No description available.

The aim of the work presented in this thesis was to establish chromatin immunoprecipitation method in our laboratory as a tool to study target genes of the nuclear receptor NHR-25 in C. elegans. Once the method is established, it will be also useful for studies of other DNA binding proteins. ChIP was performed in transiently transfected cells HEK293 and analyzed using PCR and qPCR. Although ChIP is typically used to find authentic target genes in the cell or in organisms, testing protein-DNA interactions by ChIP in transient transfection system (by transfecting both the expression vector of the protein of interest and a vector containing potential binding sequence/promoter of the protein) can be useful as it serves as a relatively quick tool to confirm the direct binding. Since the detection is by PCR, this method is sensitive yet less costly non radioactive method to analyze protein-DNA interaction. For the first step towards ChIP in C. elegans; pulling down tagged protein directly from the worm was also performed as a preparation for in vivo analysis of NHR-25 regulated genes.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Cryptococcus neoformans species Complex comprises the main agents of cryptococcosis, an important systemic mycosis of nature and opportunistic. These species are capable of expressing multiple virulence factors that contribute to the development of resistance to antifungal therapeutic use. Research seeking new effective therapy measures are necessary. Heat shock proteins (Hsp) have been shown to be a promising molecular target for medical treatment of various diseases such as various forms of cancer and atherosclerosis. Studies have demonstrated that the expression of these chaperonins is also required for the maintenance of cellular homeostasis in different fungal pathogens. From this perspective, this study aimed to investigate the effect of blocking the protein Hsp90 front of sensitivity to antifungal agents in vitro planktonic cells and sessile, production of virulence factors in vitro and in vivo virulence in Cryptococcus neoformans species Complex. Therefore, the action of yeast Hsp90 protein was blocked with radicicol drug - an antibiotic capable of inhibiting the ATPase activity of Hsp90 - and then the cultures were evaluated for in vitro susceptibility to antifungal agents for therapeutic use in planktonic cells and associated in biofilms. We also assessed the effect of inactivation of Hsp90 on the production of virulence factors capsule, melanin and proteases. In addition, the in vivo virulence was investigated in a model of experimental infection using the nematode Caenorhabditis elegans. The results revealed that radicicol inhibited the growth of planktonic cells Cryptococcus spp. (n=12) with values of minimum inhibitory concentration (MIC) ranging from 0.5 to 2 &#956;g/mL. We observed synergy between pharmacological and radicicol azoles. Furthermore, blockade of Hsp90 potentiated the effect of in vitro antifungal agents, particularly for the fluconazole and itraconazole, which MIC values were reduced by up to 64 times the face of isolated cells (n=12). Although the blockade of Hsp90 by isolated radicicol was not able to inhibit biofilm formation and biofilm formed of Cryptococcus spp. (n=12) (p>0.05), the inactivation of Hsp90 potentiated the effect of amphotericin B, azoles, especially fluconazole, when tested in combination with radicicol (p<0.05). Moreover, blockade of Hsp90 significantly reduced the volume of capsular Cryptococcus spp. (n=4) (p<0.05). However, no difference was observed in production of melanin by radicicol treated cells (n=4) (p>0.05) as well as the production of protease by strains of Cryptococcus spp. (n=4) (p>0.05). By contrast, inactivation of Hsp90 decreased virulence Cryptococcus spp. in vitro and in vivo in C. elegans (p<0.0001) as well as maximize the effect of fluconazole in vitro and in vivo. Therefore, Hsp90 is an important target for the development of antifungal strategies. However, further studies are needed to better understand the role of Hsp90 in fungal pathogens of the genus Cryptococcus spp. / O Complexo Cryptococcus neoformans/C. gattii compreende os principais agentes da criptococose, uma importante micose de natureza sistÃmica e carÃter oportunista. Essas espÃcies sÃo capazes de expressar mÃltiplos fatores de virulÃncia que contribuem para o surgimento de resistÃncia aos antifÃngicos de uso terapÃutico. Pesquisas que buscam novas medidas eficazes de terapia sÃo necessÃrias. ProteÃnas do choque tÃrmico (Hsp) tÃm se demonstrado um alvo molecular promissor em Ãrea mÃdica para o tratamento de diversas doenÃas, tais como diferentes tipos de cÃncer e aterosclerose. Estudos demonstram que a expressÃo dessas chaperoninas tambÃm à necessÃria para a manutenÃÃo da homeostase celular em diferentes patÃgenos fÃngicos. Nessa perspectiva, o presente estudo teve como objetivo investigar o efeito do bloqueio das proteÃnas Hsp90 frente à sensibilidade a antifÃngicos in vitro em cÃlulas planctÃnicas e sÃsseis, produÃÃo de fatores de virulÃncia in vitro e virulÃncia in vivo no Complexo Cryptococcus neoformans/C. gattii. Para tanto, a aÃÃo das proteÃnas Hsp90 fÃngicas foi bloqueada com o fÃrmaco radicicol â um antibiÃtico capaz de inibir a atividade ATPÃsica das Hsp90 â e, em seguida, as culturas foram avaliadas quanto à sensibilidade in vitro a antifÃngicos de uso terapÃutico em cÃlulas planctÃnicas e associadas em biofilmes. TambÃm foi avaliado o efeito da inativaÃÃo das Hsp90 sobre a produÃÃo dos fatores de virulÃncia cÃpsula, melanina e proteases. AlÃm disso, a virulÃncia in vivo foi investigada em modelo de infecÃÃo experimental utilizando o nematÃdeo Caenorhabditis elegans. Os resultados revelaram que o radicicol inibiu o crescimento das cÃlulas planctÃnicas de Cryptococcus spp. (n=12), com valores de concentraÃÃo inibitÃria mÃnima (CIM) que variaram de 0,5 â 2 &#956;g/mL. Observou-se sinergismo farmacolÃgico entre o radicicol e os azÃlicos. Ademais, o bloqueio das Hsp90 potencializou o efeito dos antifÃngicos in vitro, sobretudo para o fluconazol e o itraconazol, os quais tiveram os valores de CIM reduzidos em atà 64 vezes frente Ãs cÃlulas isoladas (n=12). Embora o bloqueio das Hsp90 pelo radicicol isolado nÃo tenha sido capaz de inibir a formaÃÃo de biofilme e biofilme formado de Cryptococcus spp. (n=12) (p>0,05), a inativaÃÃo das Hsp90 potencializou o efeito da anfotericina B e derivados azÃlicos, sobretudo o fluconazol, quando testados em combinaÃÃo com o radicicol (p<0,05). AlÃm disso, o bloqueio das Hsp90 reduziu significativamente o volume capsular de Cryptococcus spp. (n=4) (p<0,05). PorÃm, nÃo observou-se diferenÃa na produÃÃo de melanina por cÃlulas tratadas com o radicicol (n=4) (p>0,05) assim como na produÃÃo de proteases pelas cepas de Cryptococcus spp. (n=4) (p>0,05). Em contrapartida, a inativaÃÃo das Hsp90 diminuiu a virulÃncia de Cryptococcus spp. in vitro e in vivo em C. elegans (p<0,0001) alÃm de potencializar o efeito do fluconazol in vitro e in vivo. Portanto, as Hsp90 sÃo um importante alvo para o desenvolvimento de estratÃgias antifÃngicas. Todavia, estudos mais aprofundados sÃo necessÃrios para melhor compreender o papel das Hsp90 nos patÃgenos fÃngicos do gÃnero Cryptococcus spp.

Identification of genetic basis of Hybrid Incompatibility (HI) in hybrids between closely related species leads to a comprehensive understanding of speciation. Although model organism C. elegans is well-established in laboratory, it has performed little contribution to this research area, because C. elegans failed to mate with other sister species and produce viable progeny. As a sister species of C. briggsae, which is close to C. elegans, newly discovered C. nigoni made it possible to identify the genetic basis of HI in nematode species. In this study, a new species pair including C. nigoni and C. briggsae was used to study the genetic and molecular bases of HI. 96 GFP markers were randomly integrated into the genome of C. briggsae by biolistic bombardment. Next-Generation Sequencing (NGS) combined with single worm PCR were performed to identify the location of GFP markers. By tracking those markers, the genomic fragments of C. briggsae linked to GFP were backcrossed into C. nigoni. Such process was repeated for at least 15 generations and total 111 strains carrying independent introgressions were generated. Widespread HI loci were identified on a genome-wide scale for the first time in nematode species, which also supported Haldane's Rule and large X-effect theory between the two species. In this study, C. nigoni genome "cn1" was de novo assembled by using a hybrid approach, which combined Illumina synthetic long-read technology and massive parallel sequencing of Fosmid mate-pair library. Two lines of hybrid sterile males each carrying an independent introgression fragment from C. briggsae X chromosome in an otherwise C. nigoni background, demonstrate similar defects in spermatogenesis. A similar pattern of downregulated genes that are specific for spermatogenesis between the two hybrids and wild type control was observed. Importantly, the downregulated genes caused by the X chromosome introgressions are significantly enriched on autosomes, suggesting an epistatic interaction between the X chromosome and autosomes. By measuring small RNAs, the results shows that a subset of 22G RNAs specifically targeting the downregulated spermatogenesis genes are significantly upregulated in hybrids, indicating that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.

Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Hybrid incompatibility (HI) is frequently manifested as lethality or sterility in hybrid progeny between related species, and plays a key role in speciation. The genetic basis of HI has been intensively studied in model organisms such as yeast and fruit fly over decades, and "Two rules of speciation" have been observed across species. C. elegans as a nematode model organism contributes little to speciation research mainly due to lack of a close relative with which it can mate and produce viable progeny. Such limitation has recently been alleviated by identification of C. nigoni, a close relative, also termed as sister species, of C. briggsae. The two can make and produce a handful of viable hybrids. Both species are members of Elegans supergroup. Hybrid cross between the two species uncovered asymmetric hybrid incompatibilities, i.e. crossing direction-dependent hybrid male sterility and inviability. Asymmetry was also observed in F1 hybrids from reciprocal crosses exclusively in male but not female (Woodruff, Eke, Baird, Félix, & Haag, 2010). Asymmetry was also observed in backcrosses between the F1 female hybrids and the parental species. For example, F2 progeny fathered by C. briggsae suffered almost 100% embryonic lethality for both males and females, whereas those fathered by C. nigoni were partially viable and fertile. Further study of HI between these two species was initiated by investigating how C. briggsae chromosomal fragments in an otherwise pure C. nigoni genome affect fitness of hybrid worms. The hybrid worms were generated by repeatedly backcrossing C. briggsae genomic fragments each bearing a visible chromosomal-integrated marker to C. nigoni to produce introgression lines. Characterization of the introgression lines provided a detailed HI landscape of between the two species. Multiple intervals on the C. briggsae X chromosome were responsible for hybrid male inviability or sterility while most of the C. briggsae autosomes were not involved in these male phenotypes (Bi et al., 2015). RNA sequencing was performed in sterile male worms bearing independent introgressions, revealing a down-regulated gene expression pattern (Li et al., 2016). To uncover the HI mechanism underlying the asymmetric HI phenotypes exhibited in hybrids in F1 generation, I performed a genome-wide screening to identify HI loci that are responsible for the hybrid male inviability and sterility in F1 as well as hybrid breakdown in F2. By crossing between C. briggsae and C. nigoni introgression lines bearing a known C. briggsae fragment, I was able to construct hybrid animals homozygous or heterozygous for C. briggsae alleles on the introgression while those on counterpart of C. nigoni were absent. Contrasting the HI phenotypes here and those between two wild-type parents allows mapping of the loci responsible for the hybrid asymmetric phenotypes. The aggregated introgressions cover 94.6% of the C. briggsae genome, including 100% of the X chromosome. Surprisingly, I identified another two C. briggsae genomic intervals on chromosomes II and IV that can rescue the hybrid male inviability but not the male sterility in F1 fathered by C. nigoni, suggesting the involvement of differential epistatic interactions in the asymmetric hybrid male fertility and inviability. What's more, I observed that two independent C. briggsae X fragments that produce male sterility in C. nigoni as an introgression rescued hybrid male sterility in F1 fathered by C. briggsae. Backcrossing of the rescued sterile F1 male to its parental species showed that they can alleviate the F2 hybrid breakdown by a handful of viable F2 mothered by C. briggsae. Subsequent backcrossing of the rescued sterile males with C. nigoni led to the isolation of a 1.1-Mb genomic interval that specifically interacts with an X-linked introgression, which is essential for hybrid male fertility. In addition, I further identified three C. briggsae genomic intervals on chromosome I, II, and IV that produced inviability in all F1 progeny, dependent on or independent of the parent-of-origin. Taken together, I identified multiple independent interacting loci that are responsible for asymmetric HI phenotypes especially hybrid male sterility and inviability, which lays a foundation for their molecular characterization.

Gene regulation in C. elegans germ cells depend on transgenerational chromatin modification and small RNA pathways. Germline silencing mechanisms evolved to repress foreign DNA from compromising the transfer of genetic information to progeny. Effective genetic tools that circumvent the silencing machinery will facilitate studies using this model organism. Specifically, translation of heat-shock inducible transgenes is inhibited in the germline making it challenging to transiently express enzymes to modify the genome. Here, we describe a genetic screen design that can be used to identify pathways that prevent germline expression of heat-shock induced transgenes. We use split-GFP (GFP1-10 and GFP11) to confine a genetic screen to germ cells. Stable transgenic lines with germline expression of single-copy integrated GFP11 were produced using MosSCI. The insertion lines will be used in RNAi or chemical mutagenesis screens for the germline de-repression of GFP1-10 expressed under heat-shock promoters. The screen is likely to identify candidate RNAi or chromatin factors involved in repressing heat-shock expression in the germline, particularly from extrachromosomal arrays. Inducible high-level expression in the germline from extrachromosomal arrays would be a valuable tool for large-scale genome engineering.

The basic helix-loop-helix (bHbH) family-of transcription factors is important in many developmental and regulatory pathways such as cellular proliferation and differentiation, lineage commitment, sex determination, neurogenesis, myogenesis, hematopoeisis and pancreatic development. The free-living nematode Caenorhabdits elegans is an important model organism. Genetics studies of a gene in nematodes help us to better understand the functioning of hornologs in more complex organisms. These studies investigate the nematode homolog of ABF-1, CeABF-1, and its potential role in the development of C. elegans. The BLAST Database (http://www.ncbi.nlm.nih.gov) predicted a bHLH protein in C. elegans, located on cosmid ZK682.4, of 170 amino acids with an overall 51% similarity to human ABF-1. Importantly, it had a 72% similarity to human ABF-1 within the bHLH domain. Genomic and eDNA clones of CeABF-1 were isolated using whole nematodes and PCR methods. Clones were constructed that would allow us to use green fluorescent protein (GFP) to localize CeABF-1 expression. We also used RNA interference to determine the function of CeABF-1. No obvious phenotype was observed in nematodes unable to produce the CeABF-1 protein. Preliminary studies suggest that CeABF-1 is not an essential gene for development in C. elegans. Constructs were also made for protein induction and antibody studies. Further studies are necessary to determine how CeABF-1 is involved in nematode development and its interaction with other proteins.

No description available.

Neurobiologists have sought an overarching logic of circuit assembly for decades. Canonically, piecemeal approaches have led to the discovery of many genetic pathways underlying discrete steps in nervous system development. These findings have cumulatively helped us understand how neurons extend axons, form neighborhoods, and choose synaptic partners to ultimately build sophisticated circuits. Today, advances in connectomics and transcriptomics have placed us in an exciting position to begin to tackle this systemically. This entails not only studying entire circuits and nervous systems, but also entire gene families which coordinate circuit assembly in space and time. The nematode C. elegans provides us with an opportunity to study circuit assembly on both genome-wide and nervous system-wide levels. In the past, C. elegans connectomics has relied heavily on the first wiring diagrams which were established in the 1980s. There is a growing need to scale this approach and study nervous systems across development, in different genetic backgrounds, and in various environmental paradigms. In this work, we first establish transgenic and in silico tools to facilitate interrogation of a previously understudied region of the C. elegans nervous system, the largest neuropil called the “nerve ring”. Our tools – WormPsyQi and AxoPAL - help study synapses and neuronal adjacencies in a precise and high-throughput manner, therefore overcoming constraints on sample size and phenotypic space. Next, we focus on the cadherin superfamily of cell adhesion molecules (CAMs) and its implications on nervous system structure and function. Across evolution, two families of CAMs have expanded significantly with increasing nervous system complexity: cadherins and immunoglobulins (IgSFs). While many studies have described the expression and function of IgSFs, many cadherins are relatively under-studied in most neuronal contexts. Here, we present an expression atlas of all cadherins encoded by the C. elegans genome. Expression patterns are described with neuron-type spatial resolution and across larval development to define the richness and diversity of the cadherin repertoire in an entire nervous system, which has never been previously done for any model organism. Our analysis reveals interesting temporal changes and a striking dichotomy between broad- and sparse-expressing cadherins. Some of the most well-conserved cadherin subfamilies - classical cadherin, calsyntenin, fat, and flamingo - are expressed in all neuron types in C. elegans. Furthermore, when analyzed in the context of the well-established C. elegans connectome, the expression atlas unfolds a putative molecular code underlying connectivity and selective adjacency. Altogether, by studying the expression of the entire cadherin family in neuronal and non-neuronal cell types, across several stages of development, this thesis highlights previously unknown salient themes of cadherin expression patterns which likely have functional implications. In addition to characterizing expression, we generated a collection of null mutants for all C. elegans cadherins, and proceeded to characterize them. To our surprise, most single mutants are viable and show minimal obvious phenotypes; we think this will favor studying neuronal functions of these genes since early lethality in other systems has often been a limitation. We also found that the C. elegans Fat cadherin homolog, cdh-4, has several structural and behavioral phenotypes. Studying neuronal structure defects in single and compound mutants of cadherins implicated by the expression and speculative molecular code will further help delineate the roles of this gene family in various aspects of circuit assembly; these include cell positioning, axodendritic patterning, synaptic partner choice, and downstream behavior. By addressing the question of circuit assembly from multiple directions and with new tools, this thesis provides a generic workflow; we hope that it will bring C. elegans neurobiologists a few steps closer to untangling complex circuit assembly in the context of entire gene families which orchestrate it.