Orientador: Ana Claudia Pavarina / Resumo: O objetivo do estudo foi avaliar a eficácia da terapia fotodinâmica antimicrobiana (aPDT) associada a Nistatina (NYS) no tratamento de candidose oral induzida em camundongos infectados com Candida albicans resistente a fluconazol. Foram utilizados 174 camundongos Swiss fêmeas com aproximadamente 5 semanas de vida. Os animais foram imunossuprimidos com predinisolona 100 mg/kg no 1º, 5º e 13 º dias de experimento. No 2º dia, os animais foram sedados com 0,1 mL de cloridrato de clorpromazina e uma suspensão de C. albicans 107 UFC/mL foi inoculada na língua dos mesmos. Do dia 7 ao 11 os tratamentos foram realizados. No grupo aPDT foi utilizado 200 mg/L de Photodithazine (PDZ) associado à luz LED de 50 J/cm2 (grupo P+L+); no grupo (P+L-) foi utilizado apenas com PDZ; o grupo (P-L+) recebeu só luz LED e nos animais do grupo NYS o medicamento foi aplicado uma vez ao dia. Além disso, foi avaliada a combinação de duas terapias: P+L+NYS e NYS+P+L+. Um grupo recebeu apenas inoculação de C. albicans (grupo P-L-) e outro grupo de animais saudáveis (grupo CNI). Após os tratamentos, foi realizada a recuperação de C. albicans por meio de swabs estéreis. Então diluições seriadas foram realizadas e plaqueadas em placas de Petri com SDA. Após 48 horas de incubação a 37o C as colônias foram quantificadas e o número de UFC/mL foi determinado. Os camundongos foram sacrificados 24 horas e 7 dias após os tratamentos. Os resultados demonstraram que a combinação das terapias promoveu redução de 2,6 lo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to evaluate of PDZ-mediated aPDT, as well as the association of this approach with antifungal nystatin, would be effective to treat oral candidosis in mice infected with fluconazole resistant C. albicans. Were used 174 female Swiss mice of 5 weeks-old-age approximately. The animals were immunosuppressed with predinisolone 100 mg/kg on the 1st, 5th and 13th days of the experiment. On day 2, the animals were sedated with 0.1 mL of chlorpromazine hydrochloride and a suspension of C. albicans 107 CFU/mL was inoculated into the tongue. From 7 to 11 the treatments were performed. In the aPDT group, was used 200 mg/L of Photodithazine (PDZ) associated with 50 J /cm2 LED light (P+L+ group). In the (P+L-) group was used only with PDZ; the group (P-L+) received only LED light and in the animals of the NYS group the drug was applied once a day. In addition, we evaluated the combination of two therapies: P+L+NYS and NYS+P+L+. One group received only inoculation of C. albicans (P-L- group) and another group of healthy animals (CNI group). After the treatments, recovery of C. albicans was performed by sterile swabs, then serial dilutions were performed and plated on Petri dishes with SDA. After 48 hours incubation at 37 °C the colonies were quantified, and the number of CFU/mL was determined. Mice were sacrificed 24 hours and 7 days after the treatments. The results showed that the combination of the therapies promoted reduction of 2.6 log10 and 2.1 log10 f... (Complete abstract click electronic access below) / Mestre

Phagosome maturation is a dynamic process involving the engulfment and degradation of pathogens by phagocytic cells. However, several pathogens have employed mechanisms that facilitate their survival and escape from the phagosome. The fungal pathogen, Candida albicans, is capable of switching from yeast to hyphal form to facilitate its pathogenicity and escape from the phagosome. Rab GTPases are key regulators in phagosome maturation by mediating interactions with the endocytic pathway and leading to biogenesis of the phagolysosome. The temporal localisation dynamics of Rab2 on maturing phagosomes containing live C. albicans was investigated. Live-cell imaging revealed green fluorescent protein (GFP)-tagged Rab2 was recruited to C. albicans-containing phagosomes. Rab2 appeared on phagosomes within 2 min following complete engulfment of C. albicans. Rab2 persisted transiently on macrophage phagosomes and this correlated with the length of C. albicans hyphae at the time of uptake, suggesting C. albicans morphology modulates Rab2 localisation dynamics. Expression of dominant negative or dominant active Rab2 did not affect macrophage migration, the rate of engulfment or phagosome acidification during the early stages of phagosome maturation. Furthermore, altered expression of Rab2 did not interfere with C. albicans ability to escape from and kill macrophages, suggesting Rab2 is not involved in the outcome of the host-pathogen interaction. However, altered expression of Rab2 reduced the acquisition of the late-stage phagosome maturation markers, cathepsin B and LAMP1, suggesting Rab2 impacts upon phagosome-lysosome fusion. Finally, the uptake of other particles by macrophages revealed Rab2 recruitment, as well as localisation dynamics on phagosomes, may be cargo-dependent. Through the use of live-cell imaging, real-time dynamics of phagosome biogenesis in live cells was examined, offering unique insight at the host-pathogen interface.

Orientador: Jacks Jorge Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-10-26T17:47:48Z (GMT). No. of bitstreams: 1 Melo_NadjaRodriguesde_M.pdf: 3247748 bytes, checksum: ec0e60306b5c5d2873bfc5144e7f933b (MD5) Previous issue date: 1999 / Resumo: Desde a caracterização da AIDS no início dos anos oitenta, aumentou o número de pacientes imunossuprimidos com as mais variadas expressões clínicas associadas. Uma delas, a candidose bucal, infecção fúngica muito comum, assumiu posição de importância insuspeita até então causando assim intensificação dos estudos associados a esta importante doença. O presente estudo teve por objetivo determinar, longitudinalmente, por um ano, a contagem e identificação do gênero Candida na saliva total de pacientes portadores do HIV, correlacionando com aspectos clínicos e parâmetros laboratoriais. Foram avaliados 188 pacientes, 93 dos quais por um período mínimo de 1 ano, portadores do HIV e provenientes do Grupo de Pesquisa em OST (GPO) - UNICAMP, incluídos no protocolo de pesquisa multicêntrico randomizado, duplo cego, com inibidor de protease H/V (Protocolo MK-639), em combinação com outros antiretrovirais, fase aberta, que está sendo desenvolvido há três anos. O paciente desta pesquisa tinha 35,2 ::!: 8,2 anos, era do gênero masculino (64,9%), leucoderma (82,4%), com instrução primária (51,6%), e casado (43,6%) ou solteiro (37,2%). Eram classificados no grupo C3 (36,7%) ou 82 (28,2%) e com a categoria de exposição, para o gênero masculino, o homossexualismo (37,7%) ou uso de drogas injetáveis (35,2%), e para o feminino, o heterossexualismo (98,5%). Os pacientes. foram submetidos a exames clínicos e laboratoriais periódicos e padronizados para determinação de indicadores de saúde geral Ie bucal tais como situação sorológica, clínico-epidemiológica, marcadores laboratoriais, fluxo salivar e análise microbiológica da saliva. A candidose bucal foi encontrada em 32% dos pacientes e nestes, a contagem de UFC/ml foi significantemente maior (p=O,OO) do que nos que não apresentaram esta manifestação bucal. A densidade de colonização por Candida não mostrou correlação com os principais marcadores de imunidade como linfócitos CD4, linfócitos C08, carga viral, entretanto foram significativos para contagem de glóbulos brancos (p= -0,0004), dosagens de TGO e TGP (p= 0,01 e p=0,02) respectivamente. C.albicans correspondeu a 75,8% das espécies identificadas / Abstract: Since the characterization of the AIOS in 1981, it has been increasing the number of immunesuppressed patients that express the most varied associated clinical expressions. One of them, the buccal candidosis, a very common fungal infection, have assumed position unsuspicious importance until then causing the intensification of the studies associated to this important infection. The present study had for objective to determine, longitudinally, for one year, the counts and identification of the gender Candida in the whole saliva of patient carriers of HIV, correlating with clínical aspects and laboratorial parameters. They were appraised 188 patient, 94 of the which for a 1 year-old minimum time, carriers of HIV and coming of the Group of Research in OST (GPO) UNICAMP, included in the protocol of research multicentric randomized, double blinded, with HIV protease inhibitor (MK-639 Protocol), open phase, that have been done during the last three years. The medium patient of this research had 35,2 :f: 8,2 years, they were of male gender (64,9%), white (82,4%), with primary instruction (51,6%), and married (43,6%) or single (37,2%). They were classified in the group C3 (36,7%) or 82 (28,2%). The risk category was, for the male gender, the homosexuality (37,7%) or the use of injected drugs (35,2%), and for the female gender, the heterosexuality (98,5%). The patients were submitted to periodical clinical ar'!d laboratorial exams in order to determine such indicators of general and buccal health as serologic situation, laboratorial markers, I salivary flow and microbiological analysis of the saliva~ The buccal candidosis was found iA 32% of the patients and in these, the UFC/ml counts was significant larger (p=O,OO) than in those patients that didn't present this oral manifestation. The colonization density for Candida show correlation (p=0,06) with immunity markers as C04 Iymphocytes counts, but didn't show correlation with CD8 Iymphocytes counts, viral load, only significant results for white cell counts (p= -0,02)" TGO and TGP levels (both p = -0,03). C.albicans corresponded at 78,5% of the identified species. The density of colonization of the saliva, expressed in UFC/ml, for Candida showed correlation with local and systemic factors / Mestrado / Biologia e Patologia Buco-Dental / Mestre em Ciências

Submitted by Aline Chiodi Borges null (aline.borges@ict.unesp.br) on 2017-02-01T12:59:00Z No. of bitstreams: 1 TESE Aline Chiodi Borges.pdf: 2538445 bytes, checksum: b94b3366c29227ee5a2e026711c63088 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-03T19:27:38Z (GMT) No. of bitstreams: 1 borges_ac_dr_sjc.pdf: 2538445 bytes, checksum: b94b3366c29227ee5a2e026711c63088 (MD5) / Made available in DSpace on 2017-02-03T19:27:38Z (GMT). No. of bitstreams: 1 borges_ac_dr_sjc.pdf: 2538445 bytes, checksum: b94b3366c29227ee5a2e026711c63088 (MD5) Previous issue date: 2016-12-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As doenças fúngicas representam grande desafio para a área médica e odontológica devido à crescente prevalência e aumento da resistência aos antifúngicos. O plasma frio sob pressão atmosférica (PFPA) é uma mistura gasosa contendo partículas carregadas, radicais livres e radiação. Sua potencial aplicação em doenças infecciosas foi relatada, contudo a literatura carece de estudo sistemático sobre o efeito antifúngico, mecanismos de ação e potencial citotóxico. Neste trabalho foi avaliado o efeito antifúngico do PFPA sobre Candida albicans através de ensaios em células planctônicas e biofilmes, efeitos sobre a integridade de parede celular e membrana plasmática, morfogênese, produção de exoenzimas, aderência às células epiteliais e efeito no tratamento in vivo de lesões de candidose oral induzida em modelo murino. Ainda, avaliou-se o efeito do PFPA sobre culturas de Trichophyton rubrum, além de efeitos sobre capacidade de adesão de conídios. Ainda, o potencial citotóxico foi investigado usando células epiteliais. PFPA em modo de tensão contínuo (MC) foi capaz de reduzir a aderência de C. albicans às células epiteliais, modular a transição levedura-hifa na cepa SC 5314 e comprometer a viabilidade de biofilmes. PFPA-MC se mostrou citotóxico em parâmetros efetivos frente a biofilmes de C. albicans. Porém, não foi observado efeito citotóxico quando o PFPA foi utilizado em modo de tensão pulsada (MP). A exposição ao PFPA-MP reduziu a invasão de C. albicans no epitélio in vivo. O PFPA-MP foi capaz de afetar o crescimento de T. rubrum a partir de 10 minutos e de afetar a sua capacidade de aderência. Assim, conclui-se que PFPA apresenta efeito antifúngico contra C. albicans e T. rubrum e é capaz de interferir em fatores de virulência de ambos os micro-organismos. / Fungal diseases represent a great challenge to the medical and dental areas, due to the increasing prevalence and antifungal resistance. Atmospheric pressure plasma jet (APPJ) is a gaseous mixture containing charged particles, free radicals and radiation. Its potential application in infectious disease has been reported, however there is still a lack of a systematic study on the antifungal effect, mechanism of action and citotoxicc potential. The general aim of this project was to evaluate the antifungal effect of APPJ on Candida albicans in planktonic and biofilm cultures, effects on cell wall and cell membrane integrity, morphogenesis, exoenzymes production, adherence to epithelial cells and in vivo effect in the treatment of oral candidosis in murine model will be performed. The effect of APPJ on Trichophyton rubrum cultures and on adherence capability were also evaluated. The cytotoxic potential was evaluated in vitro. APPJ in continuous tension mode (CM) was able to reduce the adherence and yeast-hyphae transition in C. albicans SC 5314 and to decrease biofilm viability. APPJ-CM showed cytotoxic effect in the parameters effective to C. albicans biofilm. Conversely, no cytotoxic effect on epithelial cells were observed when pulsed (PM) plasma jet was used. In vivo tests showed that APPJ-PM was able to prevent C. albicans invasion to the epithelium. T. rubrum cultures were affectd by APPJ-PM after 15 minutes of exposure and conidia adherence was impaired by 10 minutes exposure. In conclusion, APPJ showed antifungal effect against C. albicans and T. rubrum and can also impair virulence factors in both microorganisms. / FAPESP: 2014/02354-7

Der opportunistisch humanpathogene Hefepilz Candida albicans gehört bei vielen gesunden Menschen zur mikrobiellen Schleimhautflora, kann jedoch bei abwehrgeschwächten Patienten oberflächliche Infektionen sowie auch lebensbedrohliche tiefe Organmykosen verursachen. Obwohl der Immunstatus des Wirtes für eine Infektion mit diesem Erreger von entscheidender Bedeutung ist, tragen vermutlich auch eine Reihe von Virulenzfaktoren zur Pathogenität von C. albicans bei, indem sie Besiedlung, Ausbreitung und Vermehrung der Pilzzellen unter Anpassung an die verschiedensten Wirtsnischen unterstützen. Eine für die Pathogenität von C. albicans wichtige Eigenschaft ist die Bildung sekretorischer Aspartylproteasen (SAPs), die durch eine große Familie homologer Gene codiert werden. Es wird angenommen, dass die individuellen Proteasen während der Infektion verschiedene Aufgaben erfüllen bzw. optimal an unterschiedliche Wirtsnischen angepaßt sind. Jedoch ist der Beitrag der einzelnen SAP-Gene zur Pathogenese noch weitgehend unverstanden. Da die wirtsinduzierte Aktivierung dieser Virulenzgene während bestimmter Infektionsstadien Hinweise auf ihre spezifische pathogenetische Bedeutung liefern könnte, wurde in dieser Arbeit eine Methode für C. albicans entwickelt, mit der die Induktion eines Gens während der Infektion nachgewiesen werden kann. Die Methode beruht auf einer genetischen Rekombination als Reporter einer Genexpression, was bedeutet, dass nach Induktion des zu untersuchenden Gens eine site-spezifische Rekombinase spezifisch einen Mykophenolsäure-Resistenzmarker aus dem Genom der Zelle entfernt. Da diese Deletion ein irreversibles Ereignis darstellt, das auf die jeweiligen Nachkommen vererbt wird, kann selbst eine vorübergehende Genaktivierung während eines bestimmten Infektionsstadiums bzw. in einem bestimmten Organ in einzelnen Zellen nach deren Reisolierung aus infiziertem Gewebe durch Ausplattieren auf geeignetem Indikatormedium nachgewiesen werden. Durch Analyse der Expression des SAP2-Gens wurde bestätigt, dass mit diesem Reportersystem eine biologisch signifikante Genaktivierung in C. albicans nachgewiesen werden kann. SAP2 wird in C. albicans in vitro in einem Medium induziert, das Rinderserumalbumin als alleinige Stickstoffquelle enthält, ist in anderen gängigen Labormedien jedoch reprimiert. Diese in vivo-Expressionstechnologie (IVET) wurde verwendet, um die Expression von sechs verschiedenen SAP-Genen von C. albicans, SAP1-SAP6, in unterschiedlichen Tiermodellen zu studieren. Dabei konnte gezeigt werden, dass die einzelnen Proteasegene abhängig von der Art der Infektion, d.h. lokal begrenzte Schleimhautinfektion bzw. Systeminfektion, und auch vom Infektionsstadium differentiell reguliert werden. Dabei wurden sogar die äußerst homologen Gene SAP4-SAP6, die aufgrund von in vitro erzielten Ergebnissen als hyphenspezifische Gene galten, in vivo unterschiedlich reguliert. SAP5 und SAP6, aber nicht die anderen SAP-Gene, wurden in einem Maus-Ösophagus-Schleimhautmodell signifikant aktiviert, als die C. albicans-Hyphen in das Epithel invadierten. Eine stadienspezifische Expression der SAP-Gene wurde in einem Maus-Peritonitis-Modell beobachtet. Kurz nach Inokulation der C. albicans-Hefezellen in die Bauchhöhle der Tiere, zu einem Zeitpunkt, als noch keine Ausbildung von Hyphen zu beobachten war, wurde SAP5, aber nicht SAP6 oder eines der anderen analysierten SAP-Gene in einem signifikanten Anteil der infizierenden Zellen aktiviert. Demzufolge scheint SAP5 für die Gewebeinvasion während der Schleimhautinfektion und auch für die ersten Schritte während einer disseminierenden Infektion von Bedeutung zu sein. Durch die intravenöse Infektion der Maus, bei der frühe Infektionsschritte umgangen werden, wurde gezeigt, dass SAP5 und SAP6, aber auch SAP4, während der späteren Stadien einer disseminierenden Infektion weiterhin aktiviert werden. Dagegen wurde eine Induktion des SAP2-Gens vorwiegend im Spätstadium einer systemischen Infektion beobachtet, nachdem die Pilzzellen innere Organe befallen hatten. Daher fördert SAP2 vermutlich weniger die Invasion von Geweben, dafür aber die Vermehrung der Pilze nach Organbefall, möglicherweise durch die Bereitstellung von Nährstoffen. Dabei wurde gezeigt, dass die in vivo-Regulation von SAP2 durch bestimmte Repeatstrukturen innerhalb der Promotorregion dieses Gens beeinflußt wird. Während des Verlaufs einer systemischen Infektion wurden sogar die zwei SAP2-Allele des hier untersuchten C. albicans-Modellstammes CAI4, die sich in dieser Repeatregion unterscheiden, differentiell reguliert. Das SAP2-2-Allel wurde nämlich bereits deutlich früher induziert als das Allel SAP2-1. Eine Expression von SAP1 und SAP3 konnte im Gegensatz zu den anderen SAP-Genen nur in wenigen der infizierenden Zellen nachgewiesen werden, so dass diesen Genen ein Beitrag zur Pathogenität in den hier untersuchten Infektionsmodellen nicht beigemessen werden kann. Im Verlauf einer Infektion setzt C. albicans vermutlich viele verschiedene Virulenzfaktoren gleichzeitig für eine bestmögliche Anpassung an die jeweilige Wirtsnische ein. Ob in Abhängigkeit entsprechender Wirtssignale dabei unterschiedliche Eigenschaften der Pilzzelle koordiniert reguliert werden, ist kaum erforscht, erscheint jedoch für ein besseres Verständnis der Erreger-Wirts-Auseinandersetzung von besonderem Interesse. An der Kontrolle der Hyphenbildung von C. albicans sind wenigstens zwei Signaltransduktionskaskaden beteiligt, eine MAP-Kinase-Kaskade und ein cAMP-abhängiger Signalweg, die in den Transkriptionsregulatoren CPH1 bzw. EFG1 enden. Nachdem dimorphes Wachstum für die Infektion von Bedeutung ist und die Expression der Gene SAP4-SAP6 in vitro mit der Hyphenwachstumsphase verbunden ist, wurde eine mögliche Abhängigkeit hyphenassoziierter SAP-Aktivierung von diesen Regulatoren durch die Analyse der SAP5-Expression in entsprechenden Mutanten analysiert. Sowohl in cph1- als auch in efg1-Einzelmutanten wurde eine reduzierte Aktivierung des SAP5-Gens in vivo beobachtet. Dadurch konnte gezeigt werden, dass sowohl CPH1 als auch EFG1 zur SAP5-Aktivierung während der Infektion beitragen. Da cph1-Mutanten im infizierten Gewebe wie der Wildtyp-Stamm Hyphen ausbildeten, war die Hyphenbildung allein offensichtlich nicht für eine volle SAP5-Aktivierung in vivo ausreichend. Andererseits war die SAP5-Induktion in vivo nicht von der Hyphenwachstumsphase abhängig, da eine verminderte, aber dennoch signifikante SAP5-Expression auch in den efg1-Mutanten zu beobachten war, die in den infizierten Tieren nur in der Hefephase wuchsen. In Zellen, in denen beide Regulatoren fehlten, konnte eine Induktion von SAP5 kaum nachgewiesen werden. Das bedeutet, dass diese Signalwege in C. albicans für die Kontrolle verschiedener zellulärer Programme während der Infektion wichtig sind und die Expression von unterschiedlichen Virulenzgenen koordinieren. Durch die in vivo-Analyse der Virulenzgenexpression in C. albicans konnten Einblicke in regulatorische Anpassungsmechanismen dieses Mikroorganismus an verschiedene Wirtsnischen gewonnen werden. Einzelne Mitglieder einer Virulenzgenfamilie dieses Pilzes werden während der Infektion differentiell und in Abhängigkeit vom Infektionsstadium reguliert und tragen daher vermutlich sehr spezifisch zur Pathogenese bei. Unterschiedliche Virulenzmerkmale können zudem während der Infektion koordiniert reguliert werden und dadurch gemeinsam die Anpassungsfähigkeit von C. albicans an den Wirt unterstützen. Die erzielten Erkenntnisse sollten letztlich dazu beitragen, die Pathogenität dieses wichtigen opportunistisch humanpathogenen Erregers besser verstehen zu können. / The opportunistic human pathogenic yeast Candida albicans is a member of the microflora on mucosal surfaces of many healthy people but can cause superficial infections as well as life-threatening deep organ mycoses in immunocompromised patients. Although the ability of C. albicans to cause disease largely depends on the immune status of the host, it is generally assumed that a number of virulence factors also contribute to the pathogenicity of C. albicans, thereby supporting colonization, distribution and multiplication of the fungal cells and allowing an adaption to various host niches. The production of secreted aspartic proteases (SAPs), encoded by a large family of homologous genes, plays an important role in C. albicans pathogenicity. It is likely that the individual proteases fullfill various functions during infection or are optimally adapted to different host niches. However, the contribution of single SAP genes to pathogenicity is not well understood. Because the host-induced activation of these virulence genes at a certain infection stage might give clues to their specific role in pathogenicity, an in vivo-expression technology (IVET) was developed in this work that allows the detection of gene activation in C. albicans during infection. The method is based on genetic recombination as a reporter of gene expression, resulting in the specific excision of a mycophenolic acid resistance marker from the genome by a site-specific recombinase after induction of the target gene. Because deletion of the marker represents an irreversible event that is inherited by the progeny of the corresponding cell, even a transient gene expression at a certain infection stage or in specific organs can be detected in single cells recovered from infected tissue by plating on appropriate indicator medium. The suitability of the reporter system for detecting a biologically meaningful gene activation in C. albicans was confirmed by analyzing expression of the SAP2 gene, which is induced in vitro during growth in a medium containing bovine serum albumin as the sole nitrogen source, but repressed in other commonly used laboratory media. IVET was then used to analyze the in vivo expression of six different SAP genes of C. albicans, SAP1-SAP6, in various animal models. It could be demonstrated that the individual protease genes are indeed differentially regulated, depending on the type of the infection, i.e. locally restricted mucosal infection or systemic infection, and the stage of an infection. Even the highly homologous SAP4-SAP6 genes, which from the results of in vitro experiments had been supposed to be hyphae-specific genes, were differentially regulated in vivo. SAP5 and SAP6, but not the other SAP genes, were significantly activated in a mouse model of oesophageal candidiasis when C. albicans hyphae invaded into the epithelium. A stage-specific expression of SAP genes was observed in a mouse model of Candida peritonitis. Soon after inoculation of C. albicans yeasts into the peritoneal cavity, before hyphae formation was observed, SAP5 but not SAP6 or any of the other SAP genes analyzed was activated in a significant part of the infecting cell population. Therefore, SAP5 seems to be important for tissue invasion during mucosal infection and also during the initial steps of a disseminated infection. By intravenous inoculation of C. albicans into mice, thereby bypassing the early infection stages, it was demonstrated that expression of SAP5 and SAP6, but also SAP4, continued into the later stages of a disseminated infection. In contrast, an induction of the SAP2 gene was observed predominantly in the late stages of a systemic infection, when the fungal cells had spread to deep organs. Hence, SAP2 presumably supports the multiplication of the fungal cells within the infected organs, perhaps by providing nutrient supply, rather than the invasion into host tissues. The in vivo regulation of SAP2 was shown to be influenced by certain repeat structures in the promotor region of this gene. In the C. albicans model strain CAI4 that was used in this study even the two SAP2 alleles, which differed in this repeat region, were differentially regulated during the course of a systemic infection, the SAP2-2 allele being induced at an earlier infection stage than the SAP2-1 allele. In contrast to the other SAP genes, expression of SAP1 and SAP3 could be detected in only few infecting cells, and a role in pathogenicity could not be attributed to these genes in the infection models used in this work. During the course of an infection C. albicans presumably employs many different virulence factors at the same time in order to achieve the best possible adaptation to the corresponding host niche. Yet, a question that has hardly been addressed but may enhance our understanding of the host-microbe interactions is whether different properties of the fungal cell are regulated in a coordinated fashion by specific host signals. At least two signal transduction pathways, a MAP kinase cascade and a cAMP-dependent pathway ending in the transcriptional regulators CPH1 and EFG1, respectively, control hyphae formation in C. albicans. As dimorphic growth is important for infection and because expression of the genes SAP4-SAP6 is linked to the hyphal growth form in vitro, a possible dependence of hyphae-associated SAP gene expression on these regulators was analyzed by studying SAP5 expression in corresponding signal transduction mutants. SAP5 activation in vivo was reduced in cph1 as well as in efg1 single mutants. Therefore, both CPH1 and EFG1 contribute to SAP5 activation during infection. Since the cph1 mutant formed hyphae in infected tissue as efficiently as the wild-type strain, hyphae formation alone evidently was not sufficient for full SAP5 induction in vivo. On the other hand, induction of SAP5 in vivo did not depend on the hyphal growth form, because a reduced but still significant SAP5 expression was also detected in the efg1 mutant that grew only in the yeast form in the infected animals. In cells defective in both of the regulators an induction of SAP5 was hardly detectable. Therefore, these signalling pathways are important for the control of various cellular programs during infection and coordinate the expression of different virulence genes in C. albicans. The in vivo analysis of virulence gene expression of C. albicans provided insights into regulatory adaptation mechanisms of the pathogen in various host niches. The individual members of a virulence gene family of this fungus are differentially and stage-specifically regulated during infection and thus presumably contribute very specifically to pathogenesis. Moreover, various virulence traits can be regulated in a coordinated fashion during infection and in this way together support the adaptability of C. albicans to the host. Overall, these findings enhance our understanding of the pathogenicity of this important opportunistic fungal pathogen.

Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 201-220).

Thèse (M.Sc.)--Université Laval, 2009. / Bibliogr.: f. 71-86. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Allelic expression imbalance, or AEI, is the term given to differences in the expression levels of the two alleles of a gene. AEI has been previously identified in a number of species using various techniques. Here, the genome-wide extent of allelic expression imbalance in the pathogenic yeast species, Candida albicans, was examined through use of RNA sequencing in combination with a novel computational pipeline based around the diploid reference genome. Techniques for validating these results were investigated, and the difficulties surrounding specificity and quantification are discussed. As C. albicans is a highly heterozygous species, it was hypothesised that polymorphisms within alleles lead to differences in allele expression, which are further linked to differences in allele function. The functional consequences of AEI were therefore interrogated through investigation of Gene Ontology, identification of condition specific responses in AEI, and targeted construction and phenotypic screening of heterozygous knockout strains. Together, these results strongly suggest that divergence in allele expression is not linked to differences in allele function. Investigations of the possible control mechanisms behind the differences in allele expression were considered, with a focus upon structural factors such as chromosomal location, GC content, allele length and codon usage. However, issues with establishing causality are present, and difficulties lie in distinguishing between functional differences and consequences of bias in sequencing technologies. This piece of research has advanced the understanding of gene expression mechanisms within a medically important pathogen, paving the way for further investigations into the functional consequences of allelic expression imbalance in Candida albicans.

The main aim of this project was to study Candida albicans cell wall biosynthesis in response to stress. The role of the MAPK, Ca2+/calcineurin and cAMP/PKA signal transduction pathways in regulating the C. albicans cell wall stress response was investigated. A library of mutants lacking receptors, signalling elements and transcription factors were screened for alterations in their ability to respond to a range of cell wall stressing agents, including CaCl2, Calcofluor White and caspofungin. Pretreatment of wild-type cells with CaCl2 and CFW, activates the Ca2+/calcineurin and PKC pathways, leading to an increase in chitin content, and reduced susceptibility to caspofungin. Although elevation of cell wall chitin content often resulted in decreased sensitivity to caspofungin, I show here that some strains with increased chitin levels remained sensitive to caspofungin. The results show that elevation of chitin is a common property of a range of mutants that are affected in coordinating cell wall stress pathways, but that multiple mechanisms are likely to operate in maintaining the robustness of the C. albicans cell wall. Some of the mutant strains of the MAPK, Ca2+/calcineurin and cAMP signalling pathways showed evidence of paradoxical growth, whereby less inhibition was achieved by higher concentrations of antifungal drug. The role of chitin-related genes and stress signalling pathways in regulating C. albicans paradoxical growth was also investigated. Based on these results, more detailed analyses were performed to investigate the correlations between sensitivity and resistance to caspofungin, in relation to paradoxical growth. The MAPK-Mkc1 and the calcineurin pathways played major roles in the paradoxical growth effect. There was a proportional relationship between echinocandin concentration and the chitin content of the cell wall although the chitin content did not continue to be upregulated by the highest echinocandin concentration. Different echinocandins, carbon source, cell morphology and medium composition influenced the extent of paradoxical growth effect. The existence of paradoxical growth in resistant strains such as Fks1 also highlights association of paradoxical growth with resistance mechanisms.

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