Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Long QT syndrome (LQTS) is a cardiac repolarization disorder affecting every 1:2000-1:3000 individuals. This disease is characterized by a prolonged QT interval on the surface electrocardiogram (ECG) of patients. Symptoms of LQTS range from dizziness and syncope to more severe symptoms such as seizures and sudden cardiac death (SCD). Clinical features of LQTS are a result of the precipitations of Torsades de Pointes, which is a polymorphic form of ventricular tachycardia. A number of genetic forms of LQTS have been identified with more than 700 mutations in 12 different genes leading to disease pathogenesis. However it has been estimated that approximately 25% of patients with compelling LQTS have no mutations within the known LQT genes. This proves to be problematic since treatment regimens depend on the genetic diagnosis of affected individuals. Of the known mutated genes, KCNE2 is associated with LQT6. KCNE2 encodes the beta-subunit of potassium ion channel proteins. These proteins contain cytoplasmic C-terminal domains in which many mutations have been identified. We hypothesize that genes encoding KCNE2-interacting proteins might be identified as disease-causing or modifying genes. The present study aimed to use yeast two-hybrid (Y2H) methodology to screen a pre-transformed cardiac cDNA library in order to identify putative interactors of the C-terminal of KCNE2. Through specific selection methods the number of KCNE2 ligands was reduced from 296 to 83. These interactors were sequenced and 14 were identified as putative interacting proteins. False positive ligands were excluded based on their function and subcellular location. Ultimately three strong candidate ligands were selected for further analysis: Alpha-B crystallin (CRYAB), Filamin C (FLNC) and voltage-dependent anion-selective channel protein 1 (VDAC1). Three-dimensional (3D) co-localization and co-immunoprecipitation were used to verify these proposed interactions and succeeded in doing so. The genes encoding verified interactors will be screened in our SA panel of LQT patients, to potentially identify novel LQT causative or modifying genes. Furthermore, the interactions verified in the present study may shed some light on the mechanism of pathogenesis of LQT causative mutations in KCNE2. / AFRIKAANSE OPSOMMING: Lang QT-sindroom (LQTS) is 'n hart her-polariserende siekte wat elke 1:2000-1:3000 individue affekteer. Hierdie siekte word gekenmerk deur 'n lang QT-interval op die oppervlak elektrokardiogram (EKG) van pasiënte. Simptome van LQTS wissel van duiseligheid en floutes tot meer ernstige simptome soos stuiptrekkings of aanvalle en skielike kardiale dood (SKD). Kliniese kenmerke van LQTS is 'n gevolg van die neerslag van Torsades de Pointes; 'n polimorfiese vorm van ventrikulêre tagikardie. Verskeie genetiese vorms van LQTS is geïdentifiseer met meer as 700 mutasies in 12 verskillende gene wat lei tot siekte patogenese. Dit is ergter beraam dat ongeveer 25% van pasiënte met dwingende LQTS geen mutasies in die bekend LQT gene besit nie. Dit is problematies aangesien siekte behandeling af hang van die genetiese diagnose van geaffekteerde individue. Een van die bekende gemuteerde gene is KCNE2 wat verband hou met LQT6. KCNE2 kodeer die beta-subeenheid van kalium ioonkanaal proteïene. Hierdie proteïene bevat sitoplasmiese C-terminale waarin baie mutasies alreeds geïdentifiseer is. Ons veronderstel dat gene wat proteïene kodeer wat met KCNE2 interaksie toon, geïdentifiseer kan word as siekte veroorsaakende of wysigings gene. Die huidige studie het die gis twee-hibried metode gebruik om 'n vooraf-getransformeerde hart cDNS biblioteek te sif om vermeende protein interaksies van die C-terminaal van KCNE2 te identifiseer. Deur middel van seleksie metodes is die aantal KCNE2 ligande verminder van 296 tot 83. Die identiteit van die proteïene is bekend gemaak deur volgorderbepaling waarna 14 geïdentifiseer is as proteïene wat moontlik interaksie kan toon met KCNE2. Vals positiewe ligande is uitgesluit op grond van hul funksie en subsellulêre lokasering. Drie kandidaat ligande is gekies vir verdere analise: Alfa-B crystallin (CRYAB), Filamin C (FLNC) en spanning-afhanklike anioon-selektiewe kanaal proteïen 1 (VDAC1). Drie-dimensionele (3D) mede-lokalisering en mede-immunopresipitasie tegnieke is gebruik om hierdie voorgestelde interaksies te verifieer en het geslaag om dit te doen. Die gene wat geverifieerde proteïene kodeer, sal gekeur word in ons Suid-Afrikaanse paneel van LQT pasiënte om sodoende potensieel nuwe LQT veroorsakende of wysigings gene te identifiseer. Verder kan die geverifieer interaksies in die huidige studie lig werp op die meganisme van die ontstaan van LQT veroorsakende mutasies in KCNE2. / Harry Crossley Foundation (South Africa) / Stellenbosch University / South African Council for Scientific and Industrial Research / Stella and Paul Loewenstein Charitable and Educational Trust

No description available.

Cannabinoids have been shown to alter synaptic plasticity but the mechanism by which this occurs at hippocampal CA3-CA1 synapses in vivo is not yet known. Utilizing in vivo electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) on anesthetized rats and mice as well as three lines of conditional knockout mouse models, the objective was to show a two-part mechanistic breakdown of cannabinoid-evoked CA3-CA1 long-term depression (LTD) in its induction as well as early and later-phase expression stages. It was determined that this cannabinoid-induced in vivo LTD requires cannabinoid type-1 receptors (CB1Rs) on astrocytes, but not CB1Rs on glutamatergic or GABAergic neuronal axons/terminals. Pharmacological testing determined that cannabinoid-induced in vivo LTD also requires activation of NMDA receptors (NMDAR) and subsequent postsynaptic endocytosis of AMPA receptors (AMPAR). There exists a clear role for NR2B-containing NMDARs in a persistent, transitory form, potentially related to prolonged or delayed glutamate release (possibly as a result of the astrocytic network). A key determination of the expression phase is the involvement of new protein synthesis (using translation and transcription inhibitors) – further evidence of the long-term action of the synaptic plasticity from a single cannabinoid dose.

Le facteur de transcription E4F1 fût initialement identifié comme une cible cellulaire de l'oncoprotéine virale E1A au cours de l'infection par l'adénovirus sérotype V. E4F1 est une protéine multifonctionelle essentielle au cours du développement embryonnaire précoce et joue des rôles importants dans l'équilibre entre prolifération/survie de différents types cellulaires, notamment des cellules souches. Au niveau moléculaire, E4F1 possède des activités transcriptionelles intrinsèques mais possède également une activité ubiquitine E3 ligase atypique dirigée contre d'autres facteurs de transcription tel que le suppresseur de tumeur p53. Récemment, il a été démontré qu'E4F1 régule les voies oncogéniques impliquant p53 et Rb qui jouent un rôle essentiel au cours de la sénescence cellulaire. La sénescence, qui est définie par un arrêt irréversible du cycle cellulaire, est considéré comme un mécanisme suppresseur de tumeurs essentiel au cours des phases précoces du développement tumoral. L'objectif de ma thèse a été d'évaluer le rôle d'E4F1 au cours de la sénescence cellulaire. Au travers d'études menées sur des fibroblastes humains primaires et des fibroblastes embryonnaires murins dérivés de souris génétiquement modifiées pour le gène E4F1, j'ai examiné comment la perturbation des activités d'E4F1 module l'initiation ou le maintien de la sénescence prématurée induit par l'oncogène RAS, la déplétion du membre de la famille polycomb Bmi1, ou par les dommages à l'ADN. Mes résultats suggèrent que la déplétion d'E4F1 protège partiellement contre l'induction de la sénescence alors que l'expression ectopique d'E4F1 accélère la sénescence par son implication dans la voie INK4A/ARF-p53. L'ensemble de mes résultats supportent la notion qu'E4F1 est un régulateur important de la sénescence cellulaire. / E4F1 was originally identified as a cellular target of the viral oncoprotein E1A during adenoviral infection. E4F1 is a multifunctional protein that is essential during early embryogenesis and plays important roles in the proliferation/survival balance of different cell types including stem cells. At the molecular level, E4F1 exhibits intrinsic transcriptional activities but also an ubiquitin E3 ligase function that targets other transcription factors, including the p53 tumor suppressor. Recent studies indicate that E4F1 impinge on several pathways, including the Rb and p53 pathways, that are known to influence cellular senescence, an irreversible state of cell cycle arrest that is considered to be an essential tumor suppressor mechanism during early steps of tumorigenesis. The objective of my thesis was to evaluate the roles of E4F1 during cellular senescence. Using human primary fibroblasts and mouse embryonic fibroblasts derived from genetic ally engineered mouse models, I investigated how perturbations of E4F1 activities modulated the initiation or the maintenance of premature senescence induced by oncogenic Ras, depletion of the polycomb member Bmi1 or DNA damage. My results suggest that E4F1 depletion partly protects from the induction of cellular senescence whereas ectopic expression of E4F1 accelerates premature senescence through its implication in the Ink4a/ARF-p53 pathway. Altogether, my results support the notion that E4F1 is an important regulator of cellular senescence.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

L'arthrose est une maladie articulaire dégénérative, avec une pathogenèse inconnue. Des études récentes suggèrent que l'activation du facteur de transcription du récepteur activateur de la prolifération des peroxysomes (PPAR) gamma est une cible thérapeutique pour ce maladie. Les agonistes du PPARγ inhibent l'inflammation et réduisent la synthèse des produits de dégradation du cartilage in vitro et in vivo. Cependant, des études utilisant des agonistes du PPARγ n’élucident pas les effets exacts médiés par ce gène complexe. En effet, certains de ces agonistes ont la capacité de régulariser d'autres voies de signalisation indépendantes de PPARγ, ainsi entraînant des effets secondaires graves. Afin d'obtenir une efficacité thérapeutique avec potentiellement moins de problèmes de sécurité, il est donc essentiel d'élucider, in vivo, le rôle exact de PPARγ dans la physiopathologie OA. Mon projet de thèse permettra de déterminer, pour la première fois, le rôle spécifique de PPARγ in vivo dans la physiopathologie OA. Les souris utilisées pour l’étude avaient une délétion conditionnelle du gène PPARγ dans le cartilage. Ces dernières ont été générées en employant le système LoxP/Cre. Pour tester cette hypothèse, j'ai généré deux types de souris avec une délétion au PPARγ, (a) une suppression du gène PPARγ spécifiquement dans le cartilage germinale pour l'étude de l'arthrose liée au développement et à l'âge et (b) la suppression inductible du gène PPARγ spécifiquement dans le cartilage chez la souris adulte pour les études OA. L’étude précédente dans notre laboratoire, utilisant ces souris ayant une délétion au gène PPARγ germinales, montre que ces souris présentent des anomalies du développement du cartilage. J'ai également exploré si ces souris qui présentent des défauts précoces du développement ont toutes les modifications phénotypiques dans le cartilage au cours du vieillissement. Mes résultats ont montré que les souris adultes, ayant une délétion au gène PPARγ, ont présenter un phénotype de l'arthrose spontanée associée à une dégradation du cartilage, l’hypocellularité, la fibrose synoviale. Cette étude a montré que PPARγ est un régulateur essentiel pour le cartilage, et c’est le manque (l’absence) de ce dernier qui conduit à un phénotype de l'arthrose spontanée accélérée (American Journal of Pathologie). A partir de ce but de l'étude, on n’a pas pu vérifier si ces souris présentaient l’OA spontanée en raison des défauts de développement ou à la suite de la délétion du gène PPARγ. Pour contourner les défauts de développement, j'ai généré des souris ayant une délétion du gène PPARγ spécifiquement dans le cartilage inductible avec le système Col2rTACre. Ces souris ont été soumises à modèle de la chirurgie OA (DMM: déstabilisation du ménisque médial) et les résultats révèlent que les souris PPARγ KO ont une dégradation accélérée du cartilage, une hypocellularité, une fibrose synoviale et une augmentation de l'expression des marqueurs cataboliques et des marqueurs inflammatoire. La perte de PPAR dans le cartilage articulaire est un évènement critique qui initie la dégradation de cartilage dans OA. Les études récentes suggèrent que le procès d’autophagie, une forme de survie cellulaire programmée, est altéré pendant l’OA et peut contribuer vers une protection diminuée des cellules, résultant la dégradation du cartilage. J’ai donc exploré le rôle de PPARγ dans la protection des cellules en déterminant l’effet de manque de PPARγ dans le cartilage par l’expression de mTOR (régulateur négatif principal d’autophagie) et les gènes d’autophagie durant OA. Mes résultats ont montré que les souris KO PPARγ présentent également une augmentation sur l'expression de mTOR et une diminution sur l’expression des marqueurs autophagiques en comparaison avec les chondrocytes articulaires isolés des souris contrôles OA. J'ai suggéré l'hypothèse que PPARγ contrôle la régulation de la signalisation de mTOR/autophagie, et finalement la mort des chondrocytes et l’expression des facteurs cataboliques et les facteurs inflammatoire. Pour tester cette hypothèse, j’ai fait la transfection des chondrocytes arthrosiques PPARγ-KO avec le vecteur d’expression de PPARγ pour déterminer si la restauration de l'expression de PPARγ peut sauver le phénotype des cellules PPARγ-KO OA. J'ai observé que la restauration de l'expression de PPARγ dans les cellules PPARγ-KO en présence du vecteur d'expression PPARγ, a pu considérablement régulariser négativement l'expression de mTOR et mettre en règle positivement l'expression des gènes autophagiques ainsi que le sauvetage significative de l'expression du collagène de type II et l’aggrecan et de baisser de manière significative l'expression de marqueurs cataboliques critiques et des marqueurs inflammatoires. Pour prouver que l’augmentation de la signalisation de mTOR et la diminution de l'autophagie est responsable du phénotype OA accélérée observée dans les souris PPARγ KO in vivo, j'ai généré les souris doubles KO PPARγ- mTOR inductible spécifique du cartilage en utilisant le système Col2 - rtTA -Cre et soumis ces souris à DMM modèle de l'arthrose. Mes résultants démontrent que les souris avec PPARγ- mTOR doubles KO ont été significativement protégés contre les OA DMM induites associées à une protection significative contre la destruction du cartilage, la perte de protéoglycanes et la perte de chondro-cellularité par rapport aux souris témoins. Considérant que mTOR est un répresseur majeur de l'autophagie, j'ai trouvé que l'expression de deux marqueurs de l'autophagie critiques (ULK1 et LC3B) a été significativement plus élevée dans les chondrocytes extraits les souris doubles KO PPARγ-mTOR par rapport aux souris témoins. En plus, les études de sauvetage in vitro en utilisant le vecteur d'expression PPAR et les études in vivo utilisant les souris doubles KO PPARγ- mTOR montrent que PPARγ est impliqué dans la régulation de la protéine signalant de mTOR/autophagie dans le cartilage articulaire. Ces résultats contournent PPARγ et sa signalisation en aval de mTOR/autophagie en tant que cibles thérapeutiques potentielles pour le traitement de l'arthrose. / Osteoarthritis (OA) is an age related degenerative joint disease with unknown pathogenesis. Recent studies suggest that the activation of the transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a therapeutic target for OA. Agonists of PPARγ inhibit inflammation and reduce the synthesis of cartilage degradation products both in vitro and in vivo. However, studies using agonists of PPARγ do not elucidate the exact effects mediated by this complex gene. Indeed, some of these agonists have the ability to regulate, in vivo, various other signaling pathways independent of PPARγ, resulting in serious side effects. It is therefore vital, in order to achieve therapeutic efficacy with potentially less safety concerns, to elucidate the exact in vivo role of PPARγ in OA pathophysiology. Thus, the aim of my PhD project was to determine the specific in vivo role of PPARγ in OA pathophysiology using cartilage-specific PPARγ knockout (KO) mice and subjecting these mice to surgical model of OA. I generated two separate PPARγ KO mice harboring a (a) constitutive cartilage-specific germ-line deletion of PPARγ gene for developmental and age-related OA study and (b) inducible cartilage-specific deletion of PPARγ in adult mouse specifically for OA studies using LoxP Cre system. Previous study in my laboratory using germ-line PPARγ KO mice shows that these mice exhibit cartilage developmental defects. I further explored if these mice which exhibit early developmental defects have any phenotypic changes in the articular cartilage during ageing. My results showed that adult PPARγ KO mice exhibited a spontaneous OA phenotype associated with enhanced cartilage degradation, hypocellularity, synovial fibrosis, and increased expression of catabolic and inflammatory factors. This study showed that PPARγ is a critical regulator of cartilage health, the lack of which leads to an accelerated spontaneous OA phenotype (Vasheghani et al, 2013; American Journal of Pathology). From this aim of the study, I could not ascertain if cartilage-specific germline PPARγ KO mice exhibited spontaneous OA because of developmental defects or as a result of PPARγ deficiency. To bypass the developmental defects, I then generated inducible cartilage-specific PPARγ KO mice using Col2rTACre system and subjected these mice to destabilization of medial meniscus (DMM) model of OA surgery. My results revealed that PPARγ KO mice showed accelerated cartilage degradation, hypo-cellularity, synovial fibrosis and increased expression of catabolic and inflammatory factors during OA. Loss of chondrocyte cellularity within the articular cartilage is one of the critical events that initiate the degradation of the cartilage during OA. Recent studies suggest that the process of autophagy, a form of programmed cell survival, is impaired during OA and may contribute towards decreased chondro-protection resulting in cartilage degradation. Thus, I further explored the role of PPARγ in chondro-protection by determining the effect of PPARγ deficiency in the cartilage on the expression of mTOR (master negative regulator of autophagy) and autophagy genes during OA. My results revealed that PPARγ-deficient chondrocytes exhibit significantly enhanced expression of mTOR and decreased expression of genes that initiate autophagy process compared to chondrocytes extracted from control OA mice. I then hypothesized that PPARγ controls mTOR/autophagy signaling and ultimately the fate of chondrocytes and the expression of catabolic and inflammatory factors in the articular cartilage. To test this, I transfected PPARγ KO OA chondrocytes with PPARγ expression vector to determine if restoration of PPARγ expression can rescue the phenotype of PPARγ KO OA cells. I observed that restoration of PPARγ expression in PPARγ KO cells significantly down-regulated the expression of mTOR and up-regulate the expression of autophagy genes along with significant rescue in the expression of collagen type II and aggrecan and significant down-regulation in the expression of critical catabolic and inflammatory markers. To validate our in vitro finding that enhanced mTOR signalling and resultant decrease in autophagy is responsible for accelerated OA phenotype observed in PPARγ KO mice, I generated inducible cartilage-specific PPARγ-mTOR double KO mice and subjected these mice to DMM model of OA. My results clearly demonstrate that PPARγ-mTOR double KO mice exhibit significant protection against DMM-induced OA associated with significant protection from cartilage destruction, proteoglycan loss and loss of chondro-cellularity compared with control mice. Since mTOR is a major repressor of autophagy, I found that the expression of two critical autophagy markers (ULK1 and LC3B) was significantly elevated in PPARγ-mTOR double KO mice compared to control mice. My in vitro rescue studies using PPARγ expression vector and in vivo studies using PPARγ- mTOR double KO mice clearly show that PPARγ is involved in the regulation of mTOR/autophagy signalling in the articular cartilage. Therefore, deficiency of PPARγ upregulates mTOR signalling resulting in the suppression of autophagy and decreased chondroprotection and increased catabolic activity leading to accelerated severe OA. This study for the first time provides direct evidence on the role of PPARγ in chondroprotection by modulation of mTOR/autophagy signalling in the articular cartilage. These findings outline PPARγ and its downstream signalling by mTOR/autophagy as potential therapeutic targets for the treatment of OA.

Die Detektion von Umweltsignalen und die gezielte zelluläre Reaktion ist eine zentrale und für das Überleben aller Lebewesen essentielle Fähigkeit. Candida albicans, als dominierender humanpathogener Pilz, ist hochgradig verschiedenen biochemischen und physikalischen Umweltbedingungen ausgesetzt, welche sowohl die Zellmorphologie als auch die Virulenz dieses Erregers beeinflussen. In der vorliegenden Arbeit wurde der Einfluss von Kohlendioxid, als ubiquitär vorkommendes Gasmolekül, auf die Zellmorphologie und Virulenz untersucht. Erhöhte Konzentrationen von Kohlendioxid stellen ein äußerst robustes Umweltsignal dar, welches die morphologische Transition vom Hefewachstum zum hyphalen Wachstum, einem Hauptvirulenzfaktor, in Candida albicans stimuliert. In diesem Zusammenhang wurde die Rolle der putativen Carboanhydrase Nce103 durch die Generation von knock – out Mutanten untersucht. Die Disruption von NCE103 in C. albicans führt zu einem Kohlendioxid – abhängigen Phänotyp, welcher Wachstum unter aeroben Bedingungen (ca. 0,033% CO2) nicht zulässt, jedoch unter Bedingungen mit einem erhöhten CO2 Gehalt von ca. 5% ermöglicht. NCE103 ist also für das Wachstum von C. albicans in Wirtsnischen mit aeroben Bedingungen essentiell. Durch Untersuchungen zur Enzymkinetik mittels Stopped – flow wurde in dieser Arbeit gezeigt, dass Nce103 die Funktion einer Carboanhydrase erfüllt. Die biochemische Funktion dieser Carboanhydrase besteht in der Fixation von CO2 bzw. HCO3ˉ in der Zelle zur Unterhaltung der wesentlichen metabolischen Reaktionen. Weiterhin konnte gezeigt werden, dass die Induktion hyphalen Wachstums durch CO2 in C. albicans nicht durch den Transport von CO2 mittels des Aquaporins Aqy1 beeinflusst wird. CO2 bzw. HCO3ˉ aktiviert in der Zelle direkt eine Adenylylcyclase (Cdc35), welche sich grundlegend von den bisher gut charakterisierten G-Protein gekoppelten Adenylylcylasen unterscheidet. Die Generation von cAMP beeinflusst in der Folge direkt die Transkription hyphenspezifischer Gene und nachfolgend die morphologische Transition vom Hefewachstum zum elongierten, hyphalen Wachstum. Dieser Mechanismus konnte sowohl in Candida albicans als auch in Cryptococcus neoformans nachgewiesen werden, was auf einen panfungal konservierten Signaltransduktionsmechanismus schliessen lässt. Die Inhibition dieser spezifischen Kaskade eröffnet neue Ansätze zur Entwicklung spezifischer antimykotischer Wirkstoffe. / Detection of environmental signals and subsequently directed reaction is essential for the survival of all living organisms. Candida albicans, as the predominant human fungal pathogen is exposed to severely different physical and chemical conditions, which influence cell morphology as well as virulence in human. In the present work, the influence of carbon dioxide as ubiquitous gaseous molecule on virulence and cell morphology was analysed. Elevated concentrations of carbon dioxide are a robust signal to induce the morphological transition from yeast growth to an elongated hyphal growth form, which is believed to be one of the main virulence factors in Candida albicans. The role of the putative carbonic anhydrase Nce103p in carbon dioxide signalling is reviewed by generating knockout mutant strains, which exhibited a carbon dioxide dependent phenotype. Growth under aerobic conditions (0,033 % carbon dioxide) is inhibited but feasible in 5% carbon dioxide. Therefore, Nce103p is essential for growth in host niches with aerobic conditions. Analysis of the biochemical properties of Nce103p by stopped – flow kinetics revealed carbonic anhydrase activity. It is hypothesised, that Nce103p is essential for fixation of carbon dioxide and bicarbonate within the cell in order to sustain basic metabolic reactions. Furthermore, the induction of hyphal growth was independent of aquaporine-mediated transport of carbon dioxide. Bicarbonate rather carbon dioxide activates directly the adenylyl cyclase Cdc35p generating cyclic AMP as second messenger and influencing the transcription of hyphal specific genes in Candida albicans thus promoting the morphological transition from yeast growth to elongated hyphal growth. This signal transduction cascade is present in Candida albicans as well as Cryptococcus neoformans and it is believed to be a pan fungal signal transduction cascade. The specific inhibition of carbon dioxide mediated polymorphism may serve as a new target for antifungal therapeutic agents.

No description available.

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants