<p>Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction. </p><p>We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.</p><p>Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.</p><p>TGF- &#946; and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-&#946; and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.</p><p> In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-&#946; signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.</p> / Dissertation

No description available.

Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process.

"We are using genetic and molecular techniques to study a simple model organism, C. elegans, to determine the cues involved in the formation of the nervous system. Two molecules currently being studied in the laboratory play roles in the formation of the IL2 neurons, a class of sensory neurons in C. elegans. The first gene, dig-1, influences the sensory process or dendrite and is involved in adhesion as well as potentially providing directional information during development. The second gene, mig-10, influences the axon and may be involved in a cell signal cascade. Genetic screens of C. elegans using Ethyl methyl sulfonate (EMS) as a mutagen resulted in the isolation of mutants with defects in the IL2 sensory map; sensory processes followed aberrant paths, appearing to be defasciculated. Complementation tests showed that the mutations failed to complement n1321, a known allele of dig-1; thus, these new mutations were alleles of dig-1 (Ryder unpub. results). Several of these new alleles of dig-1, including nu336 and n1480, have been further studied to elucidate the role of this gene in sensory map formation. A dig-1 candidate gene was identified that encodes a protein that is a member of the immunoglobulin super-family (IgSF). The candidate gene is predicted to be a large gene, with a transcript of approximately 45Kb. The encoded protein contains three distinct regions and is similar to the hyalectan family of proteoglycans. N terminal region 1 contains immunoglobulin and fibronectin-like domains. Central region 2 is an area that is highly repeated with a potential to have GAGs attached. C-terminal region 3 contains domains associated with adhesion. Polymerase chain reaction (PCR) products from alleles nu336 and n1480 were amplified and sequenced from the candidate gene. The DNA lesion present in the candidate gene from both alleles fit the method for how that mutation was generated. The point mutation in allele nu336 removes a potential glycosylation site. The large re-arrangement in allele n1480 truncates the transcript, suggesting that the protein is also truncated. The sequencing results along with rescuing data (R. Proenca, personal communication) showed that the candidate gene for dig-1 was the gene of interest. Each of the alleles was further studied to determine how severe that allele was by looking at the neuronal process aspect and the brood size as well as displacement of the gonad. In general, alleles with severe defects in the nervous system also had severe gonad displacement, suggesting the gene functions similarly in the two tissues. To determine if the gene was expressed at the RNA level, reverse transcriptase polymerase chain reaction (RT-PCR) was used. Most of the RT-PCRs amplified a cDNA of the appropriate size that showed dig-1 was expressed at the RNA level. RT-PCR further suggested that all three regions were in one transcript as well as confirming part of the predicted exon structure to be correct. In addition, northern analysis showed the presence of a large transcript in wildtype worms as well as a smaller truncated transcript from allele n1480. To investigate developmental differences mixed stage of RNA and embryonic RNA from wildtype animals were compared using gene specific primers. The initial RT-PCR showed potential alternative splicing occurring at the 5? end of the gene during development. To examine expression at the protein level, two recombinant proteins from dig-1 were successfully made by cloning cDNA products from the 5?and 3? end of dig-1. The constructs were sequenced and shown to be in frame. The recombinant proteins (Ant1Con1 and Ant3Con3) were mass produced and sent to a commercial source for injection into pre-screened rabbits. Western analysis showed the presence of an antibody in the serum from two of the rabbits. These antibodies should prove useful in future determination of correctness of our models of DIG-1 function. IgSF members have been shown to have many roles in nervous system development. DIG-1 could act in either an attractive or a repellent role to position sensory processes during development. DIG-1 might also change its function over time; early in development DIG-1 could be adhesive and later become repellant as more sugars are added. The gene mig-10 is involved in sensory map formation. To localize MIG-10 expression, several transgenic animals were generated by injection of two constructs that should recombine in the worm to create a MIG-10::GFP fusion protein. Ten transgenic lines were generated and screened by PCR for the presence of the correct recombinant construct. If this construct makes functional, rescuing protein, the GFP expression should reflect the expression pattern of the MIG-10 protein."

No description available.

Magíster en Ciencias de la Ingeniería, Mención Química. Ingeniera Civil en Biotecnología / La nutrición es un tema muy importante en la sociedad, ya que muchos alimentos consumidos por el ser humano difieren en su aporte nutricional, y se ve reflejado en aspectos físicos, fisiológicos y neurológicos. A partir de esto, se formuló la pregunta ¿cuáles son los efectos sistémicos de la dieta en C. elegans? Para responder a esta interrogante se utilizó como modelo de estudio biológico al nematodo bacterívoro Caenorhabditis elegans que, con una anatomía simple, posee 6 neuronas responsables de la respuesta al tacto. En el laboratorio, C elegans puede ser alimentado con una series de cepas y especies de bacterias. Alterando la dieta de C. elegans se puede afectar a un número de rasgos, incluyendo esperanza de vida, fecundidad y la tasa de bombeo faríngeo. El presente trabajo de investigación tuvo por objetivo evaluar la actividad in vivo de diferentes dietas bacterianas suplementadas con azúcares sobre el desarrollo, la fertilidad y la integridad neuronal del gusano. Para lograr lo anterior se utilizan dos mutantes de C. elegans: un modelo de degeneración neuronal (mec-4d), que corresponde a un gusano que lleva una mutación en el canal MEC-4, que se expresa sólo en las neuronas del tacto y es responsable de regular el ingreso de sodio. Esta mutación provoca que el canal se abra constitutivamente provocando la muerte progresiva en esas neuronas; y un modelo de C. elegans asociado a la vía de señalización de la insulina. En este mutante a 25°C se bloquea el receptor de insulina DAF-2/IGF-1 evitando las fosforilaciones y por tanto, permitiendo la entrada del factor de transcripción DAF-16/FOXO al núcleo incrementando la capacidad celular antioxidativa. Con los mutantes antes mencionados se hicieron ensayos de fecundidad, de crecimiento y para observar la morfología neuronal en presencia de bacterias con diferente calidad nutricional y de azúcares. Los resultados muestras que los azúcares no alteran los rasgos estudiados, ya que los efectos de las dietas en sí y las mutaciones en C. elegans tiene un impacto mayor que no se ve alterado por la presencia de monosacáridos. Por otro lado, se observa que el mutante mec-4d en presencia de dietas bacterianas de mejor calidad, la protección contra la degeneración neuronal es mayor, la tasa de desarrollo se ve acelerada y la fertilidad se ve favorecida con el aumento en la deposición de huevos. En cuanto al mutante daf-2(ts);mec-4d, se observa que la degeneración neuronal es casi completamente evitada, independiente de la calidad de la alimentación. Pero, la tasa de desarrollo no se ve influenciada, y la fertilidad se ve disminuida con esta mutación. Finalmente, C. elegans permitió estudiar con éxito los rasgos de historia de vida del nematodo influenciados por dietas bacterianas de diferente calidad nutricional. Sin embargo, es necesaria la identificación de los compuestos que hacen la diferencia.

The behaviour of individual cells must be carefully coordinated across a tissue to achieve correct function. In particular, proliferation and differentiation decisions must be precisely regulated throughout development, tissue maintenance, and repair. A better understanding of how these processes are controlled would have implications for human health; cancer is, after all, dysregulated proliferation, while regenerative medicine relies on being able to influence cell decisions accurately. To investigate such fundamental biological processes, it is common practice to use an experimentally tractable model organism. Here, we focus on the germ line of the nematode worm C. elegans, which provides opportunities to study organogenesis, tissue maintenance, and ageing effects. Despite the advantages of this organism as a biological model, certain questions about germ cell behaviour and coordination remain challenging to address in the lab. There is therefore a need for computational models of the germ line to complement experimental approaches. In this thesis, we develop a new in silico model of the C. elegans germ line. Novel aspects include working in three dimensions, covering the late larval period, and integrating a logical model of germ cell behaviour into a wider cell mechanics simulation. Our model produces a reasonable fit to wild-type germline behaviour, and provides the first cell tracking and labelling predictions for the larval period. It also suggests two new biological hypotheses: 1) that “stretching” growth plays a significant role in gonadogenesis, and 2) that a feedback mechanism acts on the germ cell cycle to prevent overproliferation. Having introduced the full model, we address some technical questions arising from our work, namely: what is the effect of applying a more physically realistic force law?; and can simulation performance be improved by changing the numerical scheme? Finally, we use in silico modelling to compare a number of hypothesised germ line maintenance mechanisms. There, our results support a model with functionally equivalent germ cells undergoing at most infrequent, transient cell cycle arrests.

Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. DSB resection is the nucleic degradation of DSB ends to expose 3’ single strand DNA (ssDNA), an intermediate required for HR. To investigate genes involved in meiosis, a forward genetic screen was performed to search for novel genes or informative new mutant alleles of known genes. Mre11 is one member of the MRX/N (Mre11-Rad50-Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in budding yeast. In Caenorhabditis elegans, evidence for the MRX/N’s role in DSB resection is limited. We isolated the first separation of function allele in C. elegans , mre 11(iow1), isolated from our forward genetic screen. The mre-11(iow1) mutants are specifically defective in meiotic DSB resection but not in DSB formation. The mre 11(iow1) mutants display chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation is greatly reduced in mre 11(iow1) mutants. Irradiation induced DSBs during meiosis fail to be repaired from the early to middle prophase I in mre 11(iow1) mutants. Our data suggest that some DSBs in mre 11(iow1) mutants are repaired by the non homologous end joining (NHEJ) pathway because removing NHEJ partially suppresses some meiotic defects conferred by mre 11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to an EXO 1 dependent form of recombination repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in early to middle meiotic prophase I and in blocking NHEJ. A reverse genetic screen and a yeast two hybrid screen were performed to search for genes with genetic and/or physical interactions with mre-11. The reverse genetic screen isolated a novel meiotic gene, nhr-2, as a partial suppressor of the meiotic defects conferred by mre-11(iow1). The yeast two hybrid screen identified kin-18 interacting with mre-11. KIN-18 is the C. elegans homolog of mammalian Thousand And One kinase (TAO) kinase. KIN-18/TAO is MAPK kinase kinase whose meiotic role was unknown. We have found that KIN-18 is essential for normal meiotic progression as kin-18 mutants exhibit accelerated meiotic recombination, ectopic germ cell differentiation, and enhanced levels of germline apoptosis. In C.elegans MPK-1 activation in late pachytene is required for physiological apoptosis (nuclei removed by apoptosis serve as nursing cells for oocytes) and oocyte differentiation. The kin-18 mutants also showed absence of MPK-1 activation and aberrant MPK-1 activation that includes ectopic activation in the wrong regions in the germline or more than one time of activation. The progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator, KSR-2, of the canonical MPK-1 signaling. Our data suggest KIN-18 affects meiotic progression by modulating the timing of MPK-1 activation. This regulation ensures the proper timing of recombination and normal apoptosis, which is required for the formation of functional oocytes. Meiosis is a conserved process; revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans will motivate hypothesis for TAO kinase’s role in the germline development in higher eukaryotes. Meiosis is a crucial for sexually reproducing organisms to maintain ploidy level from one generation to the next. Accurate chromosome segregation in the meiosis requires meiotic recombination between homologous chromosomes. Failure in recombination can lead to abnormal segregation of chromosomes in meiosis, which leads to aneuploidy. Anueploidy is a leading cause of miscarriages and attributes to chromosomal related birth defects. Meiotic recombination starts with programmed DNA double strand breaks (DSBs), followed by repair of these DSBs by homologous recombination (HR) pathway. One key step in HR is resection, a process to covert DSB ends into single strand DNA (ssDNA). To broaden our understanding of meiotic DSB resection, we used a nematode, C. elegans, as a model to investigate genes in DSB resection. We have isolated a specific mutant allele of a meiotic gene, mre-11. Our data suggest meiotic DSB resection in C. elegans requires collaboration of mre-11 and another gene exo-1; efficient resection of DSB ends is important to safeguard repair of DSB by HR against other illegitimate repair pathway. In addition, we identified a gene kin-18 by looking for genes interacting with mre-11. Characterization of kin-18 show meiotic recombination is tightly coordinated with germ cell progression. Our analysis provides significant improvement in the understanding of meiotic recombination in C. elegans. Given the high conservation of the two genes, mre-11 and kin-18, our finding may be applied to other organisms.

Platinum based chemotherapy is widely used to treat cancer. Cisplatin (diamminedichloroplatinum) combination treatments provide cure for metastatic testicular cancer and prolong survival for patients suffering from ovarian, head and neck, bladder and non small cell lung cancer. Tumors that initially respond to treatment may eventually acquire resistance, resulting in treatment failure. Cisplatin resistant cells are crossresistant to arsenite and antimonite and these metalloids are exported from bacteria by the ars-operon. In this thesis, we describe the human ArsA homolog, ASNA1, as a protein involved in a novel resistance mechanism to cisplatin, arsenite and antimonite. ASNA1 was downregulated by antisense and siRNA techniques in human melanoma and ovarian carcinoma cell lines. These cells displayed increased sensitivity to arsenite and the platinum based drugs cisplatin, carboplatin and oxaliplatin. In both melanoma and ovarian carcinoma, cisplatin resistant cells overexpressed ASNA1. Blockage of ASNA1 resulted in increased apoptosis and retarded growth, complicating further characterization of ASNA1 in human cell lines. ASNA1 also promotes insulin signaling and mediates membrane insertion of tail-anchored proteins. To explore different aspects of ASNA1 function with respect to cisplatin resistance, we used the model organism C. elegans. In the nematode C. elegans, asna-1 (rnai) treated larvae were hypersensitive to cisplatin, arsenite and antimonite. Adult asna-1 mutant worms were cisplatin sensitive and this hypersensitivity was seen even when apoptosis was blocked. Expression of human ASNA1 rescued the cisplatin hypersensitivity in asna-1 mutants, showing conservation of function. Transgene expression of mutated forms of asna-1 separated the cisplatin hypersensitivity phenotype from the insulin signaling phenotype of asna-1 mutants. Three ASNA-1 residues, His164, Cys285 and Cys288 were identified as essential for ASNA-1 promoted cisplatin resistance but not for insulin signaling. Finally, studies of the C. elegans germline revealed increased numbers of apoptotic cells in asna-1 mutants. In conclusion, C. elegans is a suitable model organism to identify and characterize cisplatin response mechanisms. A targeted therapy against ASNA1 could sensitize cisplatin resistant cells and improve outcome for cancer patients.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.