Orientador : Jose Francisco Hofling / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-03T15:09:29Z (GMT). No. of bitstreams: 1 Mondin_MagdaElizabethBaglioniGouvea_M.pdf: 3209793 bytes, checksum: c8e5deb73e22171b11dfa7389bf7d3c3 (MD5) Previous issue date: 2003 / Resumo: A cárie precoce na infância, especificamente aquela associada ao consumo de carboidratos fermentáveis em mamadeira, popularmente conhecida como síndrome da cárie de mamadeira, representa um dos mais prevalentes problemas relacionados à saúde bucal de crianças durante a primeira infância e tem demonstrado ser um fator local que favorece a proliferação de Candida spp. na saliva e biofilme dental. O presente estudo teve por objetivo determinar a prevalência de Cândidas spp. a espécie predominante e o padrão da colonização na cavidade bucal de um grupo de crianças com cárie precoce na infância e um grupo de crianças livres de cárie, relacionando aos aspectos clínicos (índice ceos e presença de lesões de cárie nos incisivos e/ou molares), além de investigar o efeito do tratamento dental sobre a prevalência desses microrganismos. Foram avaliadas inicialmente 64 crianças na faixa etária de 2 a 3 anos, incluindo 32 crianças livres de cárie (grupo GH ou Grupo controle) e 32 crianças com precoce na infância (grupo GC), que não haviam recebido tratamento dental ou orientações sobre higiene bucal anterior ao inicio do estudo. Participaram da avaliação pós-tratamento dental, 31 crianças da amostra inicial, das quais 16 do grupo GC, que terminaram efetivamente o tratamento dental e 15 do grupo GH. Nos dias determinados para cada tempo experimental, pré e pós-tratamento dental, foram coletadas amostras intrabucais (mucosa jugal, dorso da língua, palato duro, biofilme dental e saliva) de todas as crianças para determinação da contagem de UFC/ml e identificação das espécies de Cândida, através dos testes de produção do tubo germinativo, produção de clamidósporos, termotolerância, crescimento em meio presuntivo CHROMagar® Candida, fermentação e assimilação de carboidratos. Houve prevalência de Candida spp. em 71,87% das crianças com cárie precoce na infância, comparada a 6,25% das crianças livres de cárie, sendo que a contagem de UFC/rnl foi significativamente maior (p < 0,05) em crianças cb grupo GC que apresentaram índice ceos elevado e presença de lesões de cárie nos incisivos e molares. A espécie predominante em ambos os grupos foi C. albicans, seguida por C. tropicalis, com maior prevalência no biofilme dental de crianças do grupo GC e no dorso da língua de crianças do grupo GH. O tratamento dental restaurador diminui a contagem de UFC/rnl de Candida spp. na cavidade bucal de crianças com cárie precoce na infância, porém não elimina completamente esses microrganismos / Abstract: The early childhood caries, mainly associated with the consumption of sweetened fluids with fermentable carbohydrates, usually from a bottle, is one of the most significant oral health problems in children and it has been suggested to be a local factor to increase the saliva and dental biofilm incidence of Candida spp. The present investigation aimed to determine the prevalence of Candida spp., the predominant specie and the colonization standard in the mouth of a group of children with early childhood caries and a group of free- caries children in connection with clinical factors (decay index - dmfs and presence of caries lesions), furthermore to investigate the effect of the dental treatment on oral Candida levels. A total of 64 children, who were 2 to 3 years old, participated of the initial sample collection. The children were distributed into two groups, the group GC included 32 children with early childhood caries and the group GH (or control group) included 32 caries-free children. Thirty-one children, remain from initial sample, 16 children from the group GC and 15 children from the group GH, participated of the second sample collection (post-treatment dental). The children had never received any previous dental treatment or received oral hygiene instructions. Oral sample was collected from saliva, tongue surface, cheeks, hard palate and dental biofilm at baseline (before dental treatment) and after dental treatment. After the characteristic growth, counting cells (CFU/ml) a subsequent identification of the species by germ tube test, microscopic morphology, growth on CHROMagar@ Candida and carbohydrate assimilation and fermentation tests, were performed. The results showed Candida spp. was more frequently isolated in children with nursing caries (71.87%) than those caries- free children (6.25%). Candida spp. was significantly more abundant when the index dmfs were greater and the caries lesions were present in the upper incisors and molars. The specie C. albicans was the most frequently detected oral yeast species, with C. tropicalis the next frequency, with prevalence on dental biofilm of children with nursing caries and on tongue of free-caries children. Children with early childhood caries and with oral C. albicans maintained carriage even after dental treatment, however the dental treatment reduces counting cells (UFC/rnl) of oral Candida sp / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental

The human body is colonized by trillions of microbes from all three domains of life – eukaryotes, bacteria and archaea. The lower gastrointestinal tract is the most densely colonized part of the body, harbouring a diverse and dynamic community of microbes. While the importance of bacteria in this so-called microbiota is well acknowledged, the role of commensal fungi remains underexplored. The most prominent fungus of the human gastrointestinal microbiota is Candida albicans. This fungus occasionally causes life-threatening disseminated infections in individuals with debilitated immune defences. It is this “pathogenic” facet that has received the most attention from researchers in the past, leaving many aspects of its “commensal” lifestyle understudied. Using gnotobiotic mice as a model system to explore the biology of C. albicans in the mammalian gut, in this dissertation I establish the global response of the host to C. albicans monocolonization as well as the spatial distribution of the fungus in the intestine in the context of co-colonization with single gut bacterial species. The fungus elicited transcriptome changes in murine intestinal tissue, which included the activation of a reactive oxygen species-related defence mechanism and the induction of regulators of the circadian clock circuitry. Both responses have previously been described in the context of a complete bacterial microbiota. Imaging the intestine of animals monocolonized with the fungus or co-colonized with C. albicans and the gut bacteria Bacteroides thetaiotaomicron or Lactobacillus reuteri revealed that the fungus was embedded in a B. thetaiotaomicron-promoted outer mucus layer in the murine colon. The gel-like outer mucus constitutes a unique microhabitat, distinct in microbial composition from the adjacent intestinal lumen. This finding indicates that bacteria can shape the specific microhabitat occupied by the fungus in the intestine. Overall, the results described in this dissertation suggest that gnotobiotic mice constitute a valuable tool to dissect multiple aspects of the interactions among host, commensal fungi and cohabiting bacteria. / Der menschliche Körper beheimatet Billionen von Mikroorganismen aus allen drei Domänen des Lebens – Eukaryoten, Bakterien und Archaeen. Der menschliche Dickdarm ist dabei das am dichtesten besiedelte Organ des Körpers. Er beherbergt eine sehr diverse und dynamische Gemeinschaft von Mikroorganismen. Während die Relevanz der so genannten Mikrobiota weitreichend anerkannt ist, wurde die Bedeutung der kommensalen Pilze bisher noch vernachlässigt. Der am häufigsten vorkommende Pilz im menschlichen Gastrointestinaltrakt ist Candida albicans. Der Hefepilz kann vor allem in Patienten mit geschwächtem Immunsystem zum Ursprung lebensbedrohlicher Krankheiten werden. Diese Pathogenität des Pilzes wurde in den letzten Jahren ausführlich beleuchtet, wohingegen die kommensalen Aspekte des Pilzes vernachlässigt wurden. In dieser Dissertation habe ich, mit Hilfe von gnotobiotischen Mausmodellen, (i) das Leben von C. albicans im Säugetierdarm untersucht, (ii) die Reaktion des Wirts auf diese Kolonisierung ermittelt, (iii) sowie das Kolonisierungsverhalten und die Interaktionen von verschiedenen Darmbakterienspezies und C. albicans erforscht. Durch die alleinige Kolonisierung des Darms mit Candida albicans konnte ich Veränderungen im Transkriptom der Darmepithelzellen nachweisen, wie etwa die Aktivierung einer reaktiven Sauerstoffspezies-vermittelten Wirtsantwort und die Induktion von mehreren Regulatoren des zirkadianen Rhythmus. Beide dieser Wirtsreaktionen wurden zuvor nur in der Anwesenheit einer bakteriellen Mikrobiota nachgewiesen. Durch die Visualisierung der Pilzzellen im Darm während der Monokolonisierung, sowie während der Kolonisierung des Darms mit dem Pilz und den Darmbakterien, Bacteroides thetaiotaomicron oder Lactobacillus reuteri, konnten wir zeigen, dass der Pilz den von B. thetaiotaomicron neu geschaffenen Lebensraum, die äußere Mukusschicht, besiedeln kann. Diese äußere gelartige Mukusschicht ist ein einzigartiger Lebensraum, welcher sich stark von der inneren, sterilen Mukusschicht unterscheidet. Diese Erkenntnisse deuten darauf hin, dass einzelne Bakterienspezies den Lebensraum des Pilzes verändern können. Zusammenfassend konnte ich feststellen, dass die gnotobiotischen Mausmodelle sich sehr gut eignen, um die unterschiedlichen Aspekte der kommensalen Kolonisierung des Darms durch C. albicans, sowie dessen Interaktionen mit dem Wirt und den anwesenden Bakterien zu untersuchen.

The human body is laden with trillions of microorganisms that belong to all three domains of life. Some species of this microbiota subsist as harmless commensals in healthy adults, but under certain circumstances, they can cause mucosal disease or even systemic, life-threatening infections. While the bacterial members of our microbiota are heavily studied today, much less attention is afforded to eukaryotic species that colonize different mucocutaneous surfaces of the human body. This dissertation focuses on identifying regulatory circuits that enable a prominent member of these eukaryotes, C. albicans, to, on the one hand, live on a specific mammalian mucosal surface as a harmless commensal and, on the other hand, proliferate as a pathogen. Since the ultimate source of many fatal Candida infections is the gastrointestinal (GI) tract of the infected individual, this organism is particularly suited to distinguishing traits essential for the gut colonization of commensal fungi and their ability to cause disease. Sequence-specific DNA-binding proteins that regulate transcription are important to most biological processes; I thus used these proteins as starting points to gain insights into 1) how a specific transcription regulator promotes virulence in C. albicans; 2) which traits C. albicans requires to inhabit the GI tract of a specific, well-defined mouse model as a harmless commensal; and 3) how three previously undescribed transcriptional regulators contribute to the commensal colonization of the digestive tract of this mouse model. Altogether, this work advances the knowledge concerning the biology of commensal fungi in the mammalian gut and genetic determinants of fungal commensalism, as well as pathogenicity. / Der menschliche Körper wird von unzähligen Mikroorganismen aus allen drei Domänen des Lebens besiedelt. Einige Spezies dieser so genannten Mikrobiota leben mit gesunden Menschen als harmlose Kommensale, können jedoch unter bestimmten Umständen auch Erkrankungen der Schleimhäute oder sogar systemische, lebensbedrohliche Krankheiten verursachen. Der bakterielle Anteil unserer Mikrobiota wurde bereits ausgiebig untersucht. Sehr viel weniger Aufmerksamkeit haben bisher eukaryotische Organismen erlangt, die unterschiedliche Schleimhäute des menschlichen Körpers besiedeln. Ziel dieser Dissertation ist es regulatorische Kreisläufe zu identifizieren, die es einem prominenten eukaryotischen Mitglied unserer Mikrobiota, Candida albicans, ermöglichen auf der einen Seite eine spezielle mukokutane Oberfläche von Säugern zu besiedeln, und sich auf der anderen Seite als Pathogen zu verbreiten. Da man annimmt, dass viele bedrohliche Candida Infektionen ihren Ursprung im Gastrointestinaltrakt desselben Individuums haben, eignet sich dieser Organismus im speziellen um Eigenschaften zu identifizieren, die es kommensalen Pilzen ermöglicht den Darm zu besiedeln aber auch Krankheiten zu verursachen. Sequenz-spezifische DNA Bindeproteine, die die Transkription regulieren sind zentrale Akteure in den meisten biologischen Prozessen; aus diesem Grund verwende ich diese Proteine als Startpunkte um Einblicke in Folgendes zu erlangen: Zuerst, wie ein spezieller Transkriptionsregulator C. albicans‘ Virulenz beeinflusst. Dann welche Eigenschaften von C. albicans Voraussetzung für die Kolonisierung des Gastrointestinaltrakts eines klar definierten Mausmodells sind. Und zuletzt wie drei bisher nicht beschriebene Transkriptionsregulatoren zu der kommensale Kolonisierung des Verdauungstrakts dieses Mausmodells beitragen. Zusammenfassend trägt diese Arbeit dazu bei, das Wissen über die Biologie kommensaler Pilze im Säugertrakt und über genetische Determinanten zu erweitern, die zum Kommensalismus aber auch zur Pathogenität von Pilzen beitragen.

Regulation of gene expression by the control of transcription is essential for any cell to adapt to the environment and survive. Transcription regulators, i.e. sequence-specific DNA binding proteins that regulate gene expression, are central elements within the gene networks of most organisms. Transcription regulators are grouped into distinct families based on structural features that determine, to a large extent, the DNA sequence(s) that they can recognise and bind. Less is known, however, about how the DNA binding preferences can diversify within transcription regulator families during evolutionary timescales, and how such diversification can affect the biology of the organism. In this dissertation I study the SREBP (sterol regulatory element binding protein) family of transcriptional regulators in yeasts, and in Candida albicans in particular, as an experimental system to address these questions. The SREBPs are conserved from fungi to humans and represent a subgroup of basic helix-loop-helix DNA binding proteins. Early chromatin immunoprecipitation experiments with SREBPs from humans and yeasts showed that these proteins bound in vivo to the canonical DNA sequence, termed E-box, most basic helix-loop-helix proteins bind to. By contrast, most recent analysis carried out with less-studied fungal SREBPs revealed a non-canonical DNA motif to be the most overrepresented sequence in the bound regions. This study aims to establish the intrinsic DNA binding preferences of key branches of this family and to determine how the divergence in DNA binding affinities originated. To this end, I combined phylogenetic and ancestral reconstruction with extensive biochemical characterisation of key SREBP proteins. The results indicated that while the most-studied SREBPs (in mammals) indeed show preference for the E-box, a second branch of the family preferentially binds the non-E-box, and a third one is able to bind both sequences with similar affinity. The preference for one or the other DNA sequence is an intrinsic property of each protein because their purified DNA binding domain was sufficient to recapitulate their in vivo binding preference. The ancestor that gave rise to these two different types of SREBPs (the branch that binds E-box and the one that binds non-E-box DNA) appears to be a protein with a broader DNA binding capability that had a slight preference for the non-canonical motif. Thus, the results imply these two branches originated by either enhancing the original ancestral preference for non-E-box or tilting it towards the E-box DNA and flipping the preference for this sequence. The main function associated with members of the SREBP family in most eukaryotes is the control of lipid biosynthesis. I have further studied the function of these proteins in the lineage that encompasses the human associated yeast C. albicans. Strikingly, the three SREBPs present in the fungus’ genome contribute to the colonisation of the mammalian gut by regulating cellular processes unrelated to lipid metabolism. Here I describe that two of the three C. albicans SREBPs form a regulatory cascade that regulates morphology and cell wall modifications under anaerobic conditions, whereas the third SREBP has been shown to be involved in the regulation of glycolysis genes. Therefore, I posit that the described diversification in DNA binding specificity in these proteins and the concomitant expansion of targets of regulation were key in enabling this fungal lineage to associate with animals. / Für jede Zelle ist es essenziell die Transkription über die Genexpression zu regulieren, um sich an unterschiedliche Lebensbedingungen anzupassen. Regulatoren der Transkription, zum Beispiel sequenzspezifische DNA-binde Proteine, sind ein zentrales Element des Genregulationsnetzwerks in den meisten Organismen. Auf Grund ihres Aufbaus sowie der daraus resultierenden spezifischen Eigenschaften DNA zu binden, werden diese Regulatoren in unterschiedliche Familien unterteilt. Bisher ist wenig darüber bekannt, wie unterschiedlich die DNA Sequenzen sein können, welche von einer Familie von Transkriptionsregulatoren gebunden werden, wie sich diese Diversität der Bindung in der Evolution über die Zeit verändert hat und ob diese unterschiedlichen Bindeaffinitäten die Biologie eines Organismus beeinflussen. In dieser Dissertation befasse ich mich mit der Transkriptionsregulator Familie der SREBPs (sterol regulatory element binding protein) in Hefen, als Modelorganismus diente dabei Candida albicans. Die Familie der SREBPs ist vom Pilz zu den Menschen genetisch weitestgehend konserviert und repräsentiert eine Unterfamilie der Helix-loop-helix DNA-binde Proteine. Erste Chromatin-Immunpräzipitation Experimente der SREBPs in Menschen und Hefen zeigen in vivo eine Bindung an eine kanonische DNA Sequenz genannt E-box, welche von den meisten der Helix-loop-helix Proteine gebunden wird. Im Gegensatz zeigen neuere Analysen, welche mit weniger bekannten SREBPs aus Pilzen durchgeführt wurden, dass hauptsächlich nicht-kanonische DNA Sequenzen gebunden werden. Diese Arbeit versucht die Präferenzen, mit welchen einige der wichtigsten Mitglieder der Familie der SREBPs an bestimmte DNA Sequenzen binden aufzudecken und heraus zu finden wie es innerhalb dieser Gruppe zu unterschiedlichen Bindungsaffinitäten kam. Dafür wurden phylogenetische Rekonstruktionsanalysen und aufwändige biochemische Charakterisierungen einiger der Proteine der SREBP Familie durchgeführt. Die Ergebnisse zeigen, dass die meisten der bisher charakterisierten SREBPs (in Säugetieren) es vorziehen an die E-box Sequenz zu binden, ein anderer Zweig des SREBP Familienstammbaums bevorzugt hingegen die non-E-box Sequenz, ein dritter Zweig des Stammbaums ist in der Lage beide Sequenzen mit gleicher Affinität zu binden. Das Bevorzugen einer der beiden DNA Sequenzen ist eine natürliche Eigenschaft des jeweiligen Proteins, da in Experimenten die isolierte DNA-binde Domäne der Proteine ausreichend war, um die in vivo Bindepräferenzen zu replizieren. Der Ursprung dieser beiden Gruppen (der E-box bindenden Gruppe und der Gruppe die non-E-box Sequenzen bindet) liegt wahrscheinlich in einem Protein, welches beide Sequenzen binden konnte, mit einem Vorzug für die nicht-kanonische Sequenz. Dies impliziert, dass die Gruppen entstanden sind indem sich entweder eine Präferenz des Vorgängerproteins für die nicht-kanonische Sequenz durchgesetzt hat oder, dass sich eine Präferenz für die E-box bindende Sequenz durchgesetzt hat und somit die Affinität dahingehend verschoben wurde. Die Hauptfunktion der meisten Proteine der SREBP Familie in Eukaryoten ist die Kontrolle der Lipid Biosynthese. In meiner Arbeit habe ich mich auf die Erforschung der SREBPs in einer Gruppe von Organismen zugewandt, die auch den mit dem Menschen assoziierten Hefepilz Candida albicans umfasst. Erstaunlicherweise beeinflussen die drei SREBPs die im Candida albicans Genom zu finden sind, die Kolonisierung des Säugetierdarms, jedoch nicht durch die Kontrolle der Lipid Biosynthese. Im Folgenden werde ich beschreiben wie zwei der drei SREBPs aus Candida albicans eine regulatorische Kaskade bilden, welche Einfluss auf die Regulierung der Morphologie und der Zellwandzusammensetzung des Pilzes unter anaeroben Bedingungen hat, wohingegen das dritte Protein der SREBP Familie für die Regulierung der Glykolyse von Bedeutung ist. Ich habe festgestellt, dass die beschriebene Vielfalt mit der diese Proteine an bestimmte DNA Sequenzen binden und die damit einhergehende Expansion der regulierbaren Ziele ein wesentlicher Grund dafür ist, dass Organismen dieses Stammbaums erfolgreich Säugetiere kolonisieren können.

The fungus Candida albicans is a typical member of the human microbiota, where it usually behaves as a commensal. It can also become pathogenic; often causing minor superficial infections in healthy people, but also potentially fatal invasive systemic infections in immunocompromised people. Unfortunately, there is only a fairly limited set of antifungal drugs, and evolution of drug resistance threatens their efficacy. Greater understanding of the mechanisms that C. albicans uses to survive in and infect the host can uncover candidate targets for novel antifungals. Protein kinases are central to a vast array of signalling pathways which govern practically all aspects of life, and furthermore are relatively straightforward to design drugs against. As such, investigation and characterization of protein kinases in C. albicans as well as their target proteins and the pathways they govern are important targets for research. AMP-activated kinases are well conserved proteins which respond to energy stress; they are represented in yeasts by the heterotrimeric SNF1 complex, which responds primarily to the absence of glucose. In this work, the SNF1 pathway was investigated with two primary goals: identify novel targets of this protein kinase and elucidate why SNF1 is essential. Two approaches were used to identify novel targets of SNF1. In one, suppressor mutants were evolved from a strain in which SNF1 activity is reduced, which exhibits defects in carbon source utilization and cell wall integrity. This revealed a suppressor mutation within SNF1 itself, coding for the catalytic subunit of the complex – SNF1Δ311-316. The second approach screened a library of artificially activated zinc cluster transcription factors, identifying Czf1 as one such transcription factor which, upon artificial activation, restored resistance to cell wall stress in a mutant of the SNF1 pathway. Finally, a, inducible gene deletion system revealed that SNF1 is not an essential gene. / Der Pilz Candida albicans ist ein typisches Mitglied der menschlichen Mikrobiota, wo er sich normalerweise als Kommensale verhält. Als fakultativ pathogener Erreger kann er jedoch auch leichte, überfachliche Infektionen bei gesunden Menschen verursachen, sowie potenziell tödliche, invasive systemische Infektionen bei immungeschwächten Menschen. Leider gibt es nur eine recht begrenzte Anzahl von Antimykotika, und die Entwicklung von Resistenzen bedroht deren Wirksamkeit. Ein besseres Verständnis der Mechanismen, die C. albicans nutzt, um im Wirt zu überleben und ihn zu infizieren, kann mögliche Angriffspunkte für neue Antimykotika aufdecken. Proteinkinasen sind von zentraler Bedeutung für eine Vielzahl von Signalwegen, die praktisch alle Aspekte des Lebens steuern und gegen die sich zudem relativ einfach Medikamente entwickeln lassen. Daher ist die Untersuchung und Charakterisierung von Proteinkinasen in C. albicans sowie ihrer Zielproteine und der von ihnen gesteuerten Signalwege ein wichtiges Ziel für die Forschung. AMP-aktivierte Kinasen sind hoch konservierte Proteine, die auf Energiestress reagieren; sie sind in Hefen durch den heterotrimeren SNF1-Komplex vertreten, der vor allem auf das Fehlen von Glukose reagiert. In dieser Arbeit wurde der SNF1-Signalweg mit zwei primären Zielen untersucht: die Identifizierung neuer Zielproteine dieser Proteinkinase und die Klärung der Frage, warum SNF1 essentiell ist. Für die Identifikation neuer Zielproteine von SNF1 wurden zwei Ansätze verwendet. Zum einen wurde ein Stamm mit reduzierter SNF1-Aktivität, für die Entwicklung von Suppressor-Mutanten verwendet, die einen Defekte bei der Verwertung von Kohlenstoffquellen und eine eingeschränkte Zellwandintegrität aufweisen. Dabei wurde eine Suppressormutation in SNF1 selbst entdeckt, die für die katalytische Untereinheit des Komplexes – SNF1Δ311-316 - kodiert. Für den zweite Ansatz wurde eine Bibliothek von künstlich aktivierten Zink-Cluster-Transkriptionsfaktoren untersucht. Dies führte zur Identifikation von Czf1 als einen solchen Transkriptionsfaktor, der nach künstlicher Aktivierung die Resistenz gegen Zellwandstress in einer Mutante des SNF1- Signalweges wiederherstellte. Schließlich zeigte ein induzierbares Gendeletionssystem, dass SNF1 kein essentielles Gen ist.

L’homéostasie de la taille est un processus important de la prolifération cellulaire mais les mécanismes moléculaires sont mal compris. Les cellules eucaryotes doivent atteindre une taille seuil avant la division, ce qui permet de maintenir une taille constante sur le long terme. Ce processus est régulé à un point de contrôle, à la fin de la phase G1, appelé START chez les levures. Le contrôle de la taille cellulaire a été étudié chez la levure modèle Saccharomyces cerevisiae mais n’a jamais été étudié chez les levures pathogènes. Dans cette thèse, nous avons utilisé Candida albicans comme organisme modèle pour étudier la régulation de la taille chez les levures opportunistes. Nous avons criblé des collections de mutants de délétions hétérozygotes et homozygotes de C.albicans afin d’identifier des gènes régulateurs de la taille. Nous avons analysé la distribution de taille de 279 mutants homozygotes et 4 348 mutants hétérozygotes (recouvrant 90% du génome). Nous avons comparé nos résultats à différents criblages effectués sur la levure modèle S.cerevisiae. Ces comparaisons montrent que peu de régulateurs sont conservés entre C. albicans et S. cerevisiae et que la régulation de la taille est processus très plastique. Par exemple, le mutant dot6 a un phénotype petit chez C. albicans mais n’a pas de phénotype de taille chez S. cerevisiae. Nous avons montré que Dot6 est un facteur de transcription nécessaire pour l’activation des gènes de la biogénèse des ribosomes. Dot6 est également un régulateur de START et joue un rôle dans l’adaptation de la taille suivant les sources de carbone disponibles. Nous avons également mis en évidence un nouveau rôle pour la protéine kinase Hog1/p38 dans la régulation de la taille chez C. albicans en absence de stress. Ce rôle n’a jamais été démontré chez S. cerevisiae. Nous avons montré que Hog1, ainsi que toute la voie HOG, sont des régulateurs négatifs de START. Nous avons mis en évidence que Hog1 régule à la fois la croissance cellulaire via Sfp1, un régulateur majeur de la biogénèse des ribosomes et des protéines ribosomales, et le cycle cellulaire via le complexe SBF (Swi4/Swi6), des facteurs de transcription nécessaires pour la transition G1/S. Nous avons également découvert qu’Ahr1, un facteur de transcription n’ayant pas d’orthologue chez S. cerevisiae, est nécessaire pour la régulation de la taille et aussi requis pour l’adaptation de la taille en fonction des acides aminés disponibles. Nous avons montré qu’Ahr1 agit dans la voie Tor1-Sch9 et régule négativement START. En conclusion, notre travail a permis de découvrir de nouveaux régulateurs de START, de caractériser leur fonction et de les placer dans différentes voies. Comme la dérégulation de la voie Hog1/p38 est associée à des pathologies humaines, nous proposons C.albicans comme organisme modèle pour l’étude de cette voie et son implication dans l’homéostasie de la taille chez les organismes eucaryotes. / Cell size homeostasis is an important process of cell proliferation but the molecular mechanisms are poorly understood. Eukaryotic cells must reach a threshold size before entering the cell cycle, which helps to maintain a constant size over the long term. This process is regulated at the end of the G1 phase, a check point called START. Cell size control has been studied in the model yeast Saccharomyces cerevisiae but has never been studied in pathogenic fungi. In this thesis, we used Candida albicansas a model organism to study the regulation of size in pathogenic yeasts. We have screened heterozygous and homozygous deletion collections of C. albicans to identify genes that control cell size. We analyzed the size distribution of 279 homozygous mutants and 4,348 heterozygous mutants (covering 90% of the genome). We compared our results with different screens performed on the model yeast S.cerevisiae. These comparisons showed that few regulators were conserved between S. cerevisiaeand C. albicans and suggesting that the cell size regulation is evolutionary plastic. For example, dot6 mutant has a small phenotype in C. albicansbut has no size phenotype in S. cerevisiae. We have shown that Dot6 is a transcriptional factor necessary for the activation of ribosome biogenes is genes. Dot6 is also a regulator of START and plays a critical role in adapting size according to the carbon sources available in the medium. We also uncovered a novel stress-independent role of the Hog1/p38 MAPK in size regulation in C. albicansa role that has never been demonstrated in S. cerevisiae. We have shown that Hog1, as well as the entire HOG pathway, are negative regulators of START. We have shown that Hog1 regulates both growth via Sfp1, a major transcriptional regulator of ribosomal biogenesis and ribosomal proteins, and the cell cycle via the SBF complex (Swi4/Swi6), transcriptional factors necessary for the G1/S transition. We also found that Ahr1, a transcription factor with no obvious ortholog in S. cerevisiae, has a role for the adaptation of the size according to the amino acids available in the medium. We have shown that Ahr1 is a negative START regulator and is controlled by the Tor1-Sch9 pathway. In conclusion, our work has permitted to discover new regulators of START, to characterise their function and to map them in different pathways. As the Hog1/p38 pathway is linked to many human pathologies, we think that C.albicansis a useful model to study of this pathway and dissect its role in size control in eukaryotes.

Candida albicans is a polymorphic fungus that can grow as yeast (Y) and hypha (H). The Y-H morphological switching is controlled through the MAPK, Cek1p. With the prior work showing that occidiofungin prevents C. albicans from forming hypha when added at the time of Y-H switching, we aim to identify the impact of occidiofungin on signaling events associated with morphological switching specifically looking at Cek1 MAPK cascade. Results from this work have demonstrated that Cek1 MAPK is not required for occidiofungin bioactivity. Further, we report that morphologically switching cells are more sensitive to occidiofungin than their non-switching counterparts. Moreover, later stages of hyphal formation exhibit increased sensitivity towards occidiofungin suggesting occidiofungin targets hyphal initiation and/or elongation process. This work also demonstrates that addition of occidiofungin beyond a discrete time window required for hyphal initiation has minimal effect on hyphae formation and elongation.

Les buts de notre projet étaient d'étudier les effets de certains polyphenols de plantes sur la croissance, la survie, la formation du biofilm et la transition blastospore-hyphe de C. albicans, une levure responsable des candidoses humaines. Ce projet a permis de démontrer que deux polyphenols naturels de la réglisse, la licochalcone A et la glabridine, ainsi qu'un polyphenol synthétisé en laboratoire, le 4-hydroxycordoin, ont des propriétés intéressantes contre certains facteurs de virulence de C. albicans. La licochalcone A et la glabridine ont montré un effet fongicide et une action synergique avec le nystatin. La licochalcone A et le 4-hydroxycordoin ont démontré une capacité d'inhiber la formation du biofilm, alors que les trois polyphenols ont montré un effet inhibiteur sur la transition blastospore-hyphe de C. albicans. Ces molécules possèdent donc un potentiel pour le traitement ou la prévention d'infections fongiques buccales à C. albicans.

Existem poucos estudos a respeito da presença de constituintes fúngicos na circulação sanguínea de indivíduos com estomatite por dentadura (ED) (AHMAD et al., 2002), considerada uma forma localizada de candidose; o que poderia caracterizar o poder de invasão sistêmica do fungo nesta condição local, bem como um prévio reconhecimento desses antígenos por células presentes na circulação sanguínea do hospedeiro, o que poderia explicar aspectos específicos da resposta imune localizada e sistêmica. Assim sendo, este trabalho teve por objetivo avaliar a presença do fungo Candida albicans (C. albicans) no palato, na superfície interna das próteses totais superiores e no sangue de pacientes com ED, em dois momentos distintos. A população de estudo foi composta por indivíduos usuários de prótese total superior (PTS), com e sem estomatite por dentadura, avaliados e selecionados nas clínicas de graduação e pós-graduação da Disciplina de Prótese, do Departamento de Prótese, da Faculdade de Odontologia de Bauru da Universidade de São Paulo (FOB USP). Indivíduos não usuários de próteses removíveis constituíram o grupo controle. Assim o trabalho foi constituído por três grupos, cada um com 14 pacientes. As lesões de estomatite por dentadura foram diagnosticadas clinicamente e por meio de confirmação microbiológica em CHROMAgar Candida, a partir de material biológico coletado da mucosa palatal e da superfície interna da PTS. A PCR foi realizada quando da ocorrência do crescimento de colônias verdes para diferenciação das espécies C. albicans e C. dubliniensis. As amostras de sangue foram analisadas para a detecção de fragmentos de DNA responsáveis pela codificação da proteína da parede da hifa1(Hwp1) de C. albicans, utilizando a técnica da PCR. Os resultados demonstraram que nem os usuários de PTS, independentemente da presença de ED, nem os voluntários não usuários apresentaram a proteína Hwp1 no sangue, em nenhuma das amostras coletadas. A presença de fungos do gênero Candida foi mais frequente (p 0,005) entre os usuários de PTS com ED quando comparado com os outros indivíduos. Além disso, pudemos constatar que os pacientes com diagnóstico clínico e microbiológico de ED não apresentaram distribuição sanguínea de C. albicans. / There are few studies about the presence of yeast constituents in the bloodstream of patients with denture stomatitis (DE), a localizated kind of candidiasis; what could characterize the yeast systemic invasion power in this local condidition, and also previous acknowledgement of these antigens by cells of the entertainer bloodstream, and explain specific features of the immune located and systemic answer. So being, this work had as a goal to evaluate the presence of the yeast Candida albicans (C. albicans) at the palate, at the internal surface of the upper denture and in the blood of patients with DE, at two different moments. The population of study was composed by individuals both with and without upper denture, with and without stomatitis, assessed and selected in the clinics of graduation and postgraduation of the Discipline of Prosthesis, of the Department of Prosthesis, of the Faculdade de Odontologia de Bauru of the University of São Paulo (FOB USP). Individuals who are not users of removable dentures constituted the group control. So the work was constituted by three groups, each one with 14 patients. The injuries of stomatitis were diagnosed clinically and through microbiological confirmation in CHROMAgar Candida, from biological collected material of the palatal mucosa and of the internal surface of the upper denture. The PCR was carried out when the growth of green colonies for differentiation of the stain C. albicans and C. dubliniensis happened. The samples of blood were analyzed for the detection of fragments of DNA responsible for the codification of the hyphal wall protein (Hwp1) of C. albicans. The results demonstrated that not even the users of upper denture, independently of the presence of the DE, not even the volunteers who are not users presented the protein Hwp1 in the blood, in none of the collected samples. The yeast Candida presence was more frequent (p 0,005) in the group 1 when compared with the other groups. Morever, we can conclude that patients with clinic and microbiologic diagnostic have not presented bloodstream distribution of C. albicans.

Leucotrienos (LTs) são mediadores lipídicos derivados do ácido araquidônico. Existem evidências que receptores da imunidade inata interagem com receptores para LTs amplificando funções efetoras de macrófagos. Investigamos se LTs modulam a fagocitose e a atividade microbicida via receptores Manose, Dectina -1 e PTX3 em macrófagos alveolares (AMs) e os mecanismos moleculares envolvidos. Nossos resultados mostram que: 1) AMs sintetizam LTs quando fagocitam C. albicans, Zy e Zy-PTX3; 2) LTs potencializam a fagocitose de C. albicans e Zy, mas não de Zy-PTX3. Este efeito dos LTs depende: do reconhecimento via receptor manose (LTB4) e Dectina-1 (LTD4); da integridade de lipid rafts; da ação em mecanismos de polimerização de actina; do aumento dos níveis de F-actina por inativação da Cofilina-1; da ativação das LIMKs, que regulam Cofilina-1; da ativação de PKC<font face=\"Symbol\">d e PI3K3) LTs aumentam a capacidade de AMs em matar C. albicans por ativação de NADPH oxidase. Em conjunto, mostramos que LTs potencializam programas de sinalização específicos para determinados PRRs. / Leukotrienes (LTs) are lipid mediators derived from arachidonic acid. There is evidence that innate immunity receptors and leukotrienes receptors interact and amplify macrophage effector functions. We investigated if LTs receptors modulate phagocytosis and microbicidal activity mediated by Mannose receptor, Dectin-1 and PTX3 in alveolar macrophages (AMs) and the molecular mechanisms involved. Our results showed that: 1) AMs produce LTs when stimulated with C.albicans, Zy, Zy-PTX3; 2) LTs enhance phagocytosis of C.albicans and Zymosan, but not Zy-PTX3. This is dependent on: recognition via mannose receptor (LTB4) and Dectin-1 (LTD4); integrity of lipid rafts; its action on actin polimerization mechanisms; enhancement of F-actin levels by induction of Cofilin-1 inactivation; activation of LIMKs that regulate Cofilin-1; activation of PKC<font face=\"Symbol\">d and PI3K3) LTs enhance killing of C.albicans by activation of NADPH oxidase. Taken together, our results showed that LTs specifically influence signaling programs of keys PRRs.