Várias zoonoses virais acometem equídeos causando quadros neurológicos, entre as quais a raiva e as encefalites equinas do Leste (EEE) e Oeste (WEE). O diagnóstico clínico geralmente não é conclusivo, o que torna imprescindível o diagnóstico laboratorial. Dados do Laboratório de Diagnóstico de Raiva do Instituto Pasteur de São Paulo, entre os anos 2000 e 2010, mostram que aproximadamente 75% das amostras enviadas foram negativas para raiva, ressaltando a relevância da realização de um diagnóstico diferencial para as encefalites equinas causadas por alfavírus. Os objetivos do estudo foram testar a adequação do uso de multiplex hemi-nested RT-PCR para o diagnóstico de raiva, EEE e WEE em amostras de sistema nervoso central de equídeos e realizar uma análise de custo das reações de cada técnica. Foram utilizados os primers 21G, 304 e 504 dirigidos ao gene N do vírus da raiva, e os primers cM3W, M2W, nEEE e nWEE dirigidos ao gene NSP1 dos vírus da EEE e WEE. Procedeu-se a um estudo preliminar dos primers e de seu uso em uma hemi-nested RT-PCR, avaliando a temperatura ótima de anelamento, a sensibilidade e especificidade analíticas e a reprodutibilidade da técnica em amostras de campo positivas para raiva e para EEE. A partir do protocolo estabelecido na reação de hemi-nested RT-PCR, realizaram-se variações de concentração de reagentes no protocolo para a reação de multiplex hemi-nested RT-PCR. Após o estabelecimento do protocolo para esta reação, os mesmos testes para verificação da sensibilidade e especificidade analíticas e da reprodutibilidade foram realizados, comparando-se os resultados com os obtidos pela hemi-nested RT-PCR. No teste de limiar de detecção, a sensibilidade analítica foi semelhante para as duas técnicas, obtendo-se 10&#45;1,7 para os três vírus padrão CVS, EEEV e WEEV. No teste de limiar de detecção utilizando uma amostra com os três vírus verificou-se uma alta especificidade dos primers, sendo que na reação de multiplex hemi-nested RT-PCR foi possível detectar simultaneamente os três vírus padrão. Não houve diferença nas proporções de amostras detectadas como positivas para raiva obtidas pelas duas técnicas, analisando-se pelo teste exato de Fisher (P=1,0000). No entando, para amostras de campo positivas para EEE, a proporção de amostras detectadas como positivas pela hemi-nested RT-PCR foi maior do que a proporção obtida pela multiplex hemi-nested RT-PCR (P<0,0001). Apesar de não ter sido possível o uso de amostras de campo positivas para WEE nesse estudo, os resultados sugerem que seria possível a detecção pela multiplex hemi-nested RT-PCR. Estes dados sugerem que a técnica de multiplex hemi-nested RT-PCR poderia ser aplicada para detecção de raiva e WEE, mas com limitações para a detecção de EEE. Pela análise de custo dos reagentes, o valor de uma reação de multiplex hemi-nested RT-PCR é semelhante ao de uma hemi-nested RT-PCR, podendo representar uma economia de pelo menos 49,17%. / Several viral zoonoses affect the equids causing neurological diseases, including rabies and Eastern and Western equine encephalitides (EEE and WEE). Clinical diagnosis is often not conclusive, in a way that laboratory diagnosis is essential. Data from the Laboratory of Rabies Diagnosis at the Pasteur Institute of São Paulo, between 2000 and 2010, demonstrate that approximately 75% of submitted equid samples were negative for rabies, emphasizing the importance of achieving a differential diagnosis for equine encephalitis caused by alphaviruses. The aims of this study were to test the suitability of using multiplex hemi-nested RT-PCR for the diagnosis of rabies, EEE and WEE in equids central nervous system samples and to perform a cost analysis of the reactions of each technique. We used the primers 21G, 304 and 504 directed to the N gene of rabies virus, and the primers cM3W, M2W, nEEE and nWEE directed the NSP1 gene of WEE and EEE viruses. A preliminary study of the primers was carried out, as well as their use in a hemi-nested RT-PCR, evaluating the optimal annealing temperature, the analytical sensitivity and specificity and the reproducibility of the technique in positive field samples for rabies and EEE. From the protocol established for the hemi-nested RT-PCR, variations in reagents concentrations for the multiplex hemi-nested RT-PCR protocol were perfomed. After establishing the protocol for this reaction, the same tests to verify the analytical sensitivity and specificity and reproducibility were performed and the results compared to those obtained by hemi-nested RT-PCR. In the detection threshold test, the analytical sensitivity was similar for both techniques, resulting in 10&#45;1.7 for the three virus standard CVS, and EEEV WEEV. In the detection threshold test using a sample with the three viruses, a high specificity of the primers was verified and the multiplex hemi-nested RT-PCR was able to detect the three viruses simultaneously. There was no difference in the proportions of samples detected as positive for rabies obtained by both techniques, according to the Fisher exact test (P = 1.0000). However, for EEE positive field samples, the proportion of samples detected as positive by the hemi-nested RT-PCR was higher than the proportion obtained by multiplex hemi-nested RT-PCR (P <0.0001). Although it was not possible to use WEE positive field samples in this study, the results suggest that its detection would be possible by multiplex hemi-nested RT-PCR. Thus, data suggest that the multiplex hemi-nested RT-PCR technique could be applied to detect rabies and WEE, but with limitations for the EEE detection. For the analysis of reagent costs, the cost of one multiplex hemi-nested RT-PCR is similar to one hemi-nested RT-PCR, and may represent a saving of 49,17% at least.

Orientadora: Maria da Glória Buzinaro / Banca: Samir Issa Samara / Banca: Ricardo Luiz Moro de Sousa / Resumo: O presente estudo teve como objetivo determinar a ocorrência de rotavírus do grupo A e a genotipagem G e P de estirpes detectadas em bezerros de rebanhos leiteiros e gado de corte, durante o período de julho de 2006 a setembro de 2008, em propriedades rurais do Estado de São Paulo, Brasil. Foram amostrados 395 bezerros, na faixa etária entre 1 e 60 dias, independentemente da manifestação clínica de diarréia, de 18 rebanhos bovinos, sendo nove de exploração leiteira e nove de gado de corte. Por meio da técnica de eletroforese em gel de poliacrilamida (EGPA), determinou-se a ocorrência de rotavírus em 33,3% (06/18) entre os rebanhos e de 8,6% (34/395) na população amostrada. A maior freqüência de infecção foi detectada em animais com idade entre 16 e 30 dias (p < 0,01). Foram diagnosticados bezerros infectados por rotavírus tanto em animais com sinais clínicos de diarréia (22%;29/133) quanto naqueles clinicamente normais (1,9%;05/262), existindo, porém, uma correlação entre a presença da infecção e a manifestação clínica da diarréia (p<0,01). Em relação ao tipo de exploração, nos rebanhos leiteiros 0 percentual de ocorrência foi de 5,3% (14/264), enquanto que nos rebanhos de gado de corte o rotavírus teve maior ocorrência (15,3%; 20/131). A análise do perfil do genoma de rotavírus por EGPA identificou dois eletroferótipos distintos, cujas diferenças estavam localizadas na posição de migração dos segmentos 2, 3, 7, 8 e 9. A genotipagem pela reação em cadeia pela polimerase (RT-SNMPCR) das amostras de rotavírus revelou a que as estirpes circulantes nos rebanhos eram G6P[5], G10P[5], não foi possível fazer associação com o genotipo P[1]. / Abstract: The present study aimed to determine the Group A rotavirus and G/P genotyping in detected virus strains in dairy and beef cattle calves from July 2006 to September 2008 in São Paulo State-Brazil farms. 395 calves in 18 flocks (9 dairy cattle and 9 beef cattle) from 1 to 60-day-old were sampled, independently whether manifesting clinically diarrhea or not. By using Poliacrylamide Gel Electrophoresis Technique (PAGE), the rotavirus occurrence of 33.3% (06/18) among flocks and 8.6% (34/395) among the population sampled were determined. The higher infection frequency was detected in 16-to-30-day-old calves (P<0.01). Rotavirus-infected flocks rotavirus-infected were diagnosticated as much the ones having clinical symptoms of diarrhea (22%, 29/133) as the ones clinically normal (1.9%, 05/262), shawing a correlation between the presence of infection and the diarrhea manifestation (p<0.01). Related to the kind of exploration, the occurrence in dairy cattle was 5.3% (14/264) while in the beef cattle the occurrence was higher (15.3%, 20/131). The rotavirus genome profile analysis by PAGE identified two distinct electroferotypes, in which differences were located in the following segment migration positions: 2, 3, 7, 8 and 9. The genotyping of the Rotavirus sample by polymerase chain reaction (RT-snmPCR) revealed that the circulating strains among the flocks were G6P[5], G10P[5] e G?P[1]. / Mestre

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-09T17:56:17Z No. of bitstreams: 1 PDF - Geisenilma Maria Gonçalves da Rocha.pdf: 891700 bytes, checksum: 6a3a21e49105289a64a7197232dd50c3 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-03-10T17:00:05Z (GMT) No. of bitstreams: 1 PDF - Geisenilma Maria Gonçalves da Rocha.pdf: 891700 bytes, checksum: 6a3a21e49105289a64a7197232dd50c3 (MD5) / Made available in DSpace on 2016-03-10T17:00:41Z (GMT). No. of bitstreams: 1 PDF - Geisenilma Maria Gonçalves da Rocha.pdf: 891700 bytes, checksum: 6a3a21e49105289a64a7197232dd50c3 (MD5) Previous issue date: 2015-02-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The demand for colored cotton fibers has grown internationally, especially motivated by ecological appeal that management provides. In Brazil, especially in the Northeast, agricultural niches are concentrated in small areas of family-based farmers who adopt both the conventional management as the organic, generating employment and income. The cotton breeding program of Embrapa Cotton has made efforts in developing new varieties of colored fibers in order to contribute to the regional agribusiness growth, nowadays with five cultivars, with fibers of colors ranging from green to various shades of brown. The challenge for researchers, however, is to generate varieties with new shades that can contribute to move the competitive market of the textile industry, especially the natural and dyes free. To advance in this segment, it is essential to understand the molecular basis of the metabolites involved in the synthesis of the color of the fibers so that we can establish strategies for genetic improvement, both by conventional means such as by genetic engineering. It is known that flavonoids are secondary metabolites which confers color to fibers and that depending on the route of biosynthesis, various color tones can be synthesized by events cascade. Being a component of biochemical nature, it is estimated that the identification of genotypes holders of biosynthesized by associated molecular markers, directly or not, to flavonoids. This strategy can configure a useful tool to identify genotypes colored fibers, helping to reduce the time the selection procedures that take between 120 to 150 days for the character can be phenotyped. Seeking to generate information that may help with the colored cotton fiber improvement aimed this work a study of genetic and molecular approaches in colorful cotton accesses, based on analysis of divergence and molecular expression, focusing on fiber specific genes involved in flavonoid biosynthesis. For analysis of genetic diversity, twelve genotypes were phenotyped by ISSR-PCR, using 12 commercial oligonucleotides. The methods of Tocher, UPGMA and 2D Projection were adopted for cluster analysis based on the standard of 50 polymorphic bands. In light of the results, proceeded to the analysis of transcripts expression, using the genes PP2A1, C4H, DFR, ANR and ANS in cDNAs fibers collected at 8, 10 and 18 DPA (after anthesis days) from BRS Rubi, BRS Topázio, BRS 200 and V3 access. In the cluster analysis by UPGMA, there was the formation of six groups being groups B, D and E only those clustered colored materials. The results of the expression through semiquantitative RT-PCR, the amplicons were observed of approximately 200, 290, 1100, 1024 and 1067 bp for PP2A1, C4H, DFR, ANS and ANR, respectively, as expected. We observed increased expression of genes C4H, DFR and ANR in brown color genotypes, suggesting that these genes may be involved in flavonoid biosynthesis brown fibers. The results obtained in this study can be applied in the cotton breeding program aimed at obtaining varieties with new colors or new shades. / A demanda por fibras de algodão colorido tem crescido em nível internacional, motivada especialmente pelo apelo ecológico que o manejo proporciona. No Brasil, especialmente na região Nordeste, os nichos agrícolas se concentram em pequenas áreas de agricultores de base familiar, que adotam tanto o manejo convencional quanto o orgânico, gerando emprego e renda. O programa de melhoramento de algodão da Embrapa Algodão tem envidado esforços no desenvolvimento de novas cultivares de fibras coloridas com o intuito de contribuir com o crescimento do agronegócio regional, detendo atualmente cinco cultivares, com tonalidades de fibras variando do verde até várias tonalidades de marrom. O desafio dos pesquisadores, contudo, é gerar cultivares com novas tonalidades que possam contribuir para movimentar o competitivo mercado da indústria têxtil, especialmente o de fibras naturais e isentas de corantes para fixação da cor. Para avançar nesse segmento, é imprescindível que se entenda a base molecular dos metabólitos envolvidos na síntese da cor das fibras para que se possa estabelecer estratégias para o melhoramento genético, tanto por vias convencionais como por meio de engenharia genética. Sabe-se que os flavonoides são metabólitos secundários que conferem cor as fibras e que, dependendo da rota de sua biossíntese, várias tonalidades de cores podem ser sintetizadas ao final da cascata de eventos. Por ser um componente de natureza bioquímica, estima-se que a identificação de acessos detentores de diferentes biossintetizados possam ser discriminados por meio de marcadores moleculares associados, diretamente ou não, aos flavonoides. Tal estratégia pode se configurar em uma ferramenta útil para contribuir posteriormente na identificação de acessos de fibras coloridas, auxiliando a reduzir o tempo nos procedimentos de seleção, que levam entre 120 a 150 dias para que o caráter possa ser fenotipado. Com intuito de gerar informações que possam contribuir com o melhoramento de fibras de algodão colorido, objetivou-se nesse trabalho proceder um estudo envolvendo abordagens genética e molecular em acessos de algodão colorido, baseando-se em análise de divergência e de expressão molecular, focalizando em genes específicos de fibra envolvidos na biossíntese de flavonoides. Para análise da diversidade genética, doze acessos foram fenotipados por meio de PCR-ISSR, utilizando-se 12 oligonucleotídeos comerciais. Os métodos de Tocher, UPGMA e Projeção 2D foram adotados para análise de agrupamento, baseado no padrão de 50 bandas polimórficas. Em função dos resultados obtidos, procederamse as análises de expressão de transcritos, utilizando-se os genes PP2A1, C4H, DFR, ANR e ANS em cDNAs de fibras coletadas aos 8, 10 e 18 DPA (dias pós antese) dos acessos BRS Rubi, BRS topázio, BRS 200 e V3. Na análise de agrupamento via UPGMA, verificou-se a formação de seis grupos, sendo os grupos B, D e E os que aglomeraram apenas os materiais coloridos. Nos resultados da expressão via RT-PCR semiquantitativa, observaram-se amplicons de aproximadamente 200, 290, 1100, 1024 e 1067 pb para PP2A1, C4H, DFR, ANR e ANS, respectivamente, como esperado. Observou-se maior expressão dos genes C4H, DFR e ANR nos acessos de coloração marrom, sugerindo que estes genes podem estar envolvidos na biossíntese de flavonoides de fibras marrons. Os resultados obtidos neste trabalho podem ser aplicados no programa de melhoramento do algodão visando a obtenção de cultivares com novas cores ou novas tonalidades.

The CDC estimated that foodborne infections resulted in approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths per year in the United States (Mead, 1999). There are over 200 known diseases caused by viruses, bacteria, parasites, toxins, metals, or prions that can be transmitted through food. Of these illnesses caused by foodborne disease, the CDC estimates that 38.6 million cases are from identifiable pathogens and 30.9 million of these cases are caused by viruses. Hence, approximately 80% of foodborne illnesses of known etiology result from viral transmission (Mead, 1999). Viral gastrointestinal illness may be caused by virus families such as: enterovirus, rotavirus, calicivirus, astrovirus, or norovirus. These viruses are highly contagious and are spread through the fecal-oral route; transmission vehicles include contaminated food or beverages, infected food handlers, fomites or close contact with an infected individual (FDA Bad Bug Book, 2003). Until recently, there have been few studies concentrating on viruses found in or on foods. There are several technical difficulties that hinder progress in detecting viral agents from foods. One of these problems is the presence of matrix inhibitors. Substances responsible for matrix inhibition include humic acid, polysaccharides, myoglobins, metal ions, glycogen, and lipids (Monpoeho, 2001). These substances in foods produce smearing of the RT-PCR amplicon bands on agarose gels. Several methods to reduce inhibitory compounds utilize multiple toxic reagents in the procedure. In this study, varying centrifugal forces were tested at different steps of the virus extraction/concentration procedure to reduce matrix inhibitory effects for molecular detection of norovirus and poliovirus seeded onto food surfaces. This method incorporates the rapid detection capabilities of RT-PCR with the ability to reduce or eliminate matrix inhibitors present in food, by altering the centrifugal force. Results for both viruses showed that band intensity decreased as the viral concentration decreased and no one method was superior for all food matrices. This investigation showed that matrix specific modifications to the basic protocol are required to efficiently extract viruses from the surface of foods. Each food should be assessed to determine modifications to the standard method that would be optimal for viral concentration and extraction.

The activation of 1,25D-dihydroxyvitamin D3 (1,25D) is catalysed by the enzyme 25-hydroxyvitamin D-1ƒhydroxylase (CYP27B1) in the kidney, which is the primary producer of 1,25D in the body. Although the synthesis of 1,25D by CYP27B1 and the catabolism of 1,25D by 25-hydroxyvitamin D-24-hydroxylase (CYP24) also take place in the bone, the significance of the bone cell-specific metabolism of vitamin D remains largely unknown. This thesis investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone. Furthermore, CYP27B1, CYP24 and VDR mRNA expression were also identified in specific regions of the rat femur and in a number of bone cell lines, with the aim to identify the bone cell types that have the capacity to metabolise and/or to respond to vitamin D. The age-related decrease in the circulating levels of 1,25D detected in animals ranging in age from 3 weeks to 2 years old, was a direct result of a reduction in the expression of CYP27B1 mRNA and an increase in the expression of CYP24 and VDR mRNA in the kidney. In contrast, the expression of CYP27B1 and CYP24 mRNA in the bone is high from 3 to 15 weeks of age, which is the period of rapid growth and development. The expression of CYP27B1 mRNA in the bone was positively correlated with the circulating levels of calcium throughout aging, which suggests that the 1,25D produced in the bone may be involved in the mineralisation process. The positive correlation found between the expression of CYP27B1 and CYP24 mRNA in the bone was in contrast with the negative correlation found between the expression of these two enzymes in the kidney. This suggests that the 1,25D produced locally in the bone, rather than the 1,25D produced in the kidney, is the primary determinant of the CYP24 activity in the bone. In vitamin D-deplete animals, fed a 0.1% calcium diet (D(-)/LC), the expression of CYP27B1 mRNA was induced and the expression of CYP24 mRNA was suppressed in the kidney. In contrast, both the expression of CYP27B1 and CYP24 mRNA were low in the bones of these D(-)/LC animals. When vitamin D-deplete animals were fed a 1% calcium diet (D(-)/HC), the expression of both CYP27B1 and CYP24 mRNA was high in the bone, which was in direct contrast with the low expression of these genes detected in the kidney. Besides this, a positive correlation was found between the expression of CYP27B1 mRNA in the bone, serum calcium levels and bone mineral volume (BV/TV) in the epiphysis, which supports the findings for the age study that the locally produced 1,25D may be involved in the promotion of bone mineralisation. Although serum PTH levels was positively correlated with the expression of CYP27B1 mRNA in the kidneys of hypocalcaemic animals, there was no such relationship detected between the levels of serum PTH and the expression of CYP27B1 mRNA in the bone. This finding suggests that the regulation of the expression of CYP27B1 mRNA in the bone is different from the regulation found in the kidney. The identification of CYP27B1 mRNA in osteoblasts-like cells, taken together with the associations between serum calcium and CYP27B1 mRNA expression in the previous studies, suggests that 1,25D produced in osteoblasts may play a significant role in the bone mineralisation process. The detection of CYP27B1 mRNA expression in a number of bone marrow cells suggests that locally produced 1,25D may also play a role in the growth and differentiation of hematopoietic cells. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2003.

La progression naturelle des tumeurs consiste en une extension locale, puis à distance (métastase) par migration de cellules dans le sang et la lymphe vers des sites secondaires. Il est donc primordial de pouvoir détecter des cellules tumorales circulantes en plus de l'analyse morphologique et de l'immunocytochimie. De plus, deux technologies (cytométrie en flux et RT-PCR quantitative en temps réel) sont adaptées pour une analyse automatisée, rapide et sensible d'une très faible quantité de cellules. Le but de notre travail a été de mettre au point des systèmes de détection pour l'identification de cellules cancéreuses dans les épanchements péritonéaux et dans le sang. L'étude des biomarqueurs moléculaires apparaît comme une approche complémentaire intéressante pour améliorer l'efficacité du diagnostic dans ce type d'échantillons biologiques. Nous nous sommes intéressés à la mise en évidence de nouveaux marqueurs tumoraux qui pourront être utilisés pour le diagnostic précoce et le pronostic des cancers en utilisant les nouvelles techniques de biologie moléculaire. Il est probable que l'utilisation de multiples marqueurs moléculaires puisse permettre d'évoquer plus particulièrement certains types de cancers. Nous avons pu mettre en place une technique de PCR quantitative en temps réel, nettement plus sensible que la cytologie classique, et nous avons appliqué cette technique à l'étude de marqueurs tumoraux dans les liquides d'épanchement, mais aussi dans le sang pour rechercher et doser l'ARN messager. Nos résultats montrent que la cytométrie en flux adaptée à des lignées cellulaires ne l'est pas pour des prélèvements cliniques. Par la PCR quantitative, il a été possible de quantifier le niveau d'expression des marqueurs tumoraux étudiés en utilisant des plasmides de référence qui ont été préparés pour chaque gène. Plusieurs marqueurs permettent de différencier des épanchements malins et des épanchements bénins, mais surtout les antigènes CLDN4 et Ep-CAM étaient significativement plus élevés (68% et 57%, respectivement) chez les patients avec épanchements malins. L'ARN messager circulant de la CLDN4 était détectable et significativement plus élevée dans les sérums de patients atteints de cancer du sein (64% p<0,05). Les résultats indiquent que l'utilisation d'une combinaison de marqueurs comportant laclaudine 4 est plus susceptible de détecter des cellules malignes et d'être utiles pour le suivi de patients

Chez les insectes, un seul et unique gène code la sous-unité principale α des canaux sodium dépendant du potentiel (Nav). De plus, quatre à cinq gènes codent les sous-unités auxiliaires β. Bien que la blatte, Periplaneta americana, soit un modèle en neurophysiologie, il n'existe aucune donnée sur les structures moléculaires des canaux Nav de cette espèce. L'objectif de cette thèse était de caractériser les canaux Nav exprimés au niveau de la chaine nerveuse (CN), du dernier ganglion abdominal (DGA) et des DUM neurones octopaminergiques. Tout d'abord, nous avons mis en évidence que la diversité moléculaire de la sousunité principale est générée par épissage alternatif au niveau de trois régions du canal. Les combinaisons d'exons majoritaires identifiées parmi les ADNc de CN et du DGA sont l'exon A seul (boucle L1), la combinaison EFHG1129 (boucle L2) et l'exon G1 (IIIS3-S4). En dépit de cette diversité moléculaire, nous n'avons isolé qu'un seul ADNc de 6153 pb codant une sous-unité principale dénommée PaNav1. La combinaison d'exons alternatifs caractérisant cette isoforme (J, A, E, F, G1129, H, G1) s'est alors avérée représentative des ADNc les plus exprimés dans la CN et le DGA. Par contraste, dans les neurones DUM, nous n'avons identifié qu'une seule population d'ADNc codant chaque région citée précédemment. Par ailleurs, une analyse bioinformatique à partir des génomes séquencés d'insecte a permis de montrer que les exons optionnels I et F sont les moins conservés entre les différents ordres d'insecte. Parallèlement à la caractérisation des sous-unités principales classiques, nous avons découvert un nouveau gène codant des sous-unités α atypiques nommées PaFPC1 et PaFPC2. Ces dernières comptent 1153 résidus d'acide aminé dont huit qui les distinguent. Elles partagent seulement 59% d'identité protéique avec PaNav1. L'absence du motif moléculaire impliqué dans l'inactivation des canaux Nav suggère que ces nouvelles sousunités possèdent des propriétés électrophysiologiques originales. Une analyse phylogénétique détaillée a permis de montrer que PaFPC1 se branche à la base des sousunités α des canaux Nav d'insecte. Cette découverte montre alors pour la première fois l'existence d''un événement de duplication du gène des canaux Nav chez les insectes. Finalement, nous avons clonés deux populations d'ADNc codant des sous-unités auxiliaires nommées PaTEH1.1 et PaTEH1.2. Grâce à la récente mise à disposition du génome de plusieurs espèces d'insecte, nous avons pu conduire une étude phylogénétique clarifiant la situation des sous-unités auxiliaires de canaux Nav au sein de ce taxon. De plus, une étude électrophysiologique préliminaire dans des ovocytes de xénope a permis de montrer que PaTEH1.1 se comporte comme une protéine chaperonne. C'est à dire qu'elle augmente significativement l'expression des sous-unités principales d'insecte. Ainsi, nos résultats originaux offrent un nouveau regard sur les canaux Nav et ouvrent de nouvelles perspectives quant à la compréhension des acteurs moléculaires à la base de l'excitabilité membranaire chez les insectes.

Bovine Spongiform Encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) which affect many species. From 1986 more than 184,000 cattle in the UK have been confirmed to be infected with this disease, and in Canada total losses to the economy reached $6 billion. This study examines the gene expression in three major innate immunity components: complement system, toll-like receptors, interleukins, and selected proteins of their signaling pathways. Quantitative real time polymerase chain reaction analyses were performed on caudal medulla samples to identify differentially expressed genes between non-exposed and orally challenged animals. In general, immune genes were down-regulated in comparison to non-challenged animals during first 12 months of disease with a tendency to be up-regulated at terminal stage of BSE. The results from this study provide a basis for further research on the mechanisms modifying immune responses and altering progression of the disease. / Animal Science

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor. In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study. In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene. In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

The use of fossil fuels presents a particularly interesting challenge - our society strongly depends on coal and oil, but we are aware that their use is damaging the environment. Currently, this awareness is gaining momentum, and pressure to evolve towards an energetically cleaner planet is very strong. Molecular hydrogen (H2) is an environmentally suitable energy carrier that could initially supplement or even substitute fossil fuels. Ideally, the primary energy source to produce hydrogen gas should be renewable, and the process of conversion back to energy without polluting emissions, making this cycle environmentally clean. Photoconversion of water to hydrogen can be achieved using the following strategies: 1) the use of photochemical fuel cells, 2) by applying photovoltaics, or 3) by promoting production of hydrogen by photosynthetic microorganisms, either phototrophic anoxygenic bacteria and cyanobacteria or eukaryotic green algae. For photobiological H2 production cyanobacteria are among the ideal candidates since they: a) are capable of H2 evolution, and b) have simple nutritional requirements - they can grow in air (N2 and CO2), water and mineral salts, with light as the only energy source. As this project started, a vision and a set of overall goals were established. These postulated that improved H2 production over a long period demanded: 1) selection of strains taking in consideration their specific hydrogen metabolism, 2) genetic modification in order to improve the H2 evolution, and 3) cultivation conditions in bioreactors should be exmined and improved. Within these goals, three main research objectives were set: 1) update and document the use of cyanobacteria for hydrogen production, 2) create tools to improve molecular biology work at the transcription analysis level, and 3) study cell division in cyanobacteria. This work resulted in: 1) the publication of a review on hydrogen evolution by cyanobacteria, 2) the development of tools to assist understanding of transcription, and 3) the start of a new fundamental research approach to ultimately improve the yield of H2 evolution by cyanobacteria.