DSS+STEC model, demonstrating its utility for future delineation of the impacts of Shiga toxins on gastrointestinal host responses to STEC.

Human cytomegalovirus (HCMV) is a betaherpesvirus which establishes a lifelong persistent infection, underpinned by its ability to establish latent infection in early myeloid lineage cells, in the infected host. Although well controlled by a healthy immune system, HCMV causes pathological and life threatening disease in individuals with a compromised or immature immune response, which can come from primary HCMV infection or reactivation of latent infection. Although progress is being made in understanding the mechanisms by which HCMV maintains latency and reactivates, a better understanding is essential towards the aim of targeting and killing latently infected cells. In this thesis, I will present evidence that the HCMV-encoded chemokine receptor homologue US28, which is expressed during latent infection of CD14+ monocytes, is necessary for maintaining HCMV latency in these monocytes and, in the absence of US28 protein expression, HCMV undergoes lytic infection. US28 expression was found to attenuate cellular signalling pathways in latently infected cells; in particular, MAP kinase and NFκB. Interestingly, deletion of the US28 gene or inhibition of the US28 protein resulted in the expression of lytic antigens which allowed detection of infected monocytes by the immune system. This observation may lead to a potential new immunotherapeutic strategy against latent HCMV. Having demonstrated that US28 protein is expressed on the surface of latently infected monocytes, I tested whether a new fusion-toxin protein, called F49A-FTP, which binds US28 protein, could be used to target and kill latently infected cells. I developed a protocol for treating latently infected monocytes with F49A-FTP which resulted in a significant reduction in virus reactivation after monocyte differentiation to dendritic cells. I was also able to show that this treatment kills CD34+ progenitor cells, which were experimentally latently infected with HCMV, as well as latently infected monocytes from a healthy, seropositive blood donor. Finally, during my investigations into the role of US28 during HCMV latency, a mass spectrometry screen was performed to measure changes in cellular protein expression when US28 protein is expressed in isolation, in THP-1 monocyte-like cell line. This identified CTCF, a transcription factor which appears to be modified by US28 in THP-1 cells. I showed that CTCF has a repressive effect on the HCMV MIEP, and that CTCF likely plays a role in HCMV latency. In summary, this work provides insights into the role of US28 during HCMV latency, and proposes potential novel therapeutic strategies to kill latently infected cells.

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont responsables d'infections humaines, allant d'une diarrhée aqueuse bénigne pouvant se compliquer en syndrome hémolytique et urémique (SHU), parfois mortel. La transmission de STEC à l'Homme s'effectue principalement par l'ingestion d'aliments contaminés. Le principal facteur de virulence des STEC est le gène stx (codant la Shiga-toxine), localisé dans le génome d'un prophage. La thèse a été centrée sur les STEC O26:H11, deuxième sérotype à l'origine de SHU dans le monde, et premier retrouvé dans les fromages au lait cru.Le premier objectif était de caractériser génétiquement les STEC O26:H11 et leurs phages Stx (variants du gène stx et sites d'insertion des phages Stx) afin d'identifier d'éventuelles différences entre ces souches selon leur origine. La majorité des souches alimentaires et bovines étudiées possèdent le variant stx1a et leurs phages Stx sont intégrés dans wrbA et yehV. Les souches humaines possèdent les variants stx1a et stx2a en proportions équivalentes. Leurs phages Stx sont aussi intégrés dans wrbA et yehV mais à la différence des souches alimentaires et bovines, le site yecE a été identifié comme site d'insertion. Toutes les souches humaines qui possédaient un phage Stx2a intégré dans wrbA et yecE ont causé des SHU ; ce qui pourrait être un indicateur de haute virulence. Des études ont montré que des souches E. coli Attachant/Effaçant (AEEC) O26:H11 sont isolées à partir d'aliments identifiés comme « stx+ » par PCR. En dehors de l'absence du gène stx, ces souches AEEC sont similaires aux STEC. Leur caractérisation a montré ici que la majorité d'entre elles ont leurs sites d'insertion intacts, caractéristique compatible avec une perte de phage Stx par excision spontanée. La stabilité des phages Stx a donc été évaluée chez les STEC O26:H11. La présence/absence de phages Stx a été quantifiée par PCRq pour chaque souche au niveau de la population bactérienne entière montrant que les STEC ont la capacité de perdre leurs phages Stx. En revanche, cette instabilité n'est pas liée aux sites d'insertion. Plusieurs essais visant à introduire des phages Stx dans les souches AEEC pour les convertir en STEC ont été réalisés mais les résultats ont montré qu'il était difficile d'infecter ces souches. L'étude du taux d'induction des phages Stx in vitro chez les STEC O26:H11 a montré, qu'en présence de mitomycine C, les phages Stx2 étaient plus inductibles que les phages Stx1. En revanche, il n'a pas été mis en évidence de différences en fonction de l'origine des souches testées et du site d'intégration des phages Stx. L'analyse morphologique de quelques phages Stx a montré que le type Stx1 ou Stx2 n'était pas lié à une forme spécifique de phage. L'étude des stress relatifs à la technologie fromagère a montré que le stress salin et le stress oxydatif, lié à la libération potentielle d'H2O2 par d'autres bactéries, entrainaient l'induction des phages Stx. Comme des souches AEEC sont fréquemment isolées à partir d'aliments qualifiés de « stx+» par PCR, le processus analytique d'isolement des STEC a aussi été étudié. La production de phages Stx lors de la phase d'enrichissement semble possible à partir d'un aliment contaminé. En revanche, aucun composant de cette méthode, testé individuellement, n'a pu être identifié comme inducteur des phages Stx. Ces travaux ont permis d'acquérir des connaissances sur la diversité des phages Stx issus de STEC O26:H11 isolées chez l'Homme et dans la filière laitière. Des différences au niveau des variants du gène stx mais aussi des sites d'insertion des phages Stx ont pu être observées en fonction de l'origine des souches. De plus les niveaux d'induction des phages Stx diffèrent selon le variant du gène stx. Ces différences pourraient refléter l'existence de clones distincts, aux potentiels de virulence différents, circulant dans les aliments, chez les bovins et les patients / Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild diarrhea to hemolytic-uremic syndrome (HUS), sometimes with fatal outcome. Transmission of STEC to humans occurs mainly through the ingestion of contaminated food. The main virulence factor of STEC is the stx gene (encodes Shiga-toxin) located in the genome of a prophage. The PhD thesis was focused on STEC O26:H11, which is the second serotype causing HUS in the world, and the first one found in raw milk cheeses. The first objective was to characterize genetically STEC O26:H11 strains and their Stx phages (stx gene subtypes and insertion sites of Stx phages) in order to identify any differences between these strains according to their origin. The majority of the investigated food and bovine strains possessed stx1a subtype and their Stx phages were integrated into wrbA and yehV. Human strains possessed the stx1a and stx2a subtypes in equivalent proportions. Their Stx phages were also integrated into wrbA and yehV but unlike food and bovine strains, yecE site were identified as insertion site. All the human strains carrying an Stx2a phage integrated into wrbA and yecE caused HUS, which could indicate a high virulence. Studies showed that Attaching/Effacing E. coli (AEEC) O26:H11 strains were isolated from foods identified as "stx +" by PCR. Except for the absence of stx gene, these AEEC strains are similar to STEC. Their characterization showed, in this study, that the majority of them had intact insertion sites, in agreement with a possible loss of Stx phage by spontaneous excision. The stability of Stx phages was evaluated in STEC O26:H11. Presence/absence of Stx phages was quantified for each strain in the total bacterial population, showing that STEC were capable of losing their Stx phages. However, this instability was not related to the insertion sites. Several attempts to introduce Stx phages in AEEC strains in order to convert them into STEC were conducted but the results showed that it was difficult to infect these strains. The induction rate of Stx phages in vitro in STEC O26:H11 showed that, with mitomycin C, the Stx2 phages were more inducible than Stx1 phages. However no difference was found with the origin of the strains tested and the Stx phage integration site used. The morphological analysis of some Stx phages showed that Stx1 or Stx2 type was not related to a specific phage shape. Study of various stress related to the cheese-making process showed that the osmotic and oxidative stress related to the potential release of H2O2 by other bacteria, led to the induction of Stx phages. Because AEEC strains are frequently isolated from food qualified as "stx+" by PCR, the analytical STEC isolation procedure was studied for its ability to induce Stx phages. Production of Stx phages during the enrichment phase seemed possible from contaminated food. However, none of the components of this method, tested individually, could be identified as an inducer of Stx phages. This work highlighted the diversity of Stx phages from STEC O26:H11 isolated from humans and dairy sector. Differences in stx subtypes and Stx phages insertion sites present among the STEC O26:H11 strains were observed depending on the origin of the strains. Moreover the induction levels of Stx phages differed according to the stx subtypes. These differences might reflect the existence of distinct clones, with varying virulence potential, circulating in foods, cattle and patients

As we learn more about immune cell subpopulations, we find an increasingly complex system of cells with diverse functions. L2pB1 cells are a PD-L2 positive B1a B lymphocyte subpopulation that has unusual properties and characteristics that are not fully understood by many. By creating and implementing a transgenic mouse model that allows for targeted depletion of this specific group of cells, we can further elucidate their physiological functions and roles in both healthy and diseased states. Here we demonstrate the depletion of L2pB1 cells utilizing a transgenic mouse model expressing Diphtheria Toxin Receptors on their surface. After a course of 4 injections of 25ng of Diphtheria Toxin per gram bodyweight, we observed a successful depletion of L2pB1 cell population. Further studies are underway investigating the effects of a high fat diet on these L2pB1 depleted mice. / 2017-04-01T00:00:00Z

Técnicas espectroscópicas vibracionais Raman e infravermelho foram utilizadas no estudo conformacional de algumas proteínas de interesse biológico, no que se refere a aspectos estruturais de seus esqueletos polipeptídicos e microambientes de cadeias laterais de certos resíduos de aminoácidos. O trabalho foi dividido em dois grupos, de acordo com os sistemas em estudo: a) Toxina &#947; do veneno de escorpião brasileiro Tityus serrulatus. A análise vibracional revelou que o esqueleto polipeptídico dessa proteína consiste de diferentes estruturas secundárias, com predominância de folhas &#946;, seguida por estruturas do tipo não regular e &#945;-hélice, com alguma evidência de dobras &#946;. Conformação gauche-gauche-gauche foi detectada para os segmentos CCSSCC das quatro pontes dissulfeto. A intensidade do dubleto Raman da Tyr a 853 e 828 cm-1 indicou que 4 dentre 5 resíduos de Tyr encontram-se expostos na superficie molecular. Também há indicações de que os 3 resíduos de Trp apresentem localização externa. Sob o ponto de vista qualitativo, as características conformacionais da toxina no estado sólido amorfo e em solução são virtualmente as mesmas. b) Fosfolipases A2 de pâncreas de porco e de Crotalus durissus terrificus. Mudanças confarmacionais foram detectadas para as moléculas de fosfolipase como consequência de diferentes condições experimentais tais como mudança de estado físico, presença de certas espécies iônicas e interação com um análogo de substrato e com o próprio substrato. Características conformacionais discrepantes foram observadas para a forma sólida amorfa e forma cristalina da fosfolipase pancreática. Transições conformacionais foram detectadas para a transformação zimogênio &#8594; fosfolipase A2 e diferentes conteúdos estruturais foram calculados para a forma tóxica e atóxica dessa enzima. Todas essas mudanças conformacionais envolveram basicamente a arquitetura do esqueleto polipeptídico, não afetando a conformação das cadeias laterais dos resíduos de amino ácidos. As pontes dissulfeto apresentaram consistentemente uma conformação ggg a qual não foi perturbada por nenhuma das condições experimentais empregadas. A ocorrência externa de resíduos de triptofano constituiu uma característica comum para os sistemas ensaiados, assim como a localização predominante de residuos de tirosina em microambientes hidrofílicos, provavelmente na superficie molecular. / Raman and infrared spectroscopies were used to investigate conformational features of some proteins of biological interest, in what concerns structural aspects of their polypeptide backbones and microenvironments of certains amino acid residue side-chains. The work has been divided in two groups as related to the systems studied: a) Toxin &#947; from the venom of Brazilian scorpion Tityus serrulatus. The vibrational analysis has revealed that the protein polypeptide backbone consists of different secondary structures, with predominance of &#946;-sheet, followed by unordered structure and &#945;-helix, with some evidence of &#946;-turns. A gauche-gauche-gauche (ggg) conformation for the CCSSCC fragments of the four dissulfide bridges has been detected. The intensity of the Tyr Raman doublet at 853 and 828 cm-1 indicated that 4 out of the 5 Tyr residues are exposed at the molecular surface. External localization of the 3 Trp residues has also been indicated. Under a qualitative point of view, conformational features of the toxin the amorphous solid state and in solution were virtually the same. B) Crotalus durissus terrificus and porcine pancreatic phospholipases A2. Conformational changes were detected for the phospholipases molecules as a consequence of different conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog and with the substrate itself. Amorphous and crystalline solid pancreatic phospholipases presented discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen &#8594; phospholipase A2 transformation and different secondary structures contents were observed for the toxic and the non toxic phospholipase melecules. All those structural changes heve been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a ggg conformation which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophylic environments, probably at the molecular surface.