Magister Chirurgiae Dentium (MChD) / Chlorhexidine has been established as the gold standard against which new chemical plaque control agents are tested (Jones, 1997). The addition of alcohol in a chlorhexidine mouthwash had been widely used, however the comparative efficacy of alcohol free chlorhexidine mouthwash had not fully been explored in this study, two chlorhexidine mouthwash preparations were tested to evaluate their comparative efficacy in the treatment of periodontal disease. Aims: To assess the efficacy of alcohol-free chlorhexidine mouth wash in comparison to alcohol containing chlorhexidine mouth wash. Objectives: To determine pre- and post- operative clinical parameters and microbial load in the management of patients with chronic periodontitis. Methodology: A double blinded randomised control trial was conducted. Patients diagnosed with active chronic periodontitis were included in the study and randomised to either a test (chlorhexidine without alcohol) or control group (chlorhexidine with alcohol). A total of 50 patients were selected for the study. Results: The Wilcoxon Signed Rank test was used to test the difference between the pre-post pair per clinical indicator and Bana-Zyme. The differences between before and after treatment per indicator were significant at P<0.001 for respectively Paroex and Peridex. These values demonstrated the difference between the clinical parameters taken before the treatment and six weeks post treatment. Conclusion: Both mouth wash solutions with and without alcohol had proven to reduce the microbial load as shown by the BANA-Zyme test, with the alcohol containing solution having been more effective.

In flowering plants, apomixis is asexual reproduction by seeds. Apomixis allows the production of offspring with the same genetic characteristics as the mother plant. Fertilization is not required. Apomixis could become a tool for naturally cloning high-yielding crop hybrids through their own seed. However, apomixis does not occur in major crop plants, except for citrus. In the present study, genes that might cause apomixis in naturally occurring apomictic plants were investigated. Sexual and apomictic species of the genus Boechera were exposed to stressed and non-stressed conditions. Effects of these treatments on the expression of apomixis was then measured. Stress triggered an increase in the frequency of sexual development in apomictic plants, but continuation of sexual development to form sexual seeds did not occur. Stress also triggered alterations in the expression of stress-related genes.

La chromatine embryonnaire subit des changements nécessaires pour l’établissement d’un nouveau programme développemental. Ce travail a étudié l’organisation de l’hétérochromatine au cours du développement sous trois facettes. La première étant celle de d’hétérochromatine constitutive, à travers, l’établissement forcé de la marque H4K20me3 qui provoque un arrêt du développement préimplantatoire. Ce phénotype dépend spécifiquement de l’activité de la methyltransferase SUV4-20h2 et induit l’activation de la voie de signalisation ATR qui bloque la phase de réplication. En deuxième partie, l’hétérochromatine facultative a été le sujet d’une analyse de l’expression des protéines du complexe non-canonique PRC1 et de la modification H2AK119ub qui en résulte. Finalement, une analyse de la chromatine embryonnaire a été mise en place et a permis la détection des changements de niveau de compaction au cours du développement préimplantatoire. / Embryonic chromatin undergoes necessary changes to establish a new developmental program. This work has addressed the organization of heterochromatin in preimplantation embryos from three angles. The first part probed the absence of constitutive heterochromatin by forcing the establishment of the H4K20me3 mark which results in an embryonic arrest prior to the 2-cell stage. This phenotype is due to the specific histone methyl-transferase activity of SUV4-20h2 and is induced by ATR activation which blocks replication. In the second part, facultative heterochromatin was studied by analyzing the levels of several members of the non-canonical PRC1 complex as well as the resultant modification H2AK119ub. Finally, an analysis of the embryonic chromatin was set up and allowed for the measurement of changes in the chromatin openness during preimplantation development.

Bactérias do gênero Methylobacterium são encontradas em associação com espécies vegetais, onde são capazes de promover o crescimento, aumentar a atividade fotossintética e reduzir o ataque de patógenos ao hospedeiro. Além de conferir estas vantagens para a planta hospedeira, estas bactérias podem também produzir biopolímeros (PHA e PHB). Desta forma, o objetivo deste trabalho foi anotar o genoma de Methylobacterium mesophilicum SR1.6/6 e avaliar o seu transcriptoma em estágios iniciais de interação com Citrus sinensis. A análise do genoma mostrou que SR1.6/6 pode produzir auxina, reduzir o estresse da planta alterando os níveis de etileno, apresenta sistema de monitoramento de populacional pelo sistema quorum sensing (QS) e um metabolismo metilotrófico completo. A análise do transcriptoma evidenciou que os exsudatos radiculares de C. sinensis induzem a expressão de genes de resposta ao estresse oxidativo, seguido da indução de genes de adesão e biofilme durante a colonização da planta hospedeira. A interação entre M. mesophilicum SR1.6/6 e a planta hospedeira envolve mecanismos de reconhecimento e adaptação ao estresse, antes mesmo de ocorrer o primeiro contato físico entre a célula bacteriana e a planta hospedeira, seguido da indução de genes de biofilme bacteriano. Além disso, foi estudada uma metodologia para a realização de mutações genéticas em Methylobacterium spp. que permitirá a obtenção de mutantes relacionados com a interação com a planta. / Methylobacterium genus are found in association with plant species, where they are able to promote plant growth, increase the photosynthetic activity and reduce the incidence of pathogens to the host. In addition to providing these benefits to the host plant, these bacteria can also produce biopolymers (PHA and PHB). Thus, the aim was to annotate the genome of Methylobacterium mesophilicum SR1.6 / 6 and assess their transcriptome in the early stages of interaction with Citrus sinensis. Genome analysis showed that SR1.6 / 6 can produce auxin, reduce plant stress by altering ethylene levels, presents population monitoring system (QS) and a complete methylotrophic metabolism. The transcriptomic analysis showed that C. sinensis exudates induce the expression of genes related to oxidative stress followed by induction of adhesion and biofilm genes during colonization of the host plant. The interaction between M. mesophilicum SR1.6 / 6 and the host plant involves recognition mechanisms and adaptation to stress, even before the first physical contact occurs between the bacterial cell and the host plant, followed by the induction of bacterial biofilm genes. Furthermore, a method has been studied for carrying genetic mutations in Methylobacterium spp. allowing the obtaining of mutants related to interaction with the plant.

Les relations complexes qui unissent les insectes phytophages et les plantes peuvent être étudiées par des approches interdisciplinaires à diverses échelles. Ces études peuvent avoir un intérêt appliqué, dans l'agriculture notamment. Au cours de cette thèse, nous avons mené une étude interdisciplinaire sur la biologie de la cécidomyie des fleurs du manguier, Procontarinia mangiferae (Felt) (Diptera : Cecidomyiidae), un bio-agresseur monophage et invasif responsable de dégâts économiques majeurs dans plusieurs zones de production du monde. L'objectif était d'améliorer les connaissances sur la biologie de cette espèce à l'île de la Réunion : (1) en évaluant sa diversité génétique et les facteurs écologiques et biologiques qui peuvent expliquer la structuration génétique de ses populations ; (2) en étudiant en milieu naturel ou contrôlé les caractéristiques de sa diapause, qui lui permettent de maintenir des populations d'une saison de floraison à la suivante ; (3) en étudiant par modélisation la dispersion des femelles dans un verger lors de sa colonisation, en prenant en compte les capacités de vol et la distribution spatiale et temporelle des stades sensibles du manguier au sein du verger. Les résultats ont montré que l'espèce P. mangiferae se reproduisait à la fois sur les inflorescences et sur les jeunes feuilles, qu'elle était présente toute l'année et sur tous les sites échantillonnés sur l'île, quelles que soient les conditions culturales ou climatiques. Ses populations sont apparues structurées en deux clusters sympatriques, dont un était plus fréquent dans la zone de culture du manguier. Ensuite, nous avons prouvé l'existence d'une diapause facultative induite toute l’année, avec cependant un taux d'induction de diapause supérieur en été. Cette diapause du troisième stade larvaire se déroule dans le sol et dure entre six semaines et plus d'un an. Les températures fraiches déclenchent les émergences des individus en diapause et permettent de synchroniser l'émergence des adultes avec la période de floraison du manguier. Enfin, nous avons montré que des femelles immigrantes étaient capables de coloniser l'ensemble des arbres d'un verger. Le vol d'arrivée des femelles dans le verger et le vol actif au sein du verger se sont avérés influencés respectivement par l'abondance et par l'attractivité de la ressource. Les connaissances obtenues sur la biologie de P. mangiferae et sur ses relations avec le manguier ouvrent des pistes pour le développement de stratégies de gestion agroécologique de ce bio-agresseur. / Phytophagous insects and plants are linked by complex relationships. Insect-plant interaction researches involve several biological disciplines at different levels of analysis. These insect–plant relationships are of crucial importance from an applied point of view, notably for agriculture. In this work, an interdisciplinary approach has been used to study on the mango blossom gall midge, Procontarinia mangiferae (Felt) (Diptera: Cecidomyiidae), an invasive insect pest specific to mango causing major economic damages worldwide. The objective was to improve our knowledge of the biology of this species in the subtropical Reunion Island (i) by describing its genetic diversity and investigate the ecological and biological determinants of the genetic structure of its populations, (ii) by carrying out field and controlled experiments to understand the diapause strategies involved in maintaining populations from one flowering season to the next one, (iii) by modeling the arrival and dispersion of females within an orchard in relation with their flight capacity and with the spatial and temporal distribution of the mango susceptible resources. The results showed that the single species P. mangiferae, feeding on both inflorescences and young leaves, was present all year round and in all the sampled sites on the island, regardless of the climatic and cultural conditions. Populations in Reunion Island appeared structured into two clusters in sympatry, one cluster being more frequent in the cultivated mango area. Secondly, we demonstrated the occurrence of facultative diapause all year round, with the highest rate of diapause observed in summer. The diapause allowed a developmental arrest at the 3rd larval instar in the soil, lasting between six weeks to more than one year. The decrease of temperature at the beginning of winter triggered off the emergence of diapausing individuals and synchronized adult emergence with the mango flowering period. Thirdly, non-native female gall midges were able to colonize all the trees of an orchard. Their arrival flight and trivial flight were oriented by the abundance and by the attractiveness of the mango resources, respectively. The knowledge obtained on the biological cycle of P. mangiferae and on its relationships with the mango tree should be useful to develop new agroecological pest management strategies.

Bactérias do gênero Methylobacterium são encontradas em associação com espécies vegetais, onde são capazes de promover o crescimento, aumentar a atividade fotossintética e reduzir o ataque de patógenos ao hospedeiro. Além de conferir estas vantagens para a planta hospedeira, estas bactérias podem também produzir biopolímeros (PHA e PHB). Desta forma, o objetivo deste trabalho foi anotar o genoma de Methylobacterium mesophilicum SR1.6/6 e avaliar o seu transcriptoma em estágios iniciais de interação com Citrus sinensis. A análise do genoma mostrou que SR1.6/6 pode produzir auxina, reduzir o estresse da planta alterando os níveis de etileno, apresenta sistema de monitoramento de populacional pelo sistema quorum sensing (QS) e um metabolismo metilotrófico completo. A análise do transcriptoma evidenciou que os exsudatos radiculares de C. sinensis induzem a expressão de genes de resposta ao estresse oxidativo, seguido da indução de genes de adesão e biofilme durante a colonização da planta hospedeira. A interação entre M. mesophilicum SR1.6/6 e a planta hospedeira envolve mecanismos de reconhecimento e adaptação ao estresse, antes mesmo de ocorrer o primeiro contato físico entre a célula bacteriana e a planta hospedeira, seguido da indução de genes de biofilme bacteriano. Além disso, foi estudada uma metodologia para a realização de mutações genéticas em Methylobacterium spp. que permitirá a obtenção de mutantes relacionados com a interação com a planta. / Methylobacterium genus are found in association with plant species, where they are able to promote plant growth, increase the photosynthetic activity and reduce the incidence of pathogens to the host. In addition to providing these benefits to the host plant, these bacteria can also produce biopolymers (PHA and PHB). Thus, the aim was to annotate the genome of Methylobacterium mesophilicum SR1.6 / 6 and assess their transcriptome in the early stages of interaction with Citrus sinensis. Genome analysis showed that SR1.6 / 6 can produce auxin, reduce plant stress by altering ethylene levels, presents population monitoring system (QS) and a complete methylotrophic metabolism. The transcriptomic analysis showed that C. sinensis exudates induce the expression of genes related to oxidative stress followed by induction of adhesion and biofilm genes during colonization of the host plant. The interaction between M. mesophilicum SR1.6 / 6 and the host plant involves recognition mechanisms and adaptation to stress, even before the first physical contact occurs between the bacterial cell and the host plant, followed by the induction of bacterial biofilm genes. Furthermore, a method has been studied for carrying genetic mutations in Methylobacterium spp. allowing the obtaining of mutants related to interaction with the plant.

Intracellular symbioses between bacteria and insects are numerous, and alter the ecology and evolution of host and symbiont alike. Long-term persistence results from either exploitation (e.g., reproductive manipulations) or mutually beneficial interactions (e.g., nutritional mutualisms). The endosymbiont Hamiltonella defensa, while not essential for growth or survival of healthy aphids, protects aphids from attack by parasitoid wasps. In this thesis, I have used a variety of sequenced-based techniques to illuminate the population and genome dynamics of H. defensa and to disentangle how these factors contribute to its ability to persist and protect its hosts.I characterized the phylogenetic relationships among H. defensa strains from aphids and a whitefly using a multilocus approach. Most loci evolve in a clonal manner, and one cluster of strains may have given rise to an obligate symbiosis. Some H. defensa strains were infected with the bacteriophage APSE, which encodes putative eukaryotic specific toxins and has been suggested to be involved in protecting aphids. I sequenced the toxin locus and the flanking regions from the APSE strains and found that although the phage genome backbone was highly conserved, strains contained non-orthologous toxin-cassettes. Sequenced cassettes contained one of three putative toxin families: Shiga toxin, cytolethal distending toxin, and YD-repeat toxins. A correlation was noted that of several genetically identical H. defensa strains, the one without phage APSE encoding the YD-repeat toxin failed to protect its aphid host. This APSE strain carrying the YD-repeat toxin has since been demonstrated to be essential for protection in several related H. defensastrains.To examine additional bacterial encoded loci that might facilitate the persistence in and protection of aphids by H. defensa, I sequenced the genome of one strain and obtained partial genomes of two additional strains. These genomes exhibit a streamlined metabolism, but are littered with mobile DNA and putative virulence factors. Horizontal gene transfer, recombination and rearrangements are common, and phage and plasmids have played an important role in resorting genes. Thus, although H. defensa benefits its host, its facultative lifestyle has resulted in a pattern of genome evolution associated with reproductive parasites rather than long-term mutualists.

O peso corporal dos animais tende a ser relativamente estável durante longos períodos de tempo. Situações de restrição calórica ou aumento na ingestão de calorias levam a alterações fisiológicas compensatórias que resistem aos efeitos destas perturbações. De fato, o gasto energético aumenta em animais submetidos à dieta hipercalórica, a chamada termogênese facultativa, de modo a manter os estoques energéticos constantes. É possível que defeitos na termogênese facultativa estejam envolvidos no desenvolvimento da obesidade. O BAT, o principal sítio de termogênese facultativa, é ativado pela liberação de NE pelo Sistema Nervoso Simpático, que se liga aos receptores adrenérgicos b1, b2 e b3 expressos nos adipócitos marrons. Diversos estudos demonstram que os receptores b são importantes na proteção contra a obesidade, mas ainda não é claro qual o papel de cada isoforma neste processo. Assim sendo, o objetivo do nosso trabalho foi avaliar o papel das isoformas b1 e b2 na mediação da termogênese facultativa induzida pela dieta. Para tanto, nós tratamos camundongos com nocaute para o receptor adrenérgico b1 (KOb1) e camundongos com nocaute para o receptor b2 (KOb2) com dieta hipercalórica por 22 semanas. O peso corporal foi medido diariamente e o consumo de oxigênio foi determinado usando-se um sistema de respirometria aberto ao final do experimento. A composição corporal foi determinada pela análise da carcaça. Animais foram expostos ao frio de 4ºC por 4h e sua temperatura corporal foi medida em vários tempos e a resposta térmica do iBAT foi determinada pela infusão de NE ou agonista b adrenérgico. Além disso, foram determinados os níveis de RNAm das isoformas de receptores adrenérgicos b nos animais nocaute. Os resultados obtidos em nosso estudo mostram que os animais KOb1 e KOb2 tratados com dieta hipercalórica não desenvolvem obesidade mais severa do que os animais selvagens mas não são capazes de aumentar o consumo de oxigênio induzido pela dieta, sugerindo que estes receptores não são relevantes na termogênese induzida pela dieta. Por outro lado, nossos dados indicam que a presença do receptor b1 é exigida para termogênese induzida pelo frio, uma vez que os camundongos KOb1 são sensíveis ao frio e a capacidade termogênica do BAT destes animais em reposta à NE é bastante reduzida quando comparados com animais selvagens. A ausência do receptor b2 não piora a resposta dos animais ao frio sugerindo que esta isoforma não esteja envolvida na termogênese induzida pela dieta ou pelo frio. Os nossos achados indicam que a isoforma do receptor adrenérgico b1 é fundamental na termogênese induzida pelo frio, mas não pela dieta. Além disso, é provável que a termogênese induzida pela dieta seja regulada por mecanismos distintos da termogênese induzida pelo frio. / The body weight of animals tends to be relatively stable over long periods of time. Situations of caloric restriction or increase in intake of calories lead to compensatory physiological changes that resist the effects of these disorders. In fact, the energy expenditure increases in animals treated with diet hypercaloric called facultative thermogenesis, in order to keep to energy stock constant. Defects in this facultative thermogenesis may be related to the development of obesity. Brown adipose tissue is the main site of facultative thermogenesis and is activated by signaling of b1, b2 e b3 adrenergic receptors by Norepinephrine released by Sympathetic Nervous System. Several studies showed that the isoforms b of adrenergic receptors are important in mechanisms involved in obesity and also in promoting cold tolerance. Nonetheless, it is unclear the role of each isoform in these process. Therefore, the purpose of our study was to evaluate the role of isoforms b1 and b2 in mediate the facultative thermogenesis. For that, we fed nocaute mice for the adrenergic receptor b1 (KOb1) and nocaute mice for the adrenergic receptor b2 (KOb2) with high fat diet for 22 weeks. During treatment body weight was determined daily. By the end of the experiment oxygen consumption was measured using a system of open respirometry and body composition was determined by analysis of the carcass. We also exposed KOb1 and KOb2 animals to cold (4C). The thermogenic response of iBAT was evaluated through i.v NE infusion. The results obtained in our study showed that the animals KOb1 and KOb2 treated with high fat diet did not gain more fat when compared to wild type animals, but are unable to increase the oxygen consumption, suggesting that these receptors are not relevant in development of obesity. Furthermore, our data indicate that the presence of the b1 receptor is required for cold-induced thermogenesis, since the KOb1 mice are sensitive to cold and BAT thermogenic response is significantly impaired when compared with animals wild type. The absence of b2 receptor does not worsen the response of animals to cold suggesting that this isoform is not involved in the diet- or cold- induced thermogenesis. In conclusion, our findings indicate that the b1 isoform of the adrenergic receptor is critical in the cold-induced thermogenesis, but not in diet induced thermogenesis. Moreover, it is likely that the diet-induced thermogenesis and cold-induced thermogenesis are regulated by different mechanisms.

Listeria monocytogenes the causative agent of foodborne listeriosis is a facultative pathogen that lives as a saprophyte in soil and as an intracellular parasite in host tissues. A regulatory protein, the transcriptional activator PrfA, plays a key role in the “saprotroph to parasite” conversion of L. monocytogenes by selectively activating key virulence genes essential for infection when the bacteria enter host cells. Central to this conversion is the plastic ability of PrfA allosterically shift between two states, weakly active (“ON-OFF”, outside in the environment) and strongly active (“ON”, intracellular compartment). In this thesis, I have used the PrfA “ON-OFF” virulence switch to understand the role of virulence regulators in the adaptability of facultative parasites to a wide range of niches. Using the PrfA model, I have also examined the trade-offs between the saprotroph and virulent states of facultative pathogens and the role of plasticity in maintaining adaptation to multiple environments. Using soil as a natural environment model, I have shown that overexpression of the PrfA-dependent virulence regulon has a negative impact on environmental survival of L. monocytogenes. Then I investigated the fitness consequences of losing PrfA switchability in non-host environments. The results in in-vitro growth conditions with isogenic strains with PrfA locked in the “ON” state and in which all the genes of the virulence regulon were deleted, showed that PrfA-dependent gene overexpression causes a reduction in fitness. Our data indicate this was directly attributable to the costs associated with the overproduction of an array of unneeded proteins and not to indirect effects of hyperactive PrfA in Listeria metabolism. Finally, I used experimental evolution studies in in-vitro only conditions and in alternate in-vitro and intracellular conditions with bacteria with wild-type or “ON-locked” PrfA alleles to visualize the selective pressures acting on the PrfA switch. The results of the selection experiments showed that adaptation to the different conditions involves a rapid evolution of PrfA with mutations changing its activity according to the specific environment in which selection occurred. The findings from this thesis highlight the importance of plastic ability, evolution of properly regulated genetic systems and the role of these genetic systems in enabling organisms to maximise their fitness during the adaptation process to a specific niche.

abstract: Parental care provides many benefits to offspring. One widely realized benefit is enhanced regulation of offspring's thermal environment. The developmental thermal environment during development can be optimized behaviorally through nest site selection and brooding, and it can be further enhanced by physiological heat production. In fact, enhancement of the developmental thermal environment has been proposed as the initial driving force for the evolution of endothermy in bird and mammals. I used pythons (Squamata: Pythonidae) to expand existing knowledge of behavioral and physiological parental tactics used to regulate offspring thermal environment. I first demonstrated that brooding behavior in the Children's python (Antaresia childreni) is largely driven by internal mechanisms, similar to solitary birds, suggesting that the early evolution of the parent-offspring association was probably hormonally driven. Two species of python are known to be facultatively thermogenic (i.e., are endothermic during reproduction). I expand current knowledge of thermogenesis in Burmese pythons (Python molurus) by demonstrating that females use their own body temperature to modulate thermogenesis. Although pythons are commonly cited as thermogenic, the actual extent of thermogenesis within the family Pythonidae is unknown. Thus, I assessed the thermogenic capability of five previously unstudied species of python to aid in understanding phylogenetic, morphological, and distributional influences on thermogenesis in pythons. Results suggest that facultative thermogenesis is likely rare among pythons. To understand why it is rare, I used an artificial model to demonstrate that energetic costs to the female likely outweigh thermal benefits to the clutch in species that do not inhabit cooler latitudes or lack large energy reserves. In combination with other studies, these results show that facultative thermogenesis during brooding in pythons likely requires particular ecological and physiological factors for its evolution. / Dissertation/Thesis / Ph.D. Biology 2012