Hamburg, Universiẗat, Diss., 2004.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 179-189). Also available in electronic version.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 193-218). Also available in electronic version.

Universiẗat, Diss., 2005--Heidelberg.

Investigating the cellular processes anti-ageing compounds interact with can identify genes and pathways involved in ageing. The macrolide lactone FK506 was identified in a phenotypic screen as extending lifespan in yeast and C. elegans through an unknown mechanism. FK506 also ameliorates neurodegeneration and age-related weight gain in rodents. Here, the mechanism of action of FK506 has been investigated in two experimental systems: C. elegans and 3T3-L1 mouse adipocytes. As the general mechanisms of ageing are well conserved between C. elegans and mammals, C. elegans has been used to understand how FK506 acts at an organismal level. Firstly, the result of the phenotypic screen was confirmed. FK506 treatment induced lifespan extension in C. elegans in the presence of population crowding stress, but not in the absence of crowding. FK506 treatment inhibited neither E. coli OP50 growth nor C. elegans pharyngeal pumping, demonstrating that FK506 did not induce dietary restriction to extend lifespan. FK506 treatment increased C. elegans thrashing and pharynx pumping rates in early adulthood and delayed accumulation of gut bacteria, showing that FK506 extended healthspan. A transcriptome analysis of FK506-treated C. elegans allowed the identification of transcripts whose levels change and potential pathways by which FK506 manifests its effect. To explore this and to identify potential targets of FK506, the cellular functions required for FK506 to extend C. elegans lifespan and healthspan were investigated using RNA-seq, RNAi, genetic mutation and co-treatment with small molecule inhibitors and inducers. Interestingly, FK506 was found to have different mechanisms of action on lifespan and healthspan. The mechanism of FK506 on C. elegans thrashing rate was DAF-16 dependent, did not require population crowding stress, had a partial interaction with FUdR and autophagy, and may involve Ca²⁺ flux. The mechanism of FK506-induced C. elegans lifespan extension overlapped with dietary restriction and was dependent on calcineurin, TOR-independent regulation of autophagy and the presence of population crowding stress. FK506 may modulate body weight by influencing metabolism and/or acting on adipocytes directly. FK506-treated aged 3T3-L1 adipocytes accumulated significantly less lipid, indicating that FK506 acts directly on adipocytes. RNA-seq of FK506-treated adipocytes found that translation-associated RNAs were upregulated whilst RNAs associated with lipid metabolism were downregulated. An ER-localised FK506-binding protein was up regulated in both C. elegans and 3T3-L1 adipocytes, fkb-4 and Fkbp2 respectively. In conclusion, FK506 has been confirmed as a potential anti-ageing treatment, through its ability to extend lifespan and healthspan in C. C. elegans. In addition, FK506 has also been shown to act directly on mouse adipocytes, resulting in a reduction in lipid accumulation. This action could explain how FK506 caused weight loss in obese aged rats, restoring body mass to a healthy adult weight.

Metazoan development is a complex and tightly controlled process that not only requires precise cell fate differentiation, but also demands accurate timing of cell division and precise cell migration. Genetic regulation of cell cycle length throughout metazoans embryogenesis is largely unknown, mainly due to the technical hurdle in quantifying cell division timing during development. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study the genetic regulation of division timing because of its invariant cell lineage and widespread division asynchronies between sister cells. A combination of in toto imaging and automated cell lineaging coupled with high throughput RNAi allows genetic screening of genes involved in regulation of Asynchrony of Division between Sister cells (ADS) or cousin cells. One of the most pronounced asynchronies between cousin cells during C. elegans embryogenesis is a significant elongation of division timing in two endoderm progenitor cells, Ea and Ep (E2), versus their cousins MSa and MSp (MS2) that mainly develop into mesoderm organs. Out of a total of 822 essential and conserved genes that were perturbed by RNAi in our previous genetic screening, 53 genes are found to produce significantly reduction specifically in the E2 cell cycle length (p<0.01). Surprisingly, nearly 70% of the 53 genes are involved in mRNA production or its regulation, indicating a differential requirement of transcription for division timing between E2 and MS2. Reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. Furthermore, our systematic data on cell division timings upon perturbation of a large cohort of essential genes provide a valuable source for inferring the function of uncharacterized gene. For example, phenotypical clustering based on cell division timings suggested that an essential gene, gad-1, is likely to be involved in general transcription, which is in agreement with its further functional assays. In summary, a combining of the published data with our own demonstrates that E2 specific cell cycle elongation requires robust and earlier zygotic genome activation (ZGA) during C. elegans embryogenesis. The cell-specific elongation might be important for coordinate fate differentiation, division timing and cell migration of E2 to ensure proper intestine development.

Submitted by Tania Ivani Rokohl (tania.rokohl@uffs.edu.br) on 2018-03-14T13:09:55Z No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) / Approved for entry into archive by Diego dos Santos Borba (dborba@uffs.edu.br) on 2018-03-14T14:00:02Z (GMT) No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) / Made available in DSpace on 2018-03-14T14:00:02Z (GMT). No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) Previous issue date: 2018-01-18 / Atualmente os produtos orgânicos têm ganhado destaque por sua qualidade nutricional, segurança alimentar e pela sustentabilidade na sua produção. A produção orgânica utiliza fungicidas à base de cobre, como a calda bordalesa, que é composta por sulfato de cobre e cal hidratada. O cobre é um elemento químico natural e fundamental para os organismos vivos, porém há controvérsias em relação aos efeitos causados ao ambiente e à saúde humana. Assim, o objetivo deste estudo foi avaliar os teores de cobre em solos e sucos de laranja e uva de uma propriedade orgânica que utiliza a calda bordalesa como fungicida, e a partir das concentrações encontradas, avaliar os efeitos da exposição aguda e crônica sobre o sistema nervoso de Caenorhabditis elegans. O estudo iniciou a partir da coleta dos solos e das frutas, logo após a colheita foram extraídos os sucos de laranja e uva. Posteriormente, foram analisadas as concentrações de cobre em solos e nos sucos de laranjas e uvas através de Espectrofotometria de Absorção Atômica por Plasma de Argônio. Após a determinação dos teores de cobre nos sucos de laranjas e uvas, os C. elegans foram divididos em cinco grupos, o grupo controle recebeu água ultrapura, e os tratados com cobre receberam as doses de 0,05 mg/L, 0,1 mg/L, 0,3 mg/L e 0,7 mg/L. Após a exposição foram avaliados parâmetros comportamentais e enzimáticos de cepas selvagens de C. elegans. Os ensaios das exposições aguda e crônica dos C. elegans ao cobre demonstraram que houve alterações da atividade da Acetilcolinesterase. Na exposição aguda as doses de 0,05 mg/L, 0,1mg/L e 0,7 mg/L ativaram a enzima, resultado similar foi encontrado após exposição crônica para as doses 0,3 mg/L e 0,7 mg/L. Já as doses de 0,05 mg/L e 0,1 mg/L inibiram a enzima após exposição crônica. Os organismos expostos também mostraram alterações comportamentais, como redução nos batimentos faríngeos à exposição aguda na dose de 0,05 mg/L e 0,7 mg/L em exposição crônica e aumento no ciclo de defecação nas doses de 0,1 mg/L e 0,3 mg/L em exposição aguda e redução em exposição crônica à 0,3 mg/L. Conforme já era esperado os níveis de cobre estavam aumentados nos solos que receberam o fungicida a base de cobre, o mesmo foi encontrado para os sucos. Porém, observou-se que as doses encontradas nos sucos promoveram significativas alterações no sistema colinérgico o que repercutiu no comportamento dos vermes. Assim, recomenda-se maior critério para aplicação de calda bordalesa, considerando que o cobre por ser um metal tem potencial de bioacumulação nos sistemas biológicos. / Currently, organic products have gained attention for its nutritional quality, food safety and sustainability in its production. Organic farming uses copper-based fungicides such as a Bordeaux mixture, which is composed of hydrated lime and copper sulfate. Copper is a natural and essential chemical element for living organisms, but there are controversies regarding the effects on the environment and human health. Thus, the objective of this study was to evaluate the copper content in soils, orange and grape juice of an organic property that uses the Bordeaux mixture as fungicide, and from the concentrations found, to evaluate the effects of acute and chronic exposure on Caenorhabditis elegans nervous system. The study started from the collection of soil and fruit, after harvest were extracted the orange and grape juices. Subsequently, the copper concentrations in soil and in the juice were analysed by Atomic Absorption Spectrometry Plasma Argon. After determination of the copper concentration in the juices, the C. elegans were divided into five groups, the control group received ultrapure water, and treated with copper received the 0.05 mg/L, 0.1 mg/L, 0.3 mg/L and 0.7 mg/L. After the exposure, behavioral and enzymatic parameters of wild strains of C. elegans were evaluated. The trials of acute and chronic exposures of C. elegans to copper showed that there were changes in acetylcholinesterase activity. In the acute exposure doses of 0.05 mg/L, 0.1 mg/L and 0.7 mg/L activated the enzyme, a similar result was found after chronic exposure at doses 0.3 mg/L and 0.7 mg/L. Doses of 0.05 mg/L and 0.1 mg/L inhibited the enzyme after chronic exposure. Exposed organisms also showed behavioral changes, such as reduction in pharyngeal beats to acute exposure at a dose of 0.05 mg/L and 0.7 mg/L on chronic exposure and increase in the defecation cycle at doses of 0.1 mg/L and 0.3 mg/L on acute exposure and reduction on chronic exposure at 0.3 mg/L. As expected, copper levels were increased in soils that received the copper-based fungicide, the same was found for juices. However, it was observed that the doses found in the juices promoted significant changes in the cholinergic system, which had repercussions on the behavior of the worms. Thus, it is recommended major criterion for the application of Bordeaux mixture, considering that copper metal has to be a potential for bioaccumulation in biological systems.

Hybrid incompatibility (HI) refers to lethality, sterility and other reduction in fitness of hybrid progeny between related species which has been frequently observed in different taxa. Its mechanism has been generalized as the consequence of conflicts between genomes of the related species that have been subject to independent evolution. HI plays a critical role in speciation by reducing or preventing exchange of the genetic materials between related species, gradually leading to reproductive isolation. According to the widely accepted Dobzhansky-Muller model, HI is produced by incompatible epistatic interactions between multiple genes that are independently diverged between the parental species. HI has been intensively studied especially in Drosophila species. A number of HI loci have been mapped in various species, several of which have been molecularly cloned. However, HI remains poorly understood in other taxa. My thesis focuses on systematic characterization of HI between two sequenced nematode species, C. briggsae and C. nigoni (C. sp. 9). The former has long been established as a companion species of C. elegans for comparative study while the latter is a recently identified species that can mate with the former and produce viable and fertile hybrids in spite of their different reproduction modes, allowing one to genetically and molecularly characterize HI between nematode species for the first time. Such a study is impossible with model organism C. elegans as it cannot produce viable hybrids with any other nematode species. To facilitate genome-wide mapping of HI loci between the two nematode species, over 90 dominant visible GFP markers have been randomly integrated into C. briggsae chromosomes, permitting repeated backcrossing the marker-associated C. briggsae genomic fragments into an otherwise C. nigoni background and definitive mapping of HI loci. Genotyping of the introgressions has been achieved by C. briggsae specific PCR, with primers as close as about half a million base pairs away from each other on average. Using the cost-effective protocol for introgression and genotyping, a genome-wide HI map between the two species has been obtained based on characterization of approximately 100 independent introgression lines. A remarkable proportion of C. nigoni genome was found to be replaceable by that from C. briggsae as judged by the fact that these introgressions are viable as a homozygote. A few HI loci critical to male inviability and sterility have been narrowed down to small intervals on the X chromosome by contrasting genotypes and HI phenotypes of independent introgression strains. Strains containing these introgressions have been examined for their gonadal structures using fluorescence microscopy. Our study provides genome-wide landscape of HI between nematodes for the first time, allowing comparative studies of HI between nematode and other species.

Made available in DSpace on 2014-06-12T15:05:02Z (GMT). No. of bitstreams: 2 arquivo4517_1.pdf: 421941 bytes, checksum: d835114d9ef4fd4e72d8438bc85644cb (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho, com o isolado de Cunninghamella elegans (UCP 542), visou à avaliação do comportamento do crescimento da espécie, em presença e ausência do naftaleno, bem como a ultraestrutura e aspectos bioquímicos, através do sistema de ubiquinonas. Os resultados demonstraram a capacidade do isolado em crescer tanto em presença, como na ausência do hidrocarboneto, contudo, exibindo pequenas variações no perfil de crescimento. O estudo cromatográfico, realizado através de camada delgada e líquida de alta eficiência revelou cromatogramas com sutis diferenças no padrão de ubiquinonas. Foi observado, neste estudo, que a amostra de C. elegans, cultivada tanto em presença como em ausência de naftaleno, apresentou as ubiquinonas Q6 e ubiquinona Q9, como picos principais. Contudo, foi possível verificar que a amostra controle cultivada na ausência do naftaleno exibiu menor percentual da ubiquinona Q9 (98,1%) quando comparada com a amostra tratada (99,7%) em naftaleno. Com a utilização do método de rotina, utilizando-se microscopia eletrônica de varredura, foi possível verificar diferenças ultraestruturais da amostra de C. elegans, cultivada na presença e ausência de naftaleno. Variações relacionadas aos aspectos morfológicos, à eletrondensidade das hifas, organização micelial e presença de clamidosporos foram detectadas

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