Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Retrogenes are functional genes that are created through retroposition, whereby mature mRNA is reverse-transcribed and re-integrated into the genome. In this study, the following objectives were accomplished: (i) intrachromosomal- and interchromosomal-retroposed genes were located in H. sapiens, (ii) interchromosomal-retroposed genes were located in M. musculus, D. melanogaster, and C. elegans. To date, this is the first assay for intrachromosomal-retroposed genes in H. sapiens and interchromosomal-retroposed genes in C. elegans. Biases discovered include excess interchromosomal generation of retrogenes by chromosome X in H. sapiens, M. musculus, and D. melanogaster. Selection pressure created by the inactivation of the X chromosome during male meiosis appears to be at least partially responsible for this phenomenon. In addition, excess interchromosomal recruitment of retrogenes by chromosome X was observed in H. sapiens. The driving force appears to be an interplay between selection for female-beneficial genes and selection for male-beneficial genes. No other chromosome biases were discovered.

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Retrogenes are functional genes that are created through retroposition, whereby mature mRNA is reverse-transcribed and re-integrated into the genome. In this study, the following objectives were accomplished: (i) intrachromosomal- and interchromosomal-retroposed genes were located in H. sapiens, (ii) interchromosomal-retroposed genes were located in M. musculus, D. melanogaster, and C. elegans. To date, this is the first assay for intrachromosomal-retroposed genes in H. sapiens and interchromosomal-retroposed genes in C. elegans. Biases discovered include excess interchromosomal generation of retrogenes by chromosome X in H. sapiens, M. musculus, and D. melanogaster. Selection pressure created by the inactivation of the X chromosome during male meiosis appears to be at least partially responsible for this phenomenon. In addition, excess interchromosomal recruitment of retrogenes by chromosome X was observed in H. sapiens. The driving force appears to be an interplay between selection for female-beneficial genes and selection for male-beneficial genes. No other chromosome biases were discovered.

14-3-3 proteins have been extensively studied from yeast to mammals, and are associated with multiple roles ranging from fundamental processes such as cell cycle, apoptosis and stress response to diseases such as neurodegeneration and cancer. Indeed, 14-3-3 proteins have been suggested as possible therapeutic targets in cancer treatment. There are seven 14-3-3 genes in mammals, whereas there are only two in Caenorhabditis elegans, ftt-2 and par-5. The ftt-2 gene is expressed only in somatic lineages, whereas par-5 expression is detected in both soma and germline. Although it is known that par-5 inactivation results in sterility, the role of this gene in germline development is poorly characterized. In the present study, we use a par-5 mutation and RNA interference to characterize par-5 functions in the germline. The lack of par-5 in germ cells causes cell cycle deregulation, the accumulation of endogenous DNA damage and genomic instability. Moreover, par-5 is required for checkpoint-induced cell cycle arrest in response to DNA-damaging agents. We propose a model whereby PAR-5 regulates CDK-1 phosphorylation to prevent premature mitotic entry. Even though mammalian 14-3-3 homologs have diverged into seven genes, we verified that the basic functions of 14-3-3 in cell cycle control have been conserved in C. elegans. Therefore, this study opens a new path to investigate molecular mechanisms of 14-3-3 proteins and establishes C. elegans as a suitable system to screen for genes (RNAi libraries or mutagenesis), and drugs which can modify 14-3-3 functions. / Las proteínas 14-3-3 han sido ampliamente estudiadas desde levadura hasta mamíferos y han sido asociadas con múltiples roles en procesos como ciclo celular, apoptosis y la respuesta al estrés. Así mismo estas proteínas se han visto involucradas en enfermedades neurodegenerativas y cáncer. De hecho, las proteínas 14-3-3 han sido propuestas como posibles agentes terapéuticos en el tratamiento contra el cáncer. En mamíferos existen 7 genes que codifican para proteínas 14-3-3, mientras en Caenorhabditis elegans solo hay dos, ftt-2 and par-5. El gen ftt-2 sólo es expresado en células somáticas, mientras par-5 se expresa tanto en células somáticas como en la línea germinal. Aunque se sabe que la inactivación de par-5 puede producir esterilidad, el rol de este gen en el desarrollo de la línea germinal no ha sido caracterizado. En este estudio, se usó una mutación de par-5 y RNA interferente para caracterizar la función de par-5 en la línea germinal. La falta de par-5 en la línea germinal causa una desregulación del ciclo celular, acumulación de daño genómico e inestabilidad genómica. Además, par-5 es requerido para el arresto celular inducido por el checkpoint en respuesta a los agentes que dañan el genoma. A partir de los resultados obtenidos, se propone un modelo según el cual PAR-5 regula la fosforilación de CDK-1 para prevenir la entrada prematura en mitosis. Aunque los homólogos de 14-3-3 en humanos han divergido en 7 genes, este estudio permitió verificar que las funciones básicas de las proteínas 14-3-3 en el control ciclo celular están conservadas en C. elegans. Por lo tanto, este estudio abre un nuevo camino para estudiar las funciones moleculares de las proteínas 14-3-3 y establece C. elegans como un modelo adecuado para la búsqueda de genes y/o drogas que modifiquen la función de las proteínas 14-3-3.

This thesis is dedicated to developments and applications of metabolomics, exploiting high field NMR spectroscopy. The first part is dedicated to a general presentation of metabolomics. We also report results about the introduction of reduced dimensionality techniques for the characterization of complex mixtures, coined targeted projection NMR spectroscopy. The second part of this manuscript reports results about three different metabolomic studies carried out in human populations. The first analysis demonstrates the suitability for metabolomics of serum samples collected in the framework of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. The second study investigates a serum metabolic signature of metastatic breast cancer. The last analysis establishes potential plasma metabolic signatures for different liver pathologies, like hepatocellular carcinoma. The third part of this thesis is dedicated to the characterization of various model organisms. The first study presents a characterization of plasma and urine metabolic differences between four rat strains commonly used as controls in genetic studies. In the second study, we investigate the effects of physiological aging in Caenorhabditis elegans (C. elegans) and observe that dietary restriction buffers metabolic changes associated with aging. We further identify that perturbations in phosphocholine metabolism correlate with life expectancy. The third analysis of this part characterizes the ahr-1 C. elegans mutant, showing strong metabolic changes in ahr-1 mutants, which suggest an involvement in development and aging processes. We finally investigate in the last study the effects at the metabolic level of the interaction between an endogenous protein E4F1 and a viral protein HBx in liver cells infected by hepatitis B virus.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 148-158.

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No. of bitstreams: 1 rossoni_rd_dr_sjc.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) Previous issue date: 2017-12-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo da atividade inibitória de Lactobacillus pode contribuir na descoberta de novas estratégias terapêuticas nas infecções por Candida. Nesse contexto, o objetivo desse estudo foi isolar e identificar Lactobacillus da cavidade bucal de indivíduos livres de cárie e avaliar seu potencial de inibição de C. albicans por meio de estudos in vitro e in vivo. Primeiramente, foram avaliados os efeitos de 30 isolados clínicos de Lactobacillus sobre o número de células viáveis (UFC) em biofilme de C. albicans e sobre a formação de hifas. Os isolados que obtiveram os maiores efeitos inibitórios sobre C. albicans foram selecionados para os testes de determinação da biomassa total dos biofilmes pela absorbância do cristal violeta, análise da arquitetura dos biofilmes por microscopia eletrônica de varredura (MEV) e quantificação da expressão de genes de C. albicans (ALS3, HWP1, EFG1 e CPH1) por qPCR. Esses isolados também foram submetidos a estudos in vivo usando os modelos de Galleria mellonella e Caenorhabditis elegans. Para o estudo em G. mellonella, a infecção experimental foi avaliada pela curva de sobrevivência, quantificação da carga fúngica na hemolinfa, densidade hemocitária, quantificação da expressão gênica de peptídeos antifúngicos (Gallerymicina e Galiomicina) e monitoramento da infecção de C. albicans por análise de bioluminescência. No modelo de C. elegans, a infecção foi avaliada por meio dos ensaios de curva de sobrevivência e estudo da filamentação de C. albicans. Os resultados dos ensaios in vitro demonstraram que L. paracasei 28.4, L. rhamnosus 5.2 e L. fermentum 20.4 foram as cepas com maior atividade antimicrobiana sobre os biofilmes de C. albicans. Nessas cepas, todos os genes analisados foram regulados negativamente na associação com Lactobacillus quando comparados com o grupo controle. No estudo in vivo, a injeção de L. paracasei 28.4 em G. mellonella infectadas com C. albicans aumentou a sobrevida das larvas, o número de hemócitos e a expressão de peptídeos antifúngicos, reduzindo assim a UFC de C. albicans. Em C. elegans, L. paracasei 28.4 também foi capaz de aumentar a sobrevida dos vermes infectados com C. albicans e reduzir a filamentação. Conclui-se que L. fermentum 20.4, L. paracasei 28.4 e L. rhamnosus 5.2 tem potencial para serem usados como probióticos na cavidade bucal devido sua ação anti-biofilme e sua regulação negativa dos genes de virulência de C. albicans. L. paracasei 28.4 foi capaz de prolongar a sobrevida nos dois modelos experimentais infectados com C. albicans por apresentarem ação antifúngica e imunomodulatória. / The study of the antifungal activity of Lactobacillus may contribute to the discovery of new therapeutic strategies for Candida infections. In this context, the objective of this study was to isolate and identify Lactobacillus from the oral cavity of caries-free subjects and to evaluate its effects through in vitro and in vivo studies. First, the effects of 30 clinical isolates of Lactobacillus on the number of viable cells (CFU) in biofilms of C. albicans and on hyphae formation were evaluated. The isolates that obtained the highest inhibitory effects on C. albicans were selected for biofilm biomass determination by violet crystal absorbance, analysis of biofilm architecture by scanning electron microscopy (SEM) and quantification of the expression of C. albicans (ALS3, HWP1, EFG1 and CPH1) by real time PCR. These isolates were also submitted to in vivo studies using the Galleria mellonella and Caenorhabditis elegans models. For the study in the model of Galleria mellonella, the experimental infection was evaluated by the survival curve, quantification of the fungal load in the hemolymph, hemocitary density, the gene expression of antifungal peptides (Gallerymicin and Galiomicin) and monitoring of C. albicans infection by bioluminescence analysis. In the Caenorhabditis elegans model, the infection was evaluated by the survival curve assays and the study of C. albicans filamentation. The results of in vitro tests demonstrated that L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 were the strains with the highest antimicrobial activity on the biofilms of C. albicans. In these strains, all analyzed genes were negatively regulated in association with Lactobacillus when compared to the control group. In the in vivo study, the injection of L. paracasei 28.4 into the G. mellonella increased survival of the larvae, the number of hemocytes and the expression of antifungal peptides, thus reducing the CFU of C. albicans. In C. elegans, L. paracasei 28.4 was also able to increase the survival of worms infected with C. albicans and reduce the filamentation. We conclude that L. fermentum 20.4, L. paracasei 28.4 and L. rhamnosus 5.2 have potential to be used as probiotics in the oral cavity due to their anti-biofilm action and their negative regulation of virulence genes of C. albicans. L. paracasei 28.4 was able to prolong survival of both experimental models infected with C. albicans for having antifungal and immunomodulatory action. / 13/25181-8 / 14/12458-4

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.

145 p. Advisers: John Conery (Computer and Information Science)and Shawn Lockery (Biology) / A print copy of this title is available through the UO Libraries under the call number: SCIENCE QA76.87 .D96 2006 / This thesis makes two major contributions: it introduces a novel method for analysis of artificial neural networks and provides new models of the nematode Caenorhabditis elegans nervous system. The analysis method extracts neural network motifs,or subnetworks of recurring neuronal function, from optimized neural networks. The method first creates models for each neuron relating network stimulus to neuronal response, then clusters the model parameters, and finally combines the neurons into multi-neuron motifs based on their cluster category. To infer biological function, this analysis method was applied to neural networks optimized to reproduce C. elegans behavior, which converged upon a small number of motifs. This allowed both a quantitative exploration of network function as well as discovery of larger motifs. Neural network models of C. elegans anatomical connectivity were optimized to reproduce two C. elegans behaviors: chemotaxis (orientation towards a maximum chemical attractant concentration) and thermotaxis (orientation towards a set temperature). Three chemotaxis motifs were identified. Experimental evidence suggests that chemotaxis is driven by a differentiator motif with two important features. The first feature was a fast, excitatory pathway in parallel with one or more slow, inhibitory pathways. The second feature was inhibitory feedback on all self-connections and recurrent loops, which regulates neuronal response. Six thermotaxis motifs were identified. Every motif consisted of two circuits, each a previously discovered chemotaxis motif with most having a dedicated sensory neuron. One circuit was thermophilic (heat-seeking) and the other was cryophilic (cold-seeking). Experimental evidence suggests that the cryophilic circuit is a differentiator motif and the thermophilic circuit functions by klinokinesis. / NSF: IBN-0080068