Clostridium perfringens is a significant human pathogen, causing 250,000 cases of food poisoning in addition to several thousand potentially lethal cases of gas gangrene each year in the United States. Historically, work in this field has centered around toxin production, as C. perfringens can produce over 13 toxins. This work expands the knowledge of the starvation-response of C. perfringens, which includes several potential virulence factors, sporulation, motility and biofilm formation. Sporulation protects cells from a variety of stresses, including starvation. Efficient sporulation requires the transcriptional regulator CcpA, mediator of catabolite repression. Sporulation is repressed by glucose, but, surprisingly, in a CcpA-independent fashion. C. perfringens cells in a biofilm are resistant to a number of environmental stresses, including oxygen and antibiotics. Biofilm formation is repressed by glucose, and other carbohydrates, independently of CcpA. Gliding motility, a type four pili (TFP)-dependent phenomenon, affords C. perfringens with a mechanism for moving across a solid surface in response to carbohydrate starvation, while carbohydrates supplementation at high levels delay the initiation of the motility response. CcpA is required for the proper initiation of motility, a ccpA^-C. perfringens strain showed a considerable increase in the time to initiation of motility on lactose and galactose, and was unable to move at all in the presence of glucose. Gliding motility represents the most significant finding of this work. TFP were previously undescribed in any Gram-positive bacterial species, and this work produced genetic evidence suggesting their presence in all members of the clostridia, and physical evidence for TFP-dependent gliding motility in a second species, C. beijerinckii. / Ph. D.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases in bovines, although nonpathogenic commensal strains also exist. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. To identify genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and may be enhanced by the presence of uptake signal sequences (USS) within the genome. We hypothesized that natural transformation occurs in H. somni because its genome is over-represented with USS and contains all the necessary genes for competence, except that ComD and ComE are mutated. For natural transformation, H. somni was grown to exponential phase, and then transferred to a non-growth defined medium to induce competence. H. somni strain 2336 was successfully transformed with homologous linear DNA (lob2A) containing an antibiotic marker gene, but at low efficiency. Shuttle vector pNS3K was also naturally transformed into H. somni at low efficiency. To attempt to improve transformation efficiency, comD and comE from H. influenzae were cloned into shuttle vector pNS3K to generate the plasmid pSScomDE. Although introduction of pSScomDE into H. somni was expected to increase the number and breadth of mutants generated by natural transformation, multiple attempts to electroporate pSScomDE into H. somni were unsuccessful. A native plasmid (pHS649) from H. somni strain 649 may prove to be a more efficient shuttle vector. Due to inefficiency in generating mutants by allelic exchange, transposon (Tn) mutagenesis with EZ / Master of Science

L’un des principaux défis de la thérapie génique est d’identifier un vecteur sûr capable d’assurer un transfert efficace et une expression soutenue d’un gène d’intérêt thérapeutique dans les cellules cibles. L’émergence de vecteurs plasmidiques de nouvelles générations a permis d’atteindre ces objectifs et de considérer la thérapie génique non virale comme une alternative prometteuse aux vecteurs viraux pour le traitement de maladies génétiques ou acquises. Appartenant à ces nouvelles générations, les dérivés du vecteur pFAR4 sont des miniplasmides dépourvus de gène de résistance à un antibiotique. Leur propagation dans les cellules d’Escherichia coli est basée sur la suppression d’une mutation non-sens de type ambre introduite dans un gène essentiel de la souche productrice, permettant ainsi d’éliminer les risques associés à l’utilisation de gène de résistance à un antibiotique tout en diminuant la taille du vecteur. Le but de cette thèse est d’étudier le potentiel de ces vecteurs dans deux contextes de thérapie génique non virale : Dans une première approche, le potentiel du vecteur pFAR4 a été évalué pour l’expression d’un gène thérapeutique dans le foie de souris. Pour ce faire, un dérivé de ce vecteur exprimant le gène Sgsh à partir d’un promoteur spécifique des hépatocytes et codant la protéine sulfamidase, protéine défectueuse chez les patients souffrant de la maladie de Sanfilippo de type A, a été administré par injection hydrodynamique à des souris. Nous avons montré que le vecteur pFAR4 promeut dans le foie une expression élevée et soutenue de la sulfamidase, qui décline rapidement lorsque le gène Sgsh est administré par un vecteur contenant un gène de résistance à la kanamycine. Dans le cadre de cette étude, il a été établi que le profil d’expression obtenu avec le vecteur pFAR4 n’est pas lié à son insertion dans le génome des hépatocytes mais résulte, de par sa taille réduite, d’une protection contre les phénomènes d’extinction de transgène couramment observés in vivo avec les vecteurs conventionnels. Dans une seconde approche, le vecteur pFAR4 a été combiné à la technologie Sleeping Beauty (SB), dont l’un des constituants majeurs est la transposase hyperactive SB100X qui promeut la transposition d’un transgène, en l’excisant du plasmide qui le porte et en l’insérant dans le génome des cellules hôtes. Cette combinaison a été étudiée in vitro dans des cellules HeLa, en utilisant un transposon contenant soit le gène de résistance à la néomycine soit le gène codant la protéine fluorescente Vénus. Nous avons ainsi montré que le plasmide pFAR4 constituait un vecteur efficace pour les composants du système SB et que la combinaison pFAR4/SB conduisait à un taux de transgénèse augmenté par rapport à une association avec des plasmides conventionnels. Cette efficacité élevée résulte d’un niveau de transfection et d’un taux d’excision augmentés, tous deux favorisés par la taille réduite du plasmide. La combinaison pFAR4/SB devrait prochainement être utilisée pour transférer le gène codant le facteur anti-angiogénique PEDF (Pigment Epithelium-Derived Factor) à des cellules primaires de l’épithélium pigmentaire de la rétine ou de l’iris dans deux essais cliniques (Phase I/II) de thérapie génique ex vivo pour le traitement de la dégénérescence maculaire liée à l’âge (DMLA). / One of the main challenges in gene therapy is to identify safe vectors that promote an efficient gene delivery and a sustained therapeutic transgene expression level in targeted cells. The development of novel plasmid vectors allowed to reach these objectives and to consider non-viral gene therapy approaches as attractive alternatives to treat genetic and acquired disorders. The pFAR4 vector is a novel antibiotic-free mini-plasmid. In Escherichia coli, its propagation is based on the suppression of an amber nonsense mutation introduced into an essential gene, thus eliminating safety concerns classically attributed to antibiotic resistance markers present on conventional plasmid DNA vectors and allowing a reduction in plasmid size. The aim of this work was to investigate the potential of pFAR4 as a gene vector in two different non-viral gene therapy approaches. In a first approach, the potential of the pFAR4 vector was assessed for the expression of a therapeutic gene in mouse liver. To this end, a pFAR4 derivative expressing the Sgsh gene from a liver-specific promoter and coding the sulfamidase, an enzyme deficient in patients suffering from the Sanfilippo A disease, was tail vein hydrodynamically injected into mouse liver. We showed that the pFAR4 derivative promoted a high and prolonged sulfamidase expression which rapidly declined when the same expression cassette was delivered by a conventional plasmid containing a kanamycin resistance marker. It was established that the superior expression profile obtained with the pFAR4 derivative did not result from its integration in host genome but seemed to benefit from protection against transcriptional silencing. In a second approach, the pFAR4 vector was combined to the Sleeping Beauty transposon system that mediates transgene integration into host genomes, after its excision from a plasmid donor by the hyperactive SB100X transposase, in order to obtain a long-term expression in dividing cells. This combination was studied in vitro, delivering either the neomycin resistance gene or the fluorescent Venus protein-encoding gene into HeLa cells. We showed that the combination pFAR4/SB led to an increased transgenesis rate in comparison to the association of SB with conventional plasmids. The pFAR4/SB combination seemed to benefit from an elevated transfection efficiency and a higher excision rate, resulting from the reduced size of the pFAR4 vector. The two technologies should be soon used for the delivery of the anti-angiogenic pigment epithelium-derived factor (PEDF) gene into autologous primary pigment epithelial cells, in the context of two PhaseI/II clinical trials based on an ex vivo gene therapeutic approach for the treatment of neovascular age-related macular degeneration (nAMD).

Xanthomonas axonopodis pv citri (Xac), uma bactéria gram negativa, é a causadora da doença Cancro Cítrico que ocasiona enormes prejuizo na citricultura brasileira. Com o seqüenciamento do genoma deste patógeno foram encontradas inúmeras sequências codificadores com funções desconhecidas. Assim com a finalidade de estudos funcionais do genoma de Xac, foram gerados mutantes randômicos usando o transposon EZ::TN, que induziu disrupção aleatoria de seus genes com o intuito de avaliar que genes inativados afetam a patogenicidade da bactéria. Para o mapeamento do local de inserção do transposon foi desenvolvida uma metodologia baseada na técnica de PCR touchdown utizando oligonucleotídeos semidegenerados. A confiabilidade deste novo método foi comprovada através do mapeamento por Southem blot de alguns mutantes. Conseguiu-se mapear 90 mutantes randômicos com este método. Os testes de patogenicidade em citros mostraram mutantes com sua patogenicidade afetada observando-se variações nos sintomas da doença. Mutantes interessantes contendo uma ORF hipotética inativada ou tendo uma inserção num espaço intergênico foram achadas, sendo também detectados alguns mutantes com nocaute de genes nos seus plasmídeos pXac33 e pXac66. / Xanthomonas axonopodis pv citri (Xac), a gram-negative bacteria, is the causer of the Citrus Canker disease that produces enormous losses to the brazilian citrus sector. Many coding sequences with unknown functions were found in the genome sequence of this pathogen. Therefore, with the goal of functional studies of Xac\'s genome, random mutants using the EZ::TN transposon, have been generated, which carry aleatory disruptions of their genes, with the aim of evaluating inactived genes that potentially affect bacterial pathogenicity. A method based in the PCR touchdown technique using semidegenerate primers was developed for the mapping of the transposon insertion site. The reliability of this new method was tested by means of mapping some mutants using Southern blot. Ninety random mutants were mapped with this method. The pathogenicity tests in citrus showed mutants with their pathogenicity affected and variations in the disease symptoms were observed. Interesting mutants containing an inactive hypothetical ORF or with an insertion in intergenic regions have been found, and also some mutants with inactivated genes in their plasmids pXac33 and pXac66 were detected.

Xanthomonas axonopodis pv citri (Xac), uma bactéria gram negativa, é a causadora da doença Cancro Cítrico que ocasiona enormes prejuizo na citricultura brasileira. Com o seqüenciamento do genoma deste patógeno foram encontradas inúmeras sequências codificadores com funções desconhecidas. Assim com a finalidade de estudos funcionais do genoma de Xac, foram gerados mutantes randômicos usando o transposon EZ::TN, que induziu disrupção aleatoria de seus genes com o intuito de avaliar que genes inativados afetam a patogenicidade da bactéria. Para o mapeamento do local de inserção do transposon foi desenvolvida uma metodologia baseada na técnica de PCR touchdown utizando oligonucleotídeos semidegenerados. A confiabilidade deste novo método foi comprovada através do mapeamento por Southem blot de alguns mutantes. Conseguiu-se mapear 90 mutantes randômicos com este método. Os testes de patogenicidade em citros mostraram mutantes com sua patogenicidade afetada observando-se variações nos sintomas da doença. Mutantes interessantes contendo uma ORF hipotética inativada ou tendo uma inserção num espaço intergênico foram achadas, sendo também detectados alguns mutantes com nocaute de genes nos seus plasmídeos pXac33 e pXac66. / Xanthomonas axonopodis pv citri (Xac), a gram-negative bacteria, is the causer of the Citrus Canker disease that produces enormous losses to the brazilian citrus sector. Many coding sequences with unknown functions were found in the genome sequence of this pathogen. Therefore, with the goal of functional studies of Xac\'s genome, random mutants using the EZ::TN transposon, have been generated, which carry aleatory disruptions of their genes, with the aim of evaluating inactived genes that potentially affect bacterial pathogenicity. A method based in the PCR touchdown technique using semidegenerate primers was developed for the mapping of the transposon insertion site. The reliability of this new method was tested by means of mapping some mutants using Southern blot. Ninety random mutants were mapped with this method. The pathogenicity tests in citrus showed mutants with their pathogenicity affected and variations in the disease symptoms were observed. Interesting mutants containing an inactive hypothetical ORF or with an insertion in intergenic regions have been found, and also some mutants with inactivated genes in their plasmids pXac33 and pXac66 were detected.

Pour étudier la capacité cellulaire à s’adapter aux changements de signalisation dépendante des RTKs, nous utilisons un modèle de souris où l’expression du RTK Met sauvage peut être accrue dans un tissu spécifique. La plupart des tissus se protègent contre cette expression anormale des RTK. Mais certains types cellulaires sont sensibles aux altérations des RTKs, c’est le cas du mésenchyme du membre pendant l’embryogenèse. En effet, l’expression de certains gènes du mésenchyme est modifiée et celui-ci n’est plus accessible aux myoblastes qui le colonisent, conduisant à des déficits des muscles du membre. Chez l’adulte une augmentation de l’expression de Met dans le foie (Alb-R26Met) perturbe l’homéostasie tissulaire, conduisant à la tumorigenèse. Pour identifier des gènes qui coopèrent avec les RTKs pendant l’initiation de la tumorigenèse, nous avons combiné les souris Alb-R26Met avec le système de mutagenèse Sleeping Beauty (SB) transposon. 285 gènes putatifs liés au cancer ont été identifiés. Certains sont des proto-oncogènes ou suppresseurs de tumeurs déjà connus, validant le système. D’autres gènes n’avaient, jusqu’à présent, jamais été associés à ce processus. 9 candidats ont été fonctionnellement validés. Pour identifier des signaux assurant le maintien de la tumeur, nous avons analysé le phosphokinome, testé l’efficacité de composés et identifié de nouvelles combinaisons de drogues qui agissent en synergie pour tuer les cellules cancéreuses dérivées de Alb-R26Met. En conclusion, ces travaux montrent qu’une approche génétique non-biaisée combinée à une approche génomique permet d’identifier de nouveaux mécanismes pertinents pour la biologie du cancer. / We explore the cell competence to deal with slight changes in RTK inputs during embryogenesis and tissue homeostasis using a mouse model in which wild-type RTK Met levels can be moderately enhanced in a tissue specific manner. Most tissues buffer enhanced RTK levels thus avoiding perturbation of developmental programs and tissue homeostasis. Nevertheless, certain cell types are vulnerable to RTK levels. During embryogenesis, the limb mesenchyme is sensitive to alterations of the spatial distribution of RTKs, as illustrated by gene expression changes and by loss of accessibility to incoming myoblasts, which lead to limb muscle defects. At adulthood, liver enhanced Met levels (Alb-R26Met) perturbs tissue homeostasis, leading to tumorigenesis. To uncover new genes that cooperate with RTKs during tumour initiation, we combined Alb-R26Met mice with the Sleeping Beauty (SB) transposon mutagenesis system. 285 putative cancer-related genes have been identified. Some correspond to known proto-oncogenes or tumour suppressors, thus validating the overall strategy we employed for cancer gene discovery. Others have not been previously linked to cancer. 9 new tumour suppressors have been functionally validated, demonstrating the validity of our screen strategy. To identify signals involved in tumour maintenance, we employed a phosphokinome-guided drug screen and identified new synergistic drugs deleterious for cancer cells modelled by the Alb-R26Met genetic setting. The overall strategy and outcomes strengthen the value of combining unbiased genetic and genomic approaches to identify new mechanisms relevant for cancer biology and new therapeutic interventions.

No description available.

Orientador: Claudia Marcia Aparecida Carareto / Banca: Cristina Vieira / Banca: Douglas Silva Domingues / Resumo: Três modelos têm sido propostos para explicar o modo de expansão dos elementos de transposição nos genomas - master gene, transposon e intermediário. O modelo master gene aplica-se à situação em que existe apenas uma única sequência ativa, de uma subfamília particular, que dá origem às demais sequências, as quais não apresentam capacidade de mobilização, enquanto o modelo transposon assume que todas as sequências de uma subfamília são ativas. Por outro lado, o modelo intermediário, considera que algumas cópias apresentam capacidade de mobilização e outras permanecem inativas. Os modelos propostos podem ser relacionados aos mecanismos de transposição, copy-and-paste e cut-and-paste, característicos das classes de elementos transponíveis I e II, respectivamente. Contudo, os estudos de dispersão intragenômica se concentram em elementos da classe I sem LTRs. Com o objetivo de ampliar o entendimento da dinâmica de dispersão genômica dos elementos de transposição e buscando verificar se os modelos propostos para a dispersão dos retroposons ajustam-se aos transposons e aos retrotransposons, foram analisados o transposon Bari e o retrotransposon 412 no genoma das espécies do grupo melanogaster de Drosophila e em populações naturais de D. melanogaster e D. simulans. Assim, buscas por seqüência similares a ambos os elementos selecionados foram realizadas nos genomas seqüenciados de seis espécies do grupo melanogaster; bem como, foram amplificadas, clonadas e sequenciadas cópias presentes nas populações naturais de ambas as espécies. Foram reconstruídas as relações evolutivas entre as sequências dos genomas, como também entre aquelas das populações naturais por meio do programa Network, utilizando o algoritmo Median Joining. Nossos resultados indicam a ausência de um modelo único de dispersão para ambos os elementos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Three models have been proposed to explain the expansion of transposable elements within genomes - master gene, transposon and intermediate. The master gene model applies to situations in which there is only one active sequence of a particular subfamily, which gives rise to other sequences which have no capacity for mobilization, while the transposon model assumes that all sequences of a subfamily are active. On the other hand, the intermediate model considers that some copies have the capacity for mobilization and others remain inactive. The proposed models can be related to mechanisms of transposition, copy-and-paste and cut-and-paste, characteristic of class I and II of transposable elements, respectively. However, studies of intragenomic dispersion concentrate on elements of the class I without LTRs. Aiming at enhancing the understanding of the transposable elements genomic dynamics of dispersion and trying to verify whether the proposed models for dispersion of retroposons fit to transposons and retrotransposons, we analyzed the retrotransposon 412 and Bari transposon in the genome of the species melanogaster group of Drosophila and in natural populations of D. melanogaster and D. simulans. Thus, searches for sequences similar to both elements were done in the sequenced genomes of six species of the melanogaster group; as well as copies present in natural populations of both species were amplified, cloned and sequenced. We reconstructed the evolutionary relationships between the sequences of the genomes, as well as those amplified from samples of wild populations through the Network program, using the Median Joining algorithm. Our results indicate the absence of a single model of dispersion for both transposable elements, Bari and 412, in the different species analyzed, as well as in the populations of both species... (Complete abstract click electronic access below) / Mestre

Bovine herpesvirus-1 (BoHV-1) is an important pathogen of cattle associated with respiratory and reproductive disease and is the most common viral agent implicated in the bovine respiratory disease complex (BRDC). BRDC is an economically significant multifactorial disease of feedlot cattle estimated to cost Australian feedlot producers $AU60 million/year in lost production, therapeutics and disease management. Worldwide BRDC is attributed to cost $US2 billion to cattle industries. In an effort to limit the associated economic costs and enhance animal health and welfare of feedlot cattle, the concerted use of vaccination and diseased animal management are practiced. Numerous vaccines are available in North America and Canada however, in Australia, feedlot producers are reliant on three vaccines. These vaccines target either the bacterial or viral agents of the BRDC and encompass antibody, subunit and attenuated live BoHV-1 preparations. Live attenuated vaccines are developed by numerous methods including, deletion or disruption of certain genes. The development of an attenuated live virus vaccine was traditionally a laborious task requiring numerous rounds of in vitro purification. Contrastingly, technological advances introduced this decade, allowing the stable maintenance of the complete herpesvirus genome in bacteria as a bacterial artificial chromosome (BAC), has advanced herpes virology exponentially in that investigation and manipulation of the herpesvirus genome can be conducted independent of a cell culture system. With respect to BRDC and the generation of vaccines to combat the disease, the tools to fully utilise the potential of BoHV-1 as a live vaccine vector are now routine. It is now possible to vii construct BoHV-1 as a delivery vector by inserting appropriate antigens of those bacterial and viral pathogens implicated in the BRDC into a BAC maintained BoHV-1 genome. However, there is a significant lack of genetic information regarding BoHV-1 and inserting several antigenic sequences would expand the genome of BoHV-1 inducing non-viability. Therefore, to further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and nonessential genes required for the in vitro viability of BoHV-1. Identifying the essential and nonessential genes will establish which genes may be preferentially deleted or replaced with exogenous antigenic sequences in a BoHV-1 derived vaccine vector. To define the requirement of genes encoded by BoHV-1, random-insertion mutagenesis utilising a Tn5 transposition system and targeted gene deletion catalysed by GET recombination was employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion was determined by direct sequencing. with the essential or nonessential requirement of either transposed or deleted open reading frames (ORFs) assessed by transfection of respective BoHV- 1 BAC DNA into host cells. Of the 73 recognised ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be nonessential for virus viability in cell culture with the requirement of the two dual copy ORFs inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by Human herpesvirus 1. However, ORFs encoding for glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to Human herpesvirus-1 encoded homologues. Further analysis of clones encompassing restriction digestion profiling, one-step growth and replication kinetic analysis defined the genetic constitution and replicative capacity of the mutant clones. Thirty-three individual ORFs of the 36 defined nonessential ORF were identified as being amenable to deletion without causing significant replicative detriment to a potential BoHV-1 vaccine vector. This study has provided the foundational information required for the future development of BoHV-1 as a multivalent vaccine vector for the protection of feedlot cattle from BRDC. Furthermore, the genetic information generated in this study contributes to the general knowledge of the prototype ruminant herpesvirus, BoHV-1, and contributes to the comparative study of gene function between the large and diverse family that is Herpesviridae.