Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.

Düsseldorf, Universiẗat, Diss., 2003.

Thesis (Ph. D.)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution. 1985. / Bibliography: p. 325-332.

It is well established that cyclin and cyclin-dependent kinase (CDK) form complex that plays a central role in driving cell cycle progression. The fundamental functions of CDK and cyclin are well conserved across eukaryotes. However, gene families encoding the two type of proteins are significantly expanded in multicellular organisms compared with single-cell species. Despite intensive studies on CDK and its associated cyclin in cultured cell lines, especially in cancer cell lines, the partnership between individual CDKs and cyclins remains elusive especially in vivo. Here I present our preliminary results on establishing the molecular function of a well-conserved cyclin L encoded by cyl-1 in C. elegans. Human cyclin L was demonstrated to form a complex with both CDK11 and CDK12, but its association with the latter remains controversial. Despite a possible function in both transcription and pre-mRNA splicing as suggested by in vitro studies or in yeast, the in vivo function of cyclin L has yet been established in any species. To study cyl-1's function in vivo, we generated multiple strains each expressing a chromosomally integrated single-copy transgenes consisting of CYL-1::GFP flanked by its native regulatory sequences using miniMos technique. The transgene demonstrates ubiquitous expression in nuclei across developmental stages and cell types with few exceptions, including maturing oocytes, in which gene activity is known to be shut down, consistent with its function in transcription and splicing. Co-immunoprecipitation followed by mass spectrometry reveals that CYL-1 interacts with both CDK-11 and CDK-12 along with some other uncharacterized factors. Functional validation of these interactions is underway.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2015 / Made available in DSpace on 2015-06-02T04:09:08Z (GMT). No. of bitstreams: 1 333872.pdf: 29230818 bytes, checksum: 967eab5e412210dd14ca098ef3ace9ff (MD5) Previous issue date: 2015 / A contaminação ambiental por metais é um fator de risco para asaúde pública, sendo o sistema nervoso central (SNC) um dos alvosdestes agentes tóxicos. Durante o desenvolvimento do SNC emhumanos, esta exposição pode estar relacionada a transtornos deaparecimento tardio. O manganês (Mn) é um metal essencial, porém emexcesso pode causar uma síndrome semelhante à doença de Parkinson,chamada manganismo. Este estudo teve por objetivo investigar se aexposição perinatal ao Mn durante um período específico dodesenvolvimento altera parâmetros neuroquímicos e comportamentaisde forma persistente. Para isso foram utilizados ratos Wistar machosneonatos e o verme nematódeo Caenorhabditis elegans no estágio larvalL1. Os ratos foram expostos a solução salina (controle) ou MnCI2 viaintraperitoneal (5, 10 ou 20 mg/kg/dia) do dia pós-natal (PND) 8 ao 12.Os testes comportamentais foram realizados no PND 60-65. Os ratosapresentaram prejuízo motor avaliado no teste do rotarod. A memória decurto prazo foi prejudicada, avaliada nos testes de reconhecimento deobjeto e reconhecimento social. A discriminação olfatória não foialterada pelo Mn. A análise bioquímica foi realizada no estriado e nohipocampo no PND 14 e 70. No PND 14 o tratamento com Mn induziuaumento da atividade de catalase no estriado e de glutationa peroxidase(GPx) no hipocampo. Foi observado também aumento dos níveis detirosina hidroxilase (TH) no estriado no PND 14. Na fase adultaobservamos redução dos níveis de tiois não-proteicos (NPSH) eaumento dos níveis de proteína glial fibrilar ácida (GFAP) no estriado eaumento da atividade de catalase e de GPx no hipocampo no PND 70. Otratamento com Mn causou redução dos níveis de TH estriatal no PND70 com elevação da fosforilação na Ser31, sugerindo uma ativaçãocompensatória da enzima em resposta à redução de seu conteúdo. Parainvestigar vias de sinalização que podem participar da toxicidade do Mnforam utilizados C. elegans selvagens (N2) e mutantes com perda defunção para proteínas das vias MAPKs e AKT. Os vermes foramexpostos ao Mn por 1 h na fase larval L1 utilizando concentrações de2,5 a 100 mM Mn. As cepas com perda de função em akt-1, akt-2 e sgk-1 apresentaram maior resistência ao Mn em comparação com o N2 noteste de viabilidade. Esta resistência pode estar relacionada com aresposta antioxidante. Os vermes N2 apresentaram queda dos níveis deGSH frente à exposição ao Mn, o que não ocorreu nos mutantes paraproteínas da via AKT. A exposição ao Mn induziu aumento daexpressão do gene que codifica o fator de transcrição SKN-1 nosmutantes akt-2 e a enzima antioxidante GCS-1 nos mutantes akt-1.Notavelmente, a expressão de sod-3 encontrava-se aumentada nosvermes akt-1 independente do tratamento com Mn. Porém os neurôniosdopaminérgicos foram degenerados de forma semelhante nos vermes N2e mutantes, avaliados pelo teste comportamental basal slowing eutilizando vermes que expressam proteína verde fluorescente (GFP) nosneurônios dopaminérgicos. Estes resultados sugerem que a via desinalização da AKT participa da toxicidade induzida pelo Mn sobre C.elegans devido ao seu papel de antagonizar os fatores de transcriçãoSKN-1 e DAF-16, que são importantes para a produção de enzimasantioxidantes nos vermes. Entretanto, este efeito não está presente nosneurônios dopaminérgicos. Este estudo documenta que a exposiçãoaguda ao Mn durante um período crítico do desenvolvimento neuralinduz disfunções cognitivas e motoras que duram até a idade adulta emratos. Estas disfunções foram acompanhadas por alterações no sistemade defesa antioxidante, tanto no hipocampo quanto no estriado ealteração no conteúdo e fosforilação de TH. Este estudo demonstra aimportância das vias de sinalização intracelular para a respostaantioxidante induzida pelo metal e caracteriza AKT como umimportante ponto de investigação dentro dos mecanismos de toxicidadeinduzida pelo Mn.<br> / Abstratc: Environmental contamination by metals is a risk factor forpublic health and the central nervous system (CNS) is one of the targetsof these toxic agents. Exposure during CNS development in humans isrelated to late onset damage. Manganese (Mn) is an essential metal, butin excess can cause a syndrome similar to Parkinson's disease, calledmanganism. This study aimed to investigate whether perinatal exposureto Mn in a specific developmental period would alter neurochemical andbehavioral parameters persistently. For this we used neonate maleWistar rats and the nematode worm Caenorhabditis elegans in the L1larval stage. Rats were exposed to saline (control) or intraperitonealMnCl2 (5, 10 or 20 mg/kg/day) from postnatal day (PND) 8 to 12. Thebehavioral tests were performed on PND 60-65. Rats exhibited motorimpairment evaluated in the rotarod test. The short-term memory wasimpaired, evaluated in the object recognition and social recognitiontests. The olfactory discrimination was not affected by Mn. Biochemicalanalysis was performed in striatum and hippocampus on PND 14 and70. On PND 14 Mn treatment induced an increase in catalase activity inthe striatum and glutathione peroxidase (GPx) in the hippocampus. Itwas also observed increased levels of tyrosine hydroxylase (TH) in thestriatum on PND 14. In adulthood we observed reduction in non-proteinthiols (NPSH) levels and increased glial fibrillary acidic protein (GFAP)levels in the striatum and increased catalase and GPx activity in thehippocampus on PND 70. Treatment with Mn caused reduction ofstriatal TH levels on PND 70 with increased phosphorylation at Ser31,suggesting a compensatory activation of the enzyme in response to thereduction of its content. To investigate signaling pathways involved inMn toxicity wild type (N2) and loss of function mutant (for proteins ofthe MAPK and AKT signaling pathways) C. elegans were used. Theworms were exposed to Mn for 1 h at the L1 larval stage usingconcentrations of 2.5 to 100 mM Mn. Strains with loss of function inakt-1, akt-2 and sgk-1 had higher resistance to Mn compared to N2 inthe survival test. This resistance may be related to the antioxidantresponse. The N2 worms had decreased levels of GSH after exposure toMn, which did not occur in AKT pathway mutants. Mn exposureinduced increase in the expression of the gene that codes SKN-1transcription factor in akt-2 mutants and GCS-1 antioxidant enzyme inakt-1 mutants. Notably, the expression of sod-3 was increased in theakt-1 mutant worms independent of Mn treatment. Howeverdopaminergic neurons were similarly degenerated in N2 and mutantworms, evaluated in the basal slowing response test and using wormsexpressing green fluorescent protein (GFP) in dopaminergic neurons.These results suggest that AKT signaling pathway participates in Mninducedtoxicity in C. elegans due to its role antagonizing thetranscription factors SKN-1 and DAF-16, which are important for theproduction of antioxidant enzymes in the worms. However, this effect isnot present in the dopaminergic neurons. This study documents thatacute exposure to Mn during a critical period of neural developmentinduces cognitive and motor dysfunctions that last into adulthood inrats. These disorders are accompanied by changes in the antioxidantdefense system, both in the hippocampus and in the striatum andchanges in the content and phosphorylation of TH. This studydemonstrates the importance of intracellular signaling pathways to theantioxidant response induced by Mn and features AKT as an importantpoint of research into the mechanisms of toxicity induced by Mn.

Made available in DSpace on 2014-06-12T15:04:42Z (GMT). No. of bitstreams: 2 arquivo4505_1.pdf: 602165 bytes, checksum: 189f048264e16bdafb2f0fdd543556c6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos foram realizados sobre a influência da salinidade (0, 2, 4, 6, 8, 10 e 12% de NaCl) e temperaturas (20, 25, 30 e 35ºC) na germinação dos esporangíolos, no crescimento radial e na morfofisiologia de Cunninghamella elegans isolada de sedimentos de mangue. A germinação e o crescimento radial foram realizados no meio Hesseltine & Anderson, modificado pela adição de diferentes concentrações de NaCl, incubadas sob distintas temperaturas. As análises morfológicas foram realizadas acompanhando as alterações na forma e tamanho dos esporangíolos no microscópio de luz. Observou-se que a elevação da salinidade retardou a emissão do tubo germinativo dos esporangíolos e reduziu o crescimento radial de C. elegans A germinação e o crescimento foram também influenciados pela temperatura, sendo as temperaturas de 35ºC e 30ºC ótimas para germinação e crescimento, respectivamente. Observou-se um perfil distinto para o crescimento de C. elegans à 35ºC, verificando-se um comportamento halofílico, com crescimento superior nos meios contendo 2 e 4% de NaCl quando comparado ao controle (0% de NaCl). Com a elevação da salinidade os esporangíolos tornaram-se em sua maioria elipsóides e de tamanhos aumentados em relação ao controle, onde os mesmos apresentaram-se em maior parte globosos. Os resultados sugerem que C. elegans apresenta comportamento halotolerante e/ou halofílico, possivelmente por ser proveniente de estuário, sendo as alterações morfológicas possivelmente mediadas pelo aumento da permeabilidade celular nas diferentes concentrações salinas

Made available in DSpace on 2014-06-12T15:05:02Z (GMT). No. of bitstreams: 2 arquivo4516_1.pdf: 562141 bytes, checksum: 80763c13e25f34576b5b0840cef70297 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Considerando o potencial do gênero Cunninghamella, o presente trabalho teve como finalidade estabelecer os aspectos fisiológicos, bioquímicos e ultraestruturais de Cunninghamella elegans cultivada em meio contendo cobre. O perfil de crescimento foi estabelecido em função da biomassa, do consumo de glicose e fosfato, pH do meio, conteúdo de polifosfato, atividade de fosfatases e remoção do metal. Os resultados obtidos indicam a influência do metal sobre o crescimento como observado pelo rendimento da biomassa. O consumo das fontes de carbono e fósforo foi semelhante para a cultura tratada e controle. A análise do perfil de polifosfato permitiu verificar comportamentos distintos na ausência e presença do metal. A análise do polifosfato celular revelou que, nas amostras tratadas, o polímero é significativamente metabolizado durante o início do cultivo pelas amostras tratadas. O isolado analisado não exibiu atividade para a fosfatase ácida. Contudo, o cultivo em presença de cobre induziu variações na expressão da fosfatase alcalina. Uma diminuição significativa da atividade enzimática foi observada para a cultura tratada com o íon metálico. Através da espectrofotometria de absorção atômica observou-se a remoção do metal do meio de cultivo ao longo do crescimento. Os resultados obtidos pela ultraestrutura demonstraram que C. elegans em presença de cobre, apresentou maior eletrondensidade, hifas mais largas e de textura mais homogênea, menor número de clamidósporos e poder de ramificação. Os estudos demonstram o potencial de C. elegans, visando sua aplicação em processos de biorremediação de ambientes poluídos por cobre

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The Candida genus is full of fungi that are subtle parts of the human microbiome, but they can cause complications if they overgrow within the body—specifically the mouth and throat, the genitalia, and the entire body through infection of the bloodstream. Candida species are a rising problem for many across the world, and this can be seen in the recent threat of Candida auris hospitalizing patients and being regularly resistant to anti-fungal medications. Beyond C. auris, Candida albicans is the most common Candida species that humans must combat because it causes the most infections in humans—mostly vaginal yeast infections. C. albicans does have natural competitors that can either inhibit its growth or kill it in general, and the competition that we took advantage of was with the Alcaligenes species. Alcaligenes faecalis and Alcaligenes viscolactis have been shown to at least inhibit C. albicans growth and maybe even kill the fungus. This rate of infection from C. albicans places it at the forefront of Candida research, and we attempted to further this research by utilizing both A. faecalis and A. viscolactis to create a co-infection model for Caenorhabditis elegans—a simple nematode lifeform. It is known that A. faecalis and A. viscolactis do not commonly adversely affect humans, so little research has been done concerning their clinical effects. We were looking to find a possible answer to C. albicans infections beyond antifungal drugs because we know that antibiotic resistance is on the rise. We performed liquid assays to test the survivability of C. elegans nematodes in various bacterial/fungal circumstances. We subjected batches of C. elegans to E. coli OP50 as a control, A. faecalis, A. viscolactis, C. albicans, A. faecalis and C. albicans, and A. viscolactis and C. albicans. This procedure was followed in order to determine the viability of using the Alcaligenes species to either help the C. elegans survive the infection or prevent them from getting infected at all. After following through with the project, we found that there was a noticeable increase in the survivability of C. elegans when subjected to both one of the Alcaligenes species and C. albicans as opposed to the C. albicans alone. The data, although early, shows the possibility of Alcaligenes species being used to combat C. albicans infections in lifeforms.

Vulval differentiation in Caenorhabditis elegans is a well characterized developmental system in which three vulval precursor cells divide, generating the 22 nuclei that form the functional wild type vulva. Additionally, the combined formation of the vulva and the uterus is a model for organogenesis. Hermaphrodites homozygous for a lin-11 mutation are unable to form a functional vulva due to abnormal mitotic divisions in two of the three vulval precursor cells that contribute cells to the vulva. Laser microsurgery was used to ablate the two abnormal vulval precursor cells and other vulval precursor cells that could take on their developmental fate. These cells were believed to be responsible for the inability of hermaphrodites homozygous for a lin- 11 mutation to form a functional vulva. The results show that ablated hermaphrodites homozygous for a lin-11 mutation are rarely able to lay eggs, suggesting that there are other defects in the egg-laying apparatus in addition to the vulval precursor cells. To ensure that the ablated animals did not form a functional vulva and fail to lay eggs due to defects in the neurons regulating egg-laying, ablated lin-11 mutant animals were exposed to serotonin, imipramine or nicotine. These drugs are able to induce egglaying in wild type and ablated wild type animals. Ablated hermaphrodites homozygous for a lin-11 mutation exposed to the drug treatments were not able to lay eggs. Therefore, the abnormal secondary cells are not entirely responsible for the lack of a functional vulva and the inability to lay eggs, suggesting that either uterine cells or other vulval cells are also abnormal.