Le disulfure de diméthyle (DMDS), molécule volatile libérée par les Allium, est un fumigant prometteur en remplacement du bromure de méthyle. Par l'utilisation des techniques d'électrophysiologie (patch-clamp), de biologie moléculaire et d'imagerie calcique sur neurones d'insectes, nous avons identifié de nouvelles cibles altérées par le DMDS à une faible concentration (1 µM). Le DMDS modifie l'activité spontanée régulière des DUM neurones en des bouffées de potentiels d'action séparées par des phases de silence. Cette altération de fréquence est la conséquence d'effets sur plusieurs cibles. Nous avons montré que le DMDS décale la dépendance vis-à-vis du potentiel de l'activation et de l'inactivation du courant Na2 vers de potentiels plus négatifs, rendant la cellule plus excitable. Dans un deuxième temps nous avons mis en évidence que le DMDS induit une augmentation de la concentration en calcium intracellulaire via l'activation des canaux TRPg et une sortie de calcium des stocks intracellulaires. Cette variation de calcium module, en cloche, les courants potassium dépendants du calcium (KCa). Grâce à l'étude du mode d'action du DMDS sur DUM neurones et au développement de nouvelles techniques associant la biologie moléculaire à l'électrophysiologie, nous avons apporté de nouveaux arguments en faveur de l'existence de deux courants KCa distincts. Parallèlement, grâce à une technique d'électroencéphalographie par télémétrie sur souris, nous avons révélé que le DMDS est susceptible d'engendrer des anomalies de l'activité électroencéphalographique.

Les protéines LIM constituent une famille de molécules régulatrices possédant dans leur séquence protéique un ou plusieurs motifs en doigts de zinc – les domaines LIM – dont la fonction principale est de diriger les interactions protéine-protéine. Les protéines LIM ont ainsi la capacité d'interagir avec de multiples partenaires protéiques, et participent à l'assemblage et au maintien de certains des complexes macromoléculaires présents au sein de la cellule. La plupart des protéines LIM ont été caractérisées dans les cellules animales, où elles sont impliquées dans l'organisation du cytosquelette d'actine, ainsi que dans la régulation de la transcription. A ce jour, seules quelques protéines LIM ont été décrites chez les végétaux. La fonction de ces protéines LIM végétales reste encore à identifier. Afin de préciser le rôle des protéines LIM chez le tournesol, nous avons choisi comme modèle expérimental le protoplaste d'hypocotyle de tournesol, en raison de la possibilité de moduler son développement en fonction des conditions de culture.<br />Nous avons étudié l'expression des gènes LIM précédemment décrits chez le tournesol dans les protoplastes par RT-PCR. Nous avons détecté un transcrit pour le gène HaWLIM-1, mais pas pour les deux autres gènes HaPLIM-1 et HaPLIM-2. Des anticorps polyclonaux spécifiques de la protéine HaWLIM-1 reconnaissent en immunoblot deux polypeptides distincts, dont les masses moléculaires sont respectivement de 52 kDa et 78 kDa. Ces masses moléculaires sont nettement supérieures à la taille attendue pour la protéine HaWLIM-1. Ces résultats indiquent que la protéine n'est pas présente sous forme de monomères dans les protoplastes, mais qu'elle participe à la formation de complexes protéiques stables.<br />L'étude par immunocytologie de la localisation intracellulaire de la protéine HaWLIM-1 révèle que cette protéine est présente simultanément dans deux compartiments distincts : le noyau et le cytoplasme. Dans le noyau, elle s'accumule préférentiellement dans le nucléole, et pourrait jouer un rôle dans la régulation de la transcription des gènes des ARNr, ou dans l'assemblage des ribosomes. Dans le cytoplasme, différentes approches, incluant des expériences de double-marquage et de déstructuration de composants du cytosquelette, ont permis de mettre en évidence une très forte colocalisation de la protéine HaWLIM-1 avec les microtubules, ce qui suggère un rôle pour cette protéine dans l'organisation du cytosquelette.<br />Lors de la culture des protoplastes, le gène HaWLIM-1 s'exprime constamment, avec cependant des variations dans le niveau d'expression. Des expériences d'immunoblot utilisant les anticorps spécifiques de la protéine HaWLIM-1 indiquent que de nouveaux polypeptides, de masses moléculaires égales à 35 kDa, 42 kDa et 64 kDa, apparaissent au cours de la culture. Cette observation suggère que la protéine HaWLIM-1 possède la capacité de s'associer et de se dissocier avec de nouveaux complexes protéiques au cours du développement. La protéine HaWLIM-1 est associée aux microtubules pendant tous les stades de la division : elle est présente au niveau de la bande préprophasique à la fin de l'interphase, au niveau du fuseau mitotique pendant la mitose, et au niveau du phragmoplaste pendant la cytokinèse. Ces observations semblent indiquer que la protéine HaWLIM-1 occupe une fonction importante au niveau des microtubules.

Dans le néocortex murin, la division des cellules précurseurs a lieu dès le stade E11 et donne naissance aux premiers neurones pionniers dont les cellules de Cajal-Retzius. L'activité électrique spontanée, portée par les canaux ioniques, joue un rôle prépondérant dans le développement du système nerveux central. Comprendre la place des canaux ioniques et de la signalisation calcique dans les phases précoces de le neurogenèse était l'objectif principal de mon projet. Nous avons montré l'apparition précoce de canaux sodiques dépendants du voltage dans 55% des cellules neuronales à E13, dont les cellules de Cajal-Retzius. En parallèle, nous avons observé des activités calciques spontanées dans les cellules proliférantes et neuronales au même stade. La conception d'un logiciel d'imagerie nous a permis d'analyser statistiquement ces activités et d'identifier les canaux ioniques impliqués. Alors que les synapses ne sont pas encore formées, nous avons observé la mise en place d'activités synchrones au sein du néocortex et démontré l'existence de communications paracrines entre les cellules. De plus, nous avons identifié l'existence d'une cascade de signalisation où la dépolarisation des récepteurs glycinergiques active les canaux sodiques présents sur les neurones pionniers. Dans ces neurones, l'influx sodique entraîne une augmentation de calcium cytoplasmique via un échangeur Na+/Ca2+ puis une exocytose glutamatergique dont le libération paracrine induit l'activation d'autres cellules néocorticales. L'utilisation de la culture organotypique de cerveau nous a laissé entrevoir une implication physiologique majeure de cette cascade de signalisation dans la corticogenèse.

Tuberculosis remains a major public health issue globally, with an estimated 9.2 million new cases in 2006. A new threat to TB control is the emergence of drug resistant strains. These strains are harder to cure as standard anti-tuberculosis first line treatments are ineffective. Multi Drug Resistant Tuberculosis (MDR-TB) is defined as Mycobacterium tuberculosis that has developed resistance to at least rifampicin and isoniazid, and these strains now account for greater than 5% of worldwide cases. Mutations within the Rifampicin Resistance Determining Region (RRDR) of the rpoB gene are present in greater than 95% of strains that show rifampicin resistance by conventional drug susceptibility testing. As rifampicin mono resistance is extremely rare, and rifampicin resistance is usually associated with isoniaizd resistance, the RRDR region of the rpoB gene is a very useful surrogate marker for MDR-TB. Many molecular assays have been attempted based on this theory and have had varied levels of success. The three methods evaluated in this study are DNA sequencing of the rpoB, katG and inhA genes, the Genotype MTBDRplus line probe assay (Hain Lifesciences) and a novel method incorporating Real-Time PCR with High Resolution Melt analysis targeted at the RRDR using the Rotorgene 6000 (Corbett Lifesciences). The sensitivity for the detection of rifampicin resistance was far better using DNA sequencing or the commercially available line probe assay than detection by the Real-Time PCR method developed in this study.

The potential for private sector healthcare business in Africa has been forecasted to reach $35 billion by 2016, with South Africa being regarded as the most industrially advanced country on the continent. South Africa’s entry to modern biotechnology is fairly recent, though, with companies in the private sector still in a developmental phase, and most having limited bioproduct ranges. While considerable research has been conducted in the past to attempt to define the biotechnology environment of South Africa, as yet, a concise overview is lacking. In particular, a synopsis of the biomedical or commercial health technology environment has not been forthcoming for entrepreneurs to refer to as a ‘roadmap’. The purpose of this study was to perform a comprehensive study on the attributes that should be met for a successful, sustainable health technology venture (HTV) to be started in South Africa; while identifying the opportunities and threats that have existed in the South African market; thereby, affecting their success and sustainability to date. In this study, two phases of research were conducted. The first was a small-sampled mixed-methods (both qualitative and quantitative) study involving 21 medical devices, biogenerics, diagnostics, and contract services companies. The second was a quantitative study, involving 107 vaccines, biogenerics, therapeutics, nutraceuticals, reagents, diagnostics, medical devices, biotools, contract services and public services companies. Inferential statistical tests were conducted on the data, including Pearson’s Chi-Square, ANOVA, bivariate correlation, linear regression, logistic regression and multinomial logistic regression. From the study, the overall proportion of business sustainability for HTVs was found to be 66.7%, and at least 30% were unsustainable (or not yet at a level of sustainability). Variations were observed in the overall rate of sustainability for companies, based on their core functional classification, location, production type, size and start-up or R&D spending. By converting the observed frequencies of activity level, as an indication of sustainability, into a probability, it was possible to observe the company type that was most, and least likely to succeed in South Africa. Based on the statistical observations in this study, the HTV type most likely to succeed in South Africa, with a 63.7% probability of reaching sustainability, is a ‘vaccines’, ‘biotools’ or ‘public services’ company from Johannesburg with at least 20 employees; that has developed its goods or services internally, but manufactured externally and spent between R20 million–and–R30 million on its R&D or start-up. Conversely, least likely to succeed (3.2% probability) is a nutraceutical company from Cape Town with between six and 20 employees, that has developed and produced internally, and which has spent between R1 million–and–R10million on its start-up. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Rotavirus diarrhea is still an important cause of mortality in children worldwide, responsible each year for about 500,000 deaths. The introduction of the vaccine against the virus could lead to a reduction in morbidity and mortality associated with rotavirus. In Brazil, the Rotarix ® rotavirus vaccine, which contains the genotype G1P [8], was included in the national programme of immunization since March 2006. This project aims to evaluate clinical and epidemiological data related to infection by RVA, rotavirus genotypes prevalent. The research project is a cross-sectional study in which children with acute diarrhoea were recorded prospectively from 2010 to 2012. The location of collection of fecal samples was the Hospital of urgency of Sergipe in Pediatric Emergency sector, in Aracaju/SE. The clinical and epidemiological information was obtained through a questionnaire. The clinical severity was determined by a scoring system 20, calculated from the survey point. It was verified the episodes of acute diarrhea and stool samples collected for research and genotyping of rotavirus by ELISA and RT-PCR method.Descriptive statistical calculations were carried out to define the epidemiology of rotavirus. EIA positive results were found in 78 of the 790 samples. The most frequent genotype was G2 P [4], followed by the G8 genotype P [4], P [8] G1, G3 P [8], G1 P [6]. Mixed infections were detected G2 P [4] P [8], P [4] and G1G2 G2G8 P [4] and in general there was a cocirculação of different genotypes in the years studied, moreover, shows an alternation between the genotypes every year. The results obtained in this study observed a variability of positive cases distributed, confirming that the seasonality in the region is not remarkable. Therefore, new strains of rotavirus are emerging and associated with severe diarrhea. For this reason, monitoring is required to follow changes in the epidemiology of rotavirus. / A diarreia por rotavírus ainda é uma importante causa de mortalidade infantil em todo o mundo, sendo responsável a cada ano por cerca de 500.000 mortes. A introdução da vacina contra o vírus pode levar a uma redução da morbidade e mortalidade associadas ao rotavírus. No Brasil, a Rotarix®, a vacina contra o rotavírus, que contém o genótipo G1P[8], foi incluído no programa nacional de imunizações em março de 2006. Este projeto teve como objetivo avaliar dados clínicos, epidemiológicos e genótipos predominantes relacionados à infecção por RV-A. O projeto de pesquisa é um estudo transversal em que as crianças com diarreia aguda foram registrados prospectivamente a partir de 2010 a 2012. O local de coleta das amostras fecais foi o Hospital de Urgência de Sergipe no setor de Urgência Pediátrica, em Aracaju/SE. A gravidade clínica foi determinada por um sistema de pontuação 20, calculado a partir do ponto de questionário. Foram verificados os episódios de diarreia aguda e coletadas amostras de fezes para pesquisa e genotipagem de rotavírus pelo método ELISA e RT-PCR. Cálculos estatísticos descritivos foram realizados para definir a epidemiologia do rotavírus. Resultados positivos foram encontrados em 78 das 790 amostras. O genótipo mais frequente foi G2P[4], seguido do genótipo G8P[4], G1P[8], G3P[8], G1P[6]. Foram detectadas infecções mistas G2 P[4] P[8], G1G2 P[4] e G2G8 P[4] e de um modo geral observou-se uma cocirculação de distintos genótipos nos anos estudados, além disso, nota-se uma alternância entre os genótipos a cada ano. O resultado obtido neste estudo observou uma variabilidade dos casos positivos distribuídos, confirmando que a sazonalidade na região não é marcante. Portanto, novas cepas de rotavírus estão surgindo e associados à diarreia grave. Por esta razão, a vigilância contínua é necessária para acompanhar as mudanças na epidemiologia do rotavírus.

INTRODUÇÃO: Poucos estudos têm focado as modificações fisiológicas que ocorrem no trato genital de mulheres menopausadas infectadas pelo HIV e sua associação com a excreção genital do vírus. Nesse estudo de corte transversal, comparou-se a excreção genital do HIV em mulheres menopausadas e em idade fértil em acompanhamento em um centro especializado em São Paulo, Brasil. Investigou-se também a associação entre a excreção genital de RNA de HIV e a viremia em ambos os grupos. Fatores associados com a intensidade da excreção genital de HIV também foram pesquisados, incluindo achados ginecológicos e marcadores de progressão da infecção por HIV. MÉTODOS: 146 mulheres infectadas pelo HIV [73 menopausadas (M)/73 em idade fértil (F)] foram selecionadas em Serviço de Extensão ao Atendimento de Pacientes com HIV/Aids Casa da Aids do Hospital das Clínicas da FMUSP, São Paulo, Brasil. As mulheres menopausadas referiram tempo médio de 8,17 anos (DP=6 anos) de menopausa. A contagem de linfócitos T CD4+ foi obtida por citometria de fluxo e a quantificação do RNA do HIV no plasma e no lavado cervicovaginal (LCV) foi realizada por RT-PCR quantitativo, utilizando-se o kit Cobas Amplicor HIV-1 Monitor Test®, no método ultrasensível. Cloreto de lítio foi introduzido no tampão para obtenção do LCV e quantificado antes e depois da coleta do lavado, a fim de determinar o fator de diluição de cada amostra. A deteção do gene SRY por PCR também foi realizada a fim de eliminar amostras com eventual contaminação espermática. A prevalência de excreção genital foi estimada para ambos os grupos e os fatores associados à intensidade da excreção viral foram investigados, utilizando-se modelo de regressão linear múltipla. As variáveis com p<0,2 na análise bivariada foram incluídas na análise multivariada, assim como o grupo em estudo (M ou F). O modelo final incluiu fatores que se mostraram independentemente associados com a intensidade da excreção genital de HIV. RESULTADOS: A prevalência de excreção genital de HIV-RNA foi similar em ambos os grupos (M: 17,8%, IC 95% 9,8 28,5; F: 22%, IC 95% 13,1 33,1, p=0,678). Similarmente, a intensidade de excreção genital do HIV também não se mostrou diferente entre os grupos (mediana - M: 1,4log/mL; F: 1,4log/mL, p=0,587). A carga viral plasmática foi detectável em 34,2% das pacientes menopausadas (IC 95% 23,5 46,3) e em 42,5% entre as pacientes em idade fértil (IC 95% 31 54,6, p=0,395). Três pacientes (2 M/1 F) exibiram excreção genital de HIV-RNA na ausência de viremia detectável. Existe evidência de correlação entre a carga viral plasmática e a genital em ambos os grupos (rM: 0,658; rF: 0,684, p<0,01). Adicionalmente, o número de células CD4+ periféricas mostrou-se negativamente correlacionada à excreção genital do HIV em ambos os grupos (rM: -0,250; rF: -0,248, p<0,05). À análise multivariada, a carga viral plasmática mostrou-se independentemente associada à ocorrência de excreção genital do HIV em ambos os grupos (OR 4,03, IC 95% 2,52 6,45, p<0,001). Já a intensidade de excreção genital mostrou-se independentemente associada ao pH vaginal (p<0,001), concentração de TNF- no LCV (p=0,01), e à carga viral plasmática (p=0,001), todos com correlação positiva. CONCLUSÕES: Apesar das modificações significativas que ocorrem na mucosa vaginal da mulher menopausada, a excreção cervicovaginal do HIV parece não ser significativamente influenciada por esse estado. A carga viral plasmática e o número de células CD4+ periféricas estão correlacionadas com a excreção genital do vírus. A frequência de excreção genital mostrouse independentemente associada à intensidade de viremia. Além disso, o aumento do pH vaginal e evidência de inflamação genital, associada à concentração de TNF- no LCV, independentemente aumentam a intensidade de excreção genital nas mulheres estudadas. / BACKGROUND: Few studies have focused on physiological modifications that occur in the genital tract of HIV-infected postmenopausal women and their association with HIV cervicovaginal shedding. In this cross-sectional study we evaluated and compared HIV genital shedding among postmenopausal and fertile-aged women under care at a specialized center in Sao Paulo, Brazil, investigating the association between HIV-RNA shedding and HIV plasma viral loads in both groups. Factors associated with higher HIV shedding were also investigated, including gynaecological features and HIV disease progression markers. METHODS: 146 women living with HIV [73 postmenopausal (PM)/73 in fertile-aged (F)] were enrolled at the HIV Clinic, University of São Paulo Medical School, Brazil. Postmenopausal women referred a mean duration of 8.17y (SD=6y) since menopause. CD4+ cell counts were obtained by flow cytometry and HIV-RNA was quantified in plasma and in cervicovaginal lavages (CVL) by RT-PCR, using Cobas Amplicor HIV-1 Monitor Ultrasensitive Test. Lithium chloride was introduced into the CVL buffer and measured before and after CVL collection in order to determine the dilution factor for each specimen. SRY gene detection by PCR was also performed in all samples in order to rule out sperm contamination. Prevalence of HIV genital shedding was estimated for both groups and factors associated with the intensity of viral shedding were investigated, using a multiple linear regression model. Variables with p<0.2 in bivariate analysis were included in multivariate analysis, as well as the study group (PM and F). The final model included factors shown to be independently associated with intensity of HIV genital shedding. RESULTS: The prevalence of HIV-RNA genital shedding was similar in both groups. (PM: 17.8%, 95%CI 9.8 28.5; F: 22%, 95%CI 13.1 33.1, p=0.678). Likewise, the intensity of HIV shedding was shown not to differ between PM and F women (means - PM: 1.4log/mL; F: 1.4log/mL, p=0.587). Plasma viral loads were detectable in 34.2% of PM patients (95%CI 23.5 46.3), as compared to 42.5% among F women (95%CI 31 54.6) (p=0.395). Three patients (2 PM/1 F) exhibited HIV-RNA genital shedding in the absence of detectable viremia. We found evidence of correlation between HIV plasma viral load and HIV cervicovaginal shedding in both groups (rPM: 0.658; rF: 0.684, p<0.01). In addition, CD4+ cell counts were shown negatively correlated to HIV shedding in both groups (rPM: -0.250; rF: -0.248, p<0.05). In multivariate analysis, HIV plasma viral load was shown independently associated with occurrence of HIV genital shedding in both groups (OR 4.03, 95%CI 2.52 6.45, p<0.001). In addition, the intensity of HIV shedding was shown independently associated with vaginal pH (p<0.001), TNF- concentrations in CVL (p=0.01), and with HIV plasma viral loads (p=0.001), all of them with positive correlation. CONCLUSION: Despite the significant changes that occur in the vaginal mucosa of postmenopausal women, HIV cervicovaginal shedding does not seem to be significantly influenced by this state. Plasma viral loads and CD4+ cell counts are correlated to HIV genital shedding. The frequency of HIV genital shedding was shown independently associated with viremia intensity. Moreover, increased vaginal pH and evidence of genital inflammation associated with TNF- concentration independently enhanced the intensity of HIV shedding in postmenopausal and fertile-aged women.

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The increased frequencies of two novel B cell populations defined as IgM+ CD19+ CD27- CD21low CD38low CD24+ and IgM+ CD19+ CD27- CD21low CD38low CD24- in peripheral blood of patients with common variable immunodeficiency (CVID) compared to healthy donors were found. The aim was to search for such B cells in patients with rheumatoid arthritis (RA) and their further characterization. The production of immunoglobulin (Ig) mRNA in single B cells was analyzed using flow cytometry, single cell sorting and RT-PCR, IgVH-specific PCR, cycle sequencing and statistical analysis. The study was focused on analysis of variable regions of the heavy chains of Igs and significant differences in the usage of VH, DH and JH gene segments, mutational frequencies, distribution of silent and replacement mutations, length and composition of CDR3 regions, clonal relation and RAG gene expression in above mentioned B cell populations were found. Because of lack of the surface CD27 molecule being regarded as marker of B cells that have undergone antigen-driven germinal reactions, analyzed populations were considered as naive. However, the pattern and type of mutations suggested that these cells could represent a new type of differentiated memory/antigen- experienced B lymphocytes (in CVID less maturated) with the likely role in...

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