Myotonic Dystrophy type 1 (DM1), the most common form of adult muscular dystrophy (~1:8000) currently has no effective treatment. In DM1, expansion of a tri-nucleotide repeat in the 3' UTR of the DMPK gene results in DMPK mRNA hairpin structures, aggregating as insoluble ribonuclear foci. The resulting mis-regulation of important splicing factors, causes the inclusion of fetal exons in dozens of transcripts that contribute to the disease phenotype. In order to identify novel gene targets and kinase signalling pathways for potential therapeutics we have performed a high-throughput RNAi. RNA foci were visualized and quantified by in-situ hybridization. From our screen, we have identified a novel gene, PACT, as a modulator of foci integrity and that PACT knockdown can induce MBNL1 protein levels. The identified signalling complex represents a valid target for DM1 therapeutics. Our data further emphasizes the utility of RNAi screens in identifying disease-associated genes.

<p>Sexual development enables microbial pathogens to purge deleterious mutations from the genome and drives genetic diversity in the population. <italic>Cryptococcus neoformans</italic> is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. <italic>C. neoformans</italic> has a defined <bold>a</bold>&ndash;&alpha; opposite sexual cycle (bisexual reproduction); however, >99% of clinical and environmental isolates are of the &alpha; mating type. Interestingly, &alpha; cells can undergo &alpha;&ndash;&alpha; unisexual reproduction, even involving genotypically identical cells. A central question is why would cells mate with themselves given that sex is costly and typically serves to admix pre-existing genetic diversity from genetically divergent parents? Sexual reproduction generates abundant spores that following inhalation, they penetrate deep into the alveoli of the lung, germinate, and establish a pulmonary infection growing as budding yeast. Therefore sex has been linked with virulence; however, hyphal development has been previously associated with reduced virulence and thus the roles of morphogenesis in virulence have not been extensively analyzed. To further understand the role of unisexual reproduction in <italic>C. neoformans</italic> we will investigate the evolutionary implications of &alpha;&ndash;&alpha; mating, explore its role in pathogenesis, and we will dissect the signaling pathway that regulates sexual development.</p><p>We isolated &alpha;&ndash;&alpha; unisexual reproduction progeny from the hyperfilamentous strain XL280 and subjected to a variety of phenotypic and genotypic assays (including whole genome sequencing and CGH). We found that unisexual and bisexual reproduction frequently generates phenotypic and genotypic diversity de novo, including aneuploidy. Aneuploidy was responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments, such as the mammalian host.</p><p>Interestingly aneuploidy strains that were fluconazole resistant were as virulent as the WT parental strain XL280. Although XL280 belongs to the serotype D lineage that exhibits limited pathogenicity, in further studies we found that is hypervirulent in the murine model. It can grow inside the lung of the host, establishing a pulmonary infection, and then disseminates to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is characterized by less protective immunity and is usually observed in the highly virulent sibling species <italic>C. gattii</italic>, responsible for the Pacific Northwest outbreak. These studies: 1) provide a technological advance that will facilitate analysis of virulence genes and attributes in <italic>C. neoformans</italic> var. <italic>neoformans</italic> (serotype D), and 2) reveal the virulence potential of serotype D that is broader and more dynamic than previously appreciated.</p><p>Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. To address this question we applied an insertional mutagenesis via <italic>Agrobacterium tumefaciens</italic> transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 governs cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. In further studies we found that Znf3 is required for sex- and mitotic-induced (SIS and MIS). SIS is less efficient in <italic>znf3</italic> unilateral matings and is abolished in <italic>znf3</italic> x <italic>znf3</italic> bilateral matings, similar to the phenotypes of <italic>rdp1</italic> mutants (the RNA-dependent RNA-polymerase of RNAi pathway). Znf3 is also required for transgene-induced mitotic silencing; <italic>znf3</italic> mutations abrogate silencing of repetitive transgenes during vegetative growth. Znf3 tagged with mCherry is localized in the cytoplasm in bright, distinct foci. Co-localization of Znf3 with the P-body marker Dcp1-GFP further supports the hypothesis that Znf3 is a novel element of the RNAi pathway and operates to defend the genome during sexual development and vegetative growth. In concussion our studies provide further understanding of unisexual reproduction as an evolutionary successful strategy.</p> / Dissertation

No description available.

Ticks acquire nutrients only by a parasitic nature of feeding on animals, including humans. During this process, a wide array of pathogens is transmitted. Ticks of the Ixodidae family receive exactly one blood meal in each active developmental. Knowing the trophic dependence of tick metabolism on the host blood meal components may enable discovering processes essential for the tick physiology and development. Exploiting a membrane system of tick feeding and whole blood fractionation, we have revealed that adult ticks need to acquire host haemoglobin-derived haem so that they can produce viable larvae, and reproduce. Haem is not further catabolised in ticks, and iron is thus acquired via independent route with the host serum transferrin as a source molecule. Using RNA-seq, we compared transcriptome compositions between guts of blood- and serum-fed ticks. We identified fifteen gut transcripts that change their levels with respect to the presence/absence of dietary red blood cells. Glutathione S-transferase, one of the identified encoded molecules, shows a clear haeminresponsive expression at both transcript and protein levels. Its apparent haem-binding properties suggest that this protein is directly involved in haem homeostasis maintenance within the tick gut. The ultimate goal of such research is to identify and verify targets that, when blocked, would render the acquisition and/or distribution system of haem in ticks nonfunctional. This would represent a novel way of anti-tick interventions in veterinary and human medicine.

The aim of this master thesis was the investigation of the uncharacterized protein TbPH1, by in silico studies, determining effects of its knock-down, studying the effect of a knock-down on the cell cycle, examining its cellular localization, and finding out about possible complexes and interaction-partners.

This thesis highlights a divergent mitochondrial intermembrane assembly pathway in the parasitic protist Trypanosoma brucei. A comparative genomic study reveals the connection of Erv1 with the cytosolic iron-sulfur protein assembly (CIA) pathway in trypanosomatids. Further, the CIA machinery of T. brucei has been described using RNAi interference and other biochemical and complementation assays. Finally, part of the divergent CIA machinery has been identified in the human intestinal pathogen Giardia intestinalis by means of complementation assays in T. brucei.

No description available.

Orientadores: Michel Georges Albert Vincentz, Fabio Tebaldi Silveira Nogueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T11:23:48Z (GMT). No. of bitstreams: 1 Zanca_AlmirSamuel_M.pdf: 11034884 bytes, checksum: 0545b58df6802e07009aef761dda3003 (MD5) Previous issue date: 2009 / Resumo: RNAs não-codificantes de 20-27 nucleotídeos (nt) regulam transcricionalmente ou pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas. Dentre estes, microRNAs (miRNAs) desempenliam papel chave no desenvolvimento vegetal, observação comprovada pela avaliação fenotípica e molecular de plantas transgênicas e de mutantes defectivos na produção de tais RNAs. MiRNAs são produzidos a partir de precursores longos (pri-miRNAs), os quais são posteriormente processados por enzimas específicas, gerando o miRNA maduro (20-22 nt). O miRNA maduro, por sua vez, guia a clivagem do mRNA de genes-alvo e bloqueia a tradução de proteínas, afetando diversos aspectos do desenvolvimento. O sequênciamento de populações de RNAs regulatórios possibilitou a identificação de miRNAs conservados e específicos em diferentes espécies vegetais, embora estudos em plantas de importância econômica sejam ainda incipientes. Atualmente, existem diversos bancos públicos de sequências ESTs disponíveis. Esses bancos possuem um grande número de sequências não-codíficantes, dentre as quais podem estar presentes pri-miRNAs, os quais são também são moléculas poliadeniladas similares a mRNAs codifícantes. O banco público de ESTs de cana-de-açúcar TIGR Gene Index foi usado como base para uma busca de miRNAs. O processo criado possibilitou a identificação de 20 precursores de miRNAs, agrupados em 15 famílias distintas. No presente trabalho desenvolveu-se também ferramenta para predição de potenciais alvos para os miRNAs encontrados. As famílias de miRNAs de cana-de-áçucar e a ferramenta de predição de genes-alvo estão integrados em banco de dados que estará disponível brevemente. Análise de expressão gênica demonstrou que precursors de miRNAs de cana-de-açúcar acumulam em níveis variáveis em distintos tecidos/órgãos. Além disso, tanto o acúmulo do miRNA maduro quanto a degradação do mRNA-alvo foram avaliados para alguns casos estudados. A caracterização de um miRNA específico de monocotiledôneas (miR528) e a confirmação de seu alvo, um gene comum em angiospermas, predito pela primeira vez neste trabalho, gera um interessante questionamento sobre a regulação desse gene via miRNA apenas em monocotiledôneas / Abstract: No-coding RNAs of 20-27 nucleotides (nt) transcriptional or posttranscriptionally regulate endogenous gene expression, affecting the cellular output of transcripts and proteins. Among these RNAs, microRNAs (miRNAs) play an important role in plant development as confirmed by phenotypic and molecular evaluation of transgenic plants and knockout mutants defective in miRNA biogenesis and function. miRNAs are produced from long precursors (pri-miRNAs), which are processed by specific enzymes into the mature miRNA (20-22 nt). The mature miRNA guides the cleavage of target genes as well as impairs protein translation, affecting several development processes. Deep sequencing of small RNAs identified conserved and species-specific miRNAs. Nevertheless, studies on crops are still in their infancy. Public ESTs databases are an important source of no-coding sequences, in which we can find miRNAs precursors, which are polyadenilated RNAs as messenger RNAs. In this work, the public sugarcane EST database TIGR Gene Index was used to search conserved miRNAs. The pipeline developed in this work made possible the identification of 20 miRNAs precursors, grouped into 15 families. It was also developed a search tool for potential miRNAs targets. Sugarcane miRNAs precursors displayed tissue/organ differential expression profiles. Moreover, a new identified miRNA target was confirmed experimentally. This new target is regulated by a monocot specific miRNA, miR528. Interestingly, this miRNA target is conserved in eudicots and monocots, even though its regulation by miRNA is not. This finding raises the question of why this gene has evolved in having a miRNA-mediated posttranscriptional regulation only in monocots / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Les cils sont des organites essentiels chez la plupart des eucaryotes. Ils sont construits par un mécanisme appelé transport intraflagellaire (IFT). Dans cette thèse, nous avons étudié le rôle de l'IFT chez le protiste Trypanosoma brucei. Par une combinaison d'approches en vidéo-microscopie et en microscopie électronique, nous avons révélé que l'IFT est absent ou s'arrête après ciblage par ARNi de gènes requis pour le transport aller et retour. Dans ces conditions, nous avons démontré que l'IFT n'est pas nécessaire au maintien de la longueur du flagelle mature mais contrôle la distribution de plusieurs protéines non structurales. Les trains IFT transportent la tubuline, le constituant majeur de l'axonème. En collaboration avec l'équipe d'Esben Lorentzen, nous avons mis en évidence l'existence d'un module de liaison à la tubuline sur les protéines IFT74/IFT81. Par FIB-SEM, nous avons démontré que les trains IFT sont présents presque exclusivement sur seulement deux (4 et 7) des 9 doublets de microtubules du flagelle. L'utilisation de méthodes d'imagerie super résolutives par SIM, a permis de montrer sur cellules vivantes, l'existence de deux voies spécifiques pour le trafic IFT aller et retour. Cette restriction s'explique par la présence d'une polyglutamylation plus marquée de la tubuline au niveau de ces doublets. L'inhibition des enzymes responsables de la polyglutamylation freine l'accès des protéines IFT aux flagelles et interfère sévèrement avec la construction de l'organite. Ces travaux démontrent donc un rôle essentiel de la polyglutamylation, qui serait lu par les moteurs du transport intraflagellaire. / Cilia and flagella are essential organelles in most eukaryotes including humans. They are built by an active mechanism termed Intraflagellar Transport or IFT. During this thesis, we have investigated the role and functioning of IFT in the protist Trypanosoma brucei. Using a combination of video-microscopy and electron microscopy, we have revealed that IFT is absent or arrested upon RNAi knockdown of genes required for anterograde and retrograde transport, respectively. In these conditions, we have demonstrated that IFT is not required for maintenance of flagellum length but that IFT controls the distribution of several non-structural proteins, to the contrary of the established dogma. IFT trains transport tubulin, the main component of the axoneme. In collaboration with the team of Esben Lorentzen (MPI Munich), we have revealed the existence of a tubulin-binding domain on proteins IFT74/IFT81. Using FIB-SEM, we have demonstrated that IFT trains are present almost exclusively on only two (4 and 7) out of 9 microtubule doublets. The use of super-resolution imaging methods (work performed at the Janelia Research Institute, USA) allowed us to show for the first time in live cells the existence of two specific bidirectional paths for IFT trafficking. This restriction is explained by differential polyglutamylation on these two doublets. The inhibition of the enzymes responsible for polyglutamylation restricts the access of IFT proteins to flagella, resulting in severe impairment of flagellum elongation. This work demonstrates an essential role for polyglutamylation that could act as a “tubulin code” that would be decrypted by the motors of intraflagellar transport.

DNA and interfering RNA (RNAi) – short interfering RNA (siRNA) and micro RNA (miRNA) – are promising new cancer therapies, especially for drug resistant lines. However, they require a delivery system in vivo to prevent degradation and off target effects. Silica based nanoparticles, both solid and mesoporous, are a promising option due to their biocompatibility, ease of preparation and morphology control, reproducibility, and facile addition of functional groups including targeting ligands. After a brief introduction to cancer treatment and review of the current nanoparticle treatments undergoing clinical trials, this thesis details the many methods explored over the past ten years to fine-tune particle preparation, pore size, functionalization, and delivery strategies. The majority of both solid and mesoporous silica nanoparticles are synthesized using the sol-gel method and then various functionalization techniques are employed to load and protect the oligonucleotides. Externally loaded systems generally use a combination of polyethylenimine (PEI) and polyethylene glycol (PEG). Mesoporous silica nanoparticles internally load the DNA or RNAi, resulting in the added variable of pore size. Several groups have investigated how pore size alters loading and release kinetics to perfect this variable. Many groups have also tested ligands targeting for over expressed proteins on the intended cancer, triggered release techniques, cell-penetrating peptides in order to create a viable in vivo delivery system. By compiling the techniques employed by researchers over the past ten years, this thesis will elucidate which approaches are most promising for future research. Furthermore, overall strategies within the field are suggested to more easily compare studies and evaluate methods.