Ce travail avait pour objectif, de caractériser le poids des infections au cours de la grossesse et d’étudier la construction de l’immunité anti-PfEMP1 chez le jeune enfant. La première partie, aborde la conséquence des infections et l’efficacité des traitements chez la mère. Cette étude réalisée sur la cohorte de femmes d’une étude prospective au Bénin, a démontré l’impact des infections à bas bruit sur le taux d’hémoglobine maternel et le faible poids de l’enfant. Le TPI-SP, a montré les limites quant à sa capacité à débarrasser les femmes enceintes infectées au moment du traitement de leur parasite. Nos résultats soutiennent davantage la nécessité de trouver des moyens alternatifs de prévention qui offrent une meilleure couverture de la grossesse. La deuxième partie aborde la construction de l’immunité anti-PfEMP1 dans la première année de vie chez l’enfant. Un résultat majeur de cette étude est la démonstration que l’acquisition des anticorps contre les PfEMP1 associés aux complications du paludisme est dépendante des infections patentes de l’enfant. La troisième partie aborde les phénotypes des parasites responsables de diverses formes cliniques du paludisme chez l’enfant Africain âgé de 0 à 5 ans. Cette étude a permis de mettre en évidence un marqueur de mauvais pronostic du paludisme cérébral. Sur un deuxième volet, nous avions montré que les isolats adhérant faiblement à ICAM-1, transcrivent fortement les gènes codant pour les PfEMP1 contenant des motifs DC8. Ces résultats soulèvent la question du rôle de EPCR dans la physiopathologie du neuropaludisme. Le travail développé dans cette thèse a permis de décrire pour la première fois la construction de l’immunité anti-PfEMP1 dans la première année de vie, de mettre à jour les connaissances sur la physiopathologie du paludisme cérébral chez le jeune enfant et de dégager des pistes à explorer prioritairement dans la perspective du développement d'un vaccin contre les formes graves du paludisme. / This work aimed to characterize the burden of P. falciparum infections during pregnancy and to study the construction of the anti-PfEMP1 immunity in the early life. The first part discusses the consequence of infection and the effectiveness of treatments in the mother. This study on the cohort of women from a prospective study in Benin, demonstrated the impact of infections with low parasitemia on the maternal hemoglobin and low weight of the child. IPT-SP, has shown the limits of its ability to rid infected pregnant women in the processing of their parasite. Our results further support the need to find alternative means of prevention that provide better coverage of pregnancy. The second part treated the construction of the anti-PfEMP1 immunity in the first year of life in children. A major finding of this study is the demonstration that the acquisition of antibodies against the PfEMP1 associated with complications of malaria depends on patentes infections in children. The third part studies parasites phenotypes responsible of various clinical forms of malaria in African children aged 0-5 years. This study allowed finding a marker of poor prognosis of cerebral malaria. On a second component, we showed that isolates bind to ICAM-1, highly transcribe the genes encoding PfEMP1 containing DC8. These results raise the question of the role of EPCR in the pathophysiology of cerebral malaria. The work developed in this thesis has allowed describing for the first time the construction of the anti-PfEMP1 immunity in the first year of life, to update knowledge on the pathophysiology of cerebral malaria in young children and identify Options to be primarily from the perspective of developing a vaccine against severe forms of malaria.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Real-time quantitative polymerase chain reaction (real-time qPCR) assays are an effective technique to detect biological warfare agents and surrogate organisms. In my study, primers were designed to detect chromosomal DNA of biological warfare agent surrogates B. thuringiensis and S. marcescens (representing B. anthracis and Y. pestis, respectively) via real-time qPCR. Species-level specificity of the primers was demonstrated through comparisons with a bacterial strain panel and corroborated by qPCR data. Additionally, the primer efficacy was tested when template DNA was spiked into metagenomic DNA extracted from clinical lung microbiome samples. The results showed that while detection of B. thuringiensis or S. marcescens was still largely successful, the addition of metagenomic DNA did significantly inhibit amplification in most cases. The present study is significant not only for the design of multiple novel primer pairs able to detect bacterial agents in metagenomic DNA, but also the quantitative insight to the influence of background DNA on single species detection at low DNA concentrations.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-04T10:18:52Z No. of bitstreams: 1 texto completo.pdf: 619624 bytes, checksum: 5386063ae0816c0b88770090de34a0a0 (MD5) / Made available in DSpace on 2015-11-04T10:18:52Z (GMT). No. of bitstreams: 1 texto completo.pdf: 619624 bytes, checksum: 5386063ae0816c0b88770090de34a0a0 (MD5) Previous issue date: 2014-04-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em condições de estresse ácido, micro-organismos do gênero Salmonella spp. utilizam como estratégia de sobrevivência um complexo mecanismo de tolerância que envolve vários genes. Os genes rpoS e nlpD são responsáveis pela expressão de proteínas que promovem a proteção da célula bacteriana contra os danos provocados pelo estresse, e os genes clpP, clpX e mviA estão envolvidos na regulação dessas proteínas no interior da célula. O presente estudo tem como objetivo associar as variações na expressão desses genes com as populações de Salmonella spp. durante um estresse ácido, condição que pode ser encontrada em carcaças bovinas que sofrem a aspersão de ácidos orgânicos para descontaminação microbiológica. A partir de uma coleção composta por 79 cepas de Salmonella spp., foi realizado um agrupamento genético por restrição enzimática através da técnica PFGE utilizando-se a enzima XbaI, e detecção dos genes rpoS, nlpD, clpP, clpX e mviA. Três cepas foram selecionadas (sorotipos: S. Derby, S. Typhimurium e S. Meleagridis) e semeadas em caldo extrato de carne com diferentes valores de pH (4,0, 5,0, e 6,0), ajustados com ácido lático e ácido acético. Logo após inoculação, e após 6, 12, 24 e 48 h de incubação a 37 °C, as populações bacterianas foram monitoradas por plaqueamento direto, e a expressão dos genes rpoS, nlpD, clpP, clpX e mviA por qPCR. Os resultados revelaram que Salmonella spp. pode se adaptar aos ácidos testados (acético e lático), principalmente quando estão em pH 5,0 ou 6,0, com expressão evidente do gene rpoS. Em pH 4,0, a redução completa das populações de Salmonella spp. ocorreu após 6 ou 24 h de incubação, o que em uma situação industrial representaria um período suficiente para que as proteínas da carne promovam o aumento do pH do meio, possibilitando dessa forma a adaptação de Salmonella spp. nesse substrato. A capacidade de adaptação de Salmonella spp. em valores de pH 5,0 e 6,0, demonstrada no presente estudo, ressalta a importância de um monitoramento adequado da redução de pH promovido pela aspersão de ácidos orgânicos em carcaças bovinas durante o processamento: caso o controle de pH não seja adequado, cepas de Salmonella spp. potencialmente contaminantes podem sobreviver e se manterem ativas nos produtos cárneos finais. Adicionalmente, é necessário que ocorra um equilíbrio entre a aplicação de ácidos orgânicos em concentrações que garantam o controle de Salmonella spp., além de outros micro-organismos, e a ausência de efeitos negativos sobre qualidade sensorial dos produtos cárneos, além de descoloração e exsudação. Os resultados obtidos permitem concluir que os ácidos orgânicos avaliados no presente estudo (ácido lático e ácido acético) podem ser considerados alternativas no controle de Salmonella spp., desde que ocorra um rígido controle dos valores de pH das soluções durante a interação com esse patógeno, e demanda o desenvolvimento de estudos em sistemas cárneos para demonstração desse comportamento. / Under acid stress conditions, Salmonella spp. presents a complex mechanism of tolerance that involves multiple genes. The rpoS, nlpD genes are responsible for the proteins expression that protect the bacterial cell against damage caused by acid stress, and clpP, clpX, mviA genes are involved in the regulation of these proteins inside the cell. The aim of this study was to associate the variations in these genes expression and Salmonella spp. populations when subjected to acid stress condition, a situation usually observed in bovine carcasses after cleaning procedures with organic acids spraying in slaughterhouse environment. Considering a culture collection composed by 79 Salmonella spp. strains, their fingerprinting were obtained by PFGE using XbaI, and all strains were subjected to PCR reactions to detect rpoS, nlpD, clpP, clpX, mviA genes. Based on the obtained data, three strains were selected (serotypes: S. Derby, S. Typhimurium and S. meleagridis) and inoculated in meat extract broth with different pH values (4.0, 5.0, and 6.0), adjusted with lactic acid and acetic acid. Immediately after inoculation, and after 6, 12, 24 and 48 h of incubation at 37 °C, the bacterial populations were monitored by direct plating, and the expression of rpoS, nlpD, clpP, clpX and mviA genes by qPCR . The results showed that Salmonella spp. can adapt to the tested acids (acetic and lactic), especially at pH 5.0 and 6.0, with evident expression of the rpoS gene. When the pH was adjusted to 4.0, complete reduction of Salmonella spp. populations occurred after 6 or 24 h of incubation, but in industrial situation this period would be sufficient for the meat proteins promote an increase in pH value, enabling the adaptation of Salmonella spp. on the substrate. The adaptability of Salmonella spp. at pH 5.0 and 6.0, as demonstrated in this study, shows the importance of adequate monitoring of pH reduction promoted by the organic acids spraying in bovine carcasses during processing: if pH control is not adequate, strains of Salmonella spp. can survive and remain active in the end meat products. Additionally, the ideal concentration of organic acids for the application in meat products depends in the balance between the pathogen contamination reduction and the absence of negative effects under sensorial quality. The results indicate that organic acids evaluated in the present study (lactic acid and acetic acid) can be considered as alternatives to control Salmonella spp., since associated to a rigorous pH control of the applied solutions during interaction with this pathogen. Furthermore, it demands further studies in meat systems to demonstrate this behavior.

Vid låga mängder av DNA, vilket det ofta är vid forensiska prover, är en lyckad DNA-analys beroende av ett effektivt DNA-extraktionsprotokoll. Bland flera kommersiellt tillgängliga extraktionsmetoder för DNA har tidigare studier visat att Chelex 100 resin, som idag används på Nationellt Forensiskt Centrum i Linköping, är en snabb och enkel metod utan toxiska ämnen, långa väntetider eller större kostnader och som även ger ett högt DNA-utbyte. På teknisk mikrobiologi i Lund har olika chelex-baserade protokoll för spårsäkring och extraktion tagits fram för att optimera det aktuella protokollet på Nationellt Forensiskt Centrum. I detta arbete kombinerades spårsäkringsteknik och DNA-extraktionsprotokoll från tidigare studier med olika rör och tops, med syftet att i bästa fall generera ett protokoll som är optimalt för de vanligast förekommande brottsplatsproverna. I resultatet observerades högre DNA-utbyte med reducerade volymer lyseringsbuffert, vilket användning av rör-i-rörsystem tillät. När rör-i-rörsystem sedan kombinerades med extraktionsprotokoll innehållande SDS, observerades en ökning i DNA-utbytet. När dessa två faktorer vidare kombinerades med en tops vars skaft kan klippas rakt av ökade DNA-utbytet ytterligare. Ett byte från mikrofugrör, som används på Nationellt Forensiskt Centrum idag, till ett rör-i-rörsystem reducerar lyseringsvolymen från ca. 600 µl till 200 µl vilket är en avgörande faktor för effektivare extraktioner. / When handling low amounts of DNA, which often is the case with forensic samples, a successful DNA analysis is dependent on an effective DNA extraction protocol. Among many commercially available extraction methods for DNA, previous research has proved Chelex 100 resin, which is used at National Forensic Center in Linkoping, to be a quick and simple method without toxic substances, long waiting time or large costs, which in addition provides a high DNA exchange. At Applied Microbiology, Lund´s University, different chelex-based protocols for sampling and DNA extraction have been developed to optimize the present protocol at the National Forensic Center. In this study, sampling techniques and DNA extraction from previous studies was further combined with different tubes and swabs with the aim to, in best case, generate a protocol that is optimal for the most common crime scene samples. The results showed improved DNA yields with less extraction volume, which the tube in tube system made possible. When tube in tube system was combined with extraction protocols containing SDS, a higher DNA yield was observed. When these two factors were further combined with a cotton swab, whose shaft can be cut straight of, the DNA yield improved even more. A change from microfuge tubes, which is used at the National Forensic Center, to tube in tube system, the lysis volume could be reduced from approximately 600 µl to 200 µl which is a deciding factor for higher DNA yield.

The presented master thesis deals with the monitoring of the influence of polysaccharides which are used as an additive in the cosmetic products, on the monitored types of bacteria which are part of the skin microbiome. And it also deals with the study the effect of polysaccharides as prebiotics on selected species of bacteria that are part of the skin microbiome. Two polysaccharides were selected to determine the effects on the skin microbiome: Nanomoist and PoLevan S. The first part of the thesis focuses on the literature search which deals with skin anatomy, skin diseases and skin microbiome and its function. The experimental part is focused on monitoring changes in the quantity of selected microorganisms of the skin microbiome, before and after application of polysaccharides to the skin using qPCR. Staphylococcus epidermidis, Cutibacterium acnes, Escherichia coli and Micrococcus luteus were monitored. The PCR products were detected by agarose gel electrophoresis. The bacterium Staphylococcus epidermidis was detected on the skin to the greatest extent, especially after the application of the polysaccharides Nanomoist and PoLevan S. Thus, a positive effect of both polysaccharides on the growth of this bacterium was found.

No description available.

Microbiota becomes one of the most important subjects in biological research and numerous studies revealed that microbiota plays a broad spectrum of essential roles in different organisms. This master thesis focuses on the bacterial part of microbiota contained in mucosal tissues of wild house mice (Mus musculus musculus). Male and female samples were collected by nasal and oral cavity lavages, vaginal mucosa lavages and from urine and stool. We aimed to detect quantitative, qualitative and sex-specific differences in bacterial populations between mucosal tissues with particular focus on bacterial cycling in vaginal mucosa during the estrous cycles. Bacterial abundances were estimated by qPCR whilst bacterial diversity was detected by targeted metagenomic sequencing of the hypervariable region of the 16S rRNA gene. Significant differences were detected in bacterial abundances and alpha diversity between particular mucosal tissues. Stool samples contained the highest number of bacteria, while samples from the nasal mucosa and urine contained low amount of bacteria. The highest alpha diversity was discovered in stool samples, the least alpha diversity was found in the urine. Mucosal tissues also varied based on the bacterial composition on the level of particular genera. Detailed analysis of estrous cycles...

Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...

No description available.