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.

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.

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Cells from bacteria to man release extracellular vesicles (EV) such as microvesicles (MV) that carry signaling molecules like morphogens and miRNAs to control intercellular communication during health and disease. MV release also sculpts membranes, e.g. repairing damaged membranes to avoid cell death. HIV viruses also bud from the plasma membrane in a similar fashion. In order to determine the in vivo functions of MVs and regulate their release, we need to understand the mechanisms of MV release by plasma membrane budding (ectocytosis). The conserved phospholipid flippase TAT-5 maintains the asymmetric localization of phosphatidylethanolamine (PE) in the plasma membrane and was the only known inhibitor of ESCRT-mediated ectocytosis in C. elegans. Loss of TAT-5 lipid flipping activity increased the externalization of PE and accumulation of MVs. However, it was unclear how cells control TAT-5 activity to release the right amount of MVs at the right time, since no upstream regulators of TAT-5 were known. To identify conserved TAT-5 regulators we looked for new proteins that inhibit MV release. To do so, we first developed a degradation-based technique to specifically label MVs. We tagged a plasma membrane reporter with the endogenous ZF1 degradation tag (degron) and expressed it in C. elegans embryos. This reporter is protected from degradation inside MVs, but is degraded inside the cell. Thus, the fluorescence is selectively maintained inside MVs, creating the first MV-specific reporter. We identified four MV release inhibitors associated with retrograde recycling, including the class III PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. We found that VPS-34, BEC-1, RME-8, and redundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit MV release. Although we confirmed that PAD-1 and the GEF-like protein MON-2 are required for endosomal recycling, they only traffic TAT-5 in the absence of sorting nexin-mediated recycling. Instead, PAD-1 is specifically required for the lipid flipping activity of TAT-5 that inhibits MV release. Thus, our work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis. In addition, we uncovered redundant intracellular trafficking pathways, which affect organelle size and revealed new regulators of TAT-5 flippase activity. These newly identified ectocytosis inhibitors provide a toolkit to test the in vivo roles of MVs. In the long term, our work will help to identify the mechanisms that govern MV budding, furthering our understanding of the mechanisms that regulate disease-mediated EV release, membrane sculpting and viral budding. / Zellen von Bakterien bis zum Menschen produzieren Extrazelluläre Vesikel (EV) wie zum Beispiel Mikrovesikel (MV). MV können Signal Moleküle wie Morphogene und miRNA transportieren, welche die normale oder krankheitsbedingte interzelluläre Kommunikation kontrollieren. Bei der Produktion von MVs werden Membranen verformt, wie auch für die Reparatur von beschädigten Membranen um den Zelltod zu verhindern. Außerdem knospen HIV-Virus Partikel von der Plasma Membrane durch eine ähnliche Art und Weise. Um zu verstehen welche in vivo Funktion MV haben, müssen wir die Mechanismen der MV Knospung von der Plasma Membran (Ektozytose) verstehen. Die konservierte Phospholipid Flippase TAT-5 hält die asymmetrische Verteilung von Phosphatidylethanolamine (PE) in der Plasma Membrane aufrecht und war der einzig bekannte Inhibitor der von ESCRT Proteinen durchgeführten Ektozytose in C. elegans. Wenn die Lipid-flippende Funktion von TAT-5 verloren geht, wird PE externalisiert und MV sammeln sich außerhalb der Zelle an. Allerdings ist es unklar mit welchen Mechanismen die Aktivität von TAT-5 reguliert wird um die richtige Menge an MV zur richtigen Zeit zu produzieren, da die vorgeschalteten Regulatoren unbekannt sind. Um konservierte TAT-5 Regulatoren zu identifizieren suchten wir nach neuen Proteinen, die die Produktion von MV inhibieren. Dazu entwickelten wir eine Degradations-Technik um MV spezifisch zu kennzeichnen. Wir markierten einen fluoreszierenden Plasma Membran Marker mit dem endogenen ZF1 Degradations-Kennzeichen (Degron) und exprimierten es im C. elegans Embryo. Der Marker wird vor der Degradation geschützt, wenn er in einem MV von der Zelle ausgesondert wurde. Dadurch bleibt die Fluoreszenz speziell in MV erhalten, während sie innerhalb der Zelle abgebaut wird. Dadurch wurde die Sichtbarkeit von ausgeschütteten MV erhöht. Wir fanden vier Proteine, welche mit Protein Recycling in Verbindung gebracht werden, die die Ausschüttung von MV verhindern: Class III PI3Kiase VPS-34, Beclin1 Homolog BEC-1, DnaJ Protein RME-8 und das nicht näher charakterisierte Dopey Homolog PAD-1. Wir benutzten dieses Set an Proteinen, um zu testen ob und wie diese TAT-5 regulieren können. Wir fanden, dass Class III PI3Kinase, RME-8 und redundante Sorting Nexins für die Plasma Membran Lokalisierung von TAT-5 verantwortlich sind, was wichtig ist um die PE Asymmetrie aufrecht zu erhalten und die MV Produktion zu verhindern. Wenn auch PAD-1 und das GEF-ähnliche MON-2 für endosomales Recycling verantwortlich sind, regulieren sie die Lokalisation von TAT-5 nur in Abwesenheit von Sorting Nexins-reguliertem Transport. Zudem scheint PAD-1 direkt für die Lipid Translokations-Aktivität von TAT-5 verantwortlich zu sein. Demnach konnten wir zeigen, dass TAT-5 und PE Schlüsselregulatoren für MV Produktion sind, was weiterhin die Ansicht unterstützt, dass PE Externalisierung für die Ektozytose verantwortlich ist. Außerdem fanden wir, dass redundante intrazelluläre Transportwege für die Größe von Organellen verantwortlich sind und deckten neue TAT-5 Aktivitäts-Regulatoren auf. Diese neu aufgedeckten Ektozytose Inhibitoren könnten Werkzeuge sein um die in vivo Funktionen von MV zu testen. Längerfristig kann unsere Forschung dazu beitragen die Mechanismen der MV Produktion zu identifizieren und die Regulation während der krankheitsbedingten EV Produktion, der Membrane Reparatur und der Virus Knospung besser zu verstehen.

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Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 54-58).

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 176-190). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 176-190). Also available on the Internet.