Primary cilia are cellular organelles that protrude into the extracellular space, acting as antennas. They detect a wide range of chemical cues, including SHH and PDGF, as well as fluid flow, and they modulate downstream signalling systems, such as WNT and ERK. Due to this cue-sensing ability and the close association of the primary cilium with the centrosome the organelle is able to influence both cell cycle progression and cell migration. This work investigated the effect of mutations on two genes associated with primary cilia: Arl13b and Ift172. The effects of the HNN genotype of Arl13b and the WIM genotype of Ift172 on cell migration were assessed uniquely within the context of direct current electric fields. Both cell lines showed a decreased migratory response when compared to WT cells, despite no clear involvement of cilia in sensing the direction of the electric field. This corroborated with previous data of in vivo Arl13b cellular migration. Through the use of in utero electroporation the migratory deficits of IFT172 knock down were then confirmed in vivo in the developing mouse neocortex. Further in vitro investigation revealed a slower proliferation rate of HNN and WIM cells, though this was not confirmed in vivo after IFT172 knock down using a standard BrDU protocol. Nevertheless, further in vitro investigations revealed a wide variety of cell cycle and intracellular changes within both cell lines. The commonalities included lower numbers of cells in the S-phase and lower MAPK3 phosphorylation compared to WT, and differences such as GSK3β phosphorylation on Ser9. This work showed for the first time that ciliopathies affect galvanotaxis, and revealed fundamental commonalities in cell migration and proliferation between various ciliary mutations, as well as differences in specific signalling pathways. This will hopefully aid in developing future therapeutic interventions for ciliary diseases.

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Made available in DSpace on 2014-06-11T19:29:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-20Bitstream added on 2014-06-13T19:58:59Z : No. of bitstreams: 1 ribeiro_apd_me_arafo.pdf: 10016445 bytes, checksum: 54f364c1faa5066e22a82916fe0e1ac2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Para considerar a Terapia Fotodinâmica (PDT) como tratamento clínico da estomatite protética, é necessário conhecer tanto o potencial antifúngico, como efeito citotóxico desta terapia sobre células normais do indivíduo. Assim, o objetivo desse estudo in vitro foi avaliar a citotoxicidade da PDT antifúngica com o fotossensibilizador Photogem® associado ao LED azul e ao LED vermelho em cultura de fibroblastos L929 e células odontoblastóides MDPC-23. As células foram cultivadas (30.000 células/cm2) em placas de 24 compartimentos por 48 horas e ambos os tipos celulares foram incubados com Photogem® (0, 10, 25, 50, 100 ou 150 mg/L) e irradiados ou não pelo LED azul (460 ± 3 nm; 25,5 ou 37,5 J/cm2; 22 mW/cm2) ou LED vermelho (630 ± 3 nm; 70 ou 100 J/cm2; 25 mW/cm2) . O metabolismo celular foi determinado 0, 12 e 24 horas após a PDT utilizando o teste do metiltetrazolium (MTT), e a morfologia celular avaliada pela microscopia eletrônica de varredura (MEV). A técnica de citometria de fluxo foi utilizada para avaliar o tipo de morte celular (necrose ou apoptose) assim como estimar os níveis intracelulares das espécies reativas de oxigênio (EROs). Observou-se redução do metabolismo celular estatisticamente significante para todas as concentrações do Photogem® quando irradiadas em qualquer dose de luz, sendo essa redução de 90 a 97% tanto para as células L929 quanto MDPC-23 (ANOVA and Dunnet’s post hoc tests; p<0.05). Essa redução da atividade mitocondrial não foi dependente da concentração do fotossensibilizador e nem da dose de luz empregada. Também, foi demonstrado que a atividade mitocondrial das células submetidas a PDT não foi recuperada após 12 ou 24 horas, caracterizando um dano irreversível. A presença do Photogem® e da luz isoladamente não alterou estatisticamente a atividade mitocondrial de ambas as linhagens. As células submetidas... / In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its antifungal potential and its cytotoxicity effect on normal cells. Therefore, the purpose of this in vitro study was to evaluate the cytotoxicity of PDT with Photogem® associated to blue and red LED on L929 and MDPC-23 cell cultures. The cells (30.000 cells/cm2) were seeded in 24-well plates for 48 hours, incubated with Photogem® (10, 25, 50, 100 or 150 mg/L) and were irradiated or not with a blue LED source (460 ± 3 nm; 25.5 or 37.5 J/cm2; 22 mW/cm2) or with a red LED source (630 ± 3 nm; 75 or 100 J/cm2; 22 mW/cm2). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet’s post hoc tests; p<0.05) and cell morphology was examined by scanning electron microscopy. Flow cytometry was employed to analyze the type of PDT-induced cell death (necrosis or apoptosis) as well as to estimate intracellular production of reactive oxygen species (ROS). There was a statistically significant decrease of mitochondrial activity for all Photogem® concentrations associated to blue or red LED regardless irradiation time; this reduction ranged from 90 to 97% for both cell lines. This reduction, however, was not dependent on the photosensitizer concentration. It was also demonstrated that the mitochondrial activity of the cells submitted to PDT was not recovered after 12 or 24 hours, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death when in contact with the photosensitizer. The presence of Photogem® and exposure to blue and red LED increased the intracellular levels of ROS in both L929 and MDPC- 23 cells in a dose-dependent manner. The association of Photogem® and blue LED caused severe toxic effects on normal cell culture... (Complete abstract click electronic access below)

Orientador: Luis Carlos Spolidório / Resumo: Hormônios esteroides sexuais participam de diversos eventos celulares e moleculares, e exercem influência sobre o epitélio e tecido conjuntivo do periodonto. A testosterona (T), principal hormônio androgênico, pode ser convertida em estradiol (E2) pela ação da enzima aromatase, ou em di-hidrotestosterona (DHT) pela ação da enzima 5α-redutase. Para elucidar o impacto de andrógenos sobre as células que compõem os tecidos conjuntivo e epitelial, fibroblastos e queratinócitos foram avaliados em relação aos efeitos de diferentes concentrações de T e DHT, além da exposição ao anastrozol (ANA), flutamida (FLU), fulvestranto (FUL), e às associações farmacológicas T+ANA, T+FLU e T+FUL. Os resultados do presente estudo indicaram que, de modo geral, hormônios esteroides androgênicos exercem efeitos opostos sobre eventos celulares de fibroblastos gengivais humano e células epiteliais HaCaT em cultura celular. Enquanto a T e a DHT agem promovendo o aumento da proliferação e atividade de fibroblastos, a exposição de células HaCaT a estes mesmos andrógenos resulta em inibição ou exiguidade do crescimento celular, atividade metabólica ou a capacidade de repovoamento da área de arranhão in vitro. Além disso, o tratamento farmacológico com ANA, FLU, FUL, e suas respectivas associações à T, parece influenciar eventos celulares de fibroblastos gengivais humano e células epiteliais HaCaT in vitro. / Abstract: Sex steroid hormones take part in different cellular and molecular process and exert their functions on the epithelium and connective tissue of the periodontium. Testosterone (T), the main androgenic hormone can be converted to estradiol (E2) through the aromatase enzyme action, or into dihydrotestosterone (DHT) by 5α-reductase activity. To elucidate the impact of androgens on the cells that constitute the connective and epithelial tissues, fibroblasts and keratinocytes were evaluated under the effects of different concentrations of T and DHT, besides to be both exposed to anastrozole (ANA), flutamide (FLU), fulvestrant (FUL), and the pharmacological associations T+ANA, T+FLU and T+FUL. The results of this study indicated that, in general, androgenic steroid hormones exert opposite effects on cellular events of human gingival fibroblasts and epithelial cells. While androgens act stimulating gingival fibroblasts, in HaCaT cells androgens promotes a shortage or inhibition of cell growth and activity. Furthermore, pharmacological treatment with ANA, FLU, FUL, and their associations to T, appears to influence cellular events of human gingival fibroblasts and HaCaT cells in vitro. / Mestre

Orientador: Carlos Henrique Ribeiro Camargo / Coorientadora: Profa. Dra. Gleyce Oliveira Silva / Banca : Eduardo Bresciani / Banca : Aleteia Massula de Melo Fernandes / Resumo: O objetivo deste estudo foi avaliar in vitro o comportamento biológico celular da N-Acetilcisteína (NAC), diante da estimulação ou não pelo LPS bacteriano. Foram utilizados fibroblastos do ligamento periodontal, que ficaram em contato por 48 horas com as substâncias testadas: NAC, Hidróxido de cálcio p.a. (HC), Lipopolissacarídeo de Escheria Coli (LPS), NAC + LPS e HC + LPS. Para os grupos NAC + LPS, HC + LPS e LPS, as células foram estimuladas com 2µg/mL de LPS por 24 horas, previamente aos tratamentos descritos. Foram realizados os testes biológicos de viabilidade celular (XTT), Espécies Reativas de Oxigênio (ROS), Elisa (para as citocinas IL-6, IL-8, IL-10, IL-1β e TNF-α) e Micronúcleo (MNT). Os dados foram analisados estatisticamente pela análise descritiva e pelos testes ANOVA e Kruskall Wallis, seguido do teste Dunn (p˂0.05) para os testes de XTT, ROS e MNT, e para o teste Elisa foi realizado cálculo das médias e desvio padrão. Os resultados obtidos mostraram que NAC teve um bom comportamento, frente à agressão provocada pelo LPS, quanto à produção de ROS. HC apresentou maior viabilidade celular que NAC, embora NAC não tenha apresentado citotoxicidade. Em relação à expressão das citocinas, NAC foi capaz de reduzir o potencial inflamatório do LPS quando da análise do TNF- α e IL-1β. NAC foi capaz de reduzir a genotoxicidade do LPS, como mostrado pelo teste MNT. Concluiu-se que NAC apresentou um comportamento biológico satisfatório, pela viabilidade celular dos fibroblast... (Resumo completo, clicar acesso eletrônico abaixo) / ABSTRACT The purpose of this study was to evaluate in vitro the action of N-Acetylcysteine (NAC) and calcium hydroxide (CH) on biological activity, either without or with LPS stimulation in periodontal ligament fibroblasts (PDLF) cells. PDLF were placed in contact with NAC, CH, NAC + LPS, LPS and CH + LPS for 48 hours. PDLF were stimulated by bacterial LPS for 24 hours in NAC + LPS, LPS and CH + LPS groups, before the substances described above are applied. The LPS and NAC effect on cell viability was measured using a XTT test. Reactive oxygen species (ROS) production was evaluated using ROS/superoxide detection kit. Inflammatory cytokines (IL-6, IL-8, IL-10, IL-1β and TNF-α) were evaluated by enzyme-linked immunosorbent (ELISA) assay. Genotoxicity was measured using micronucleus test (MNT). The means and standard deviation for all tests were calculated. Data were analyzed statistically by descriptive analysis, ANOVA and Kruskall Wallis tests, followed by Dunn test (p˂.05). CH was superior to NAC on cellular viability, although NAC was not cytotoxic. The results showed that NAC was able to reduce ROS production of LPS. NAC was able to reduce the inflammatory potential of LPS by decreasing the TNF-α and IL-1β release. NAC was able to reduce the genotoxicity of LPS. It was concluded that NAC showed a satisfactory biological activity, presenting a minimal effect on cell viability of PDLF cells and reducing ROS production and inflammatory potential provoked by LPS, and to decrease its genotoxicity / Mestre

Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...

Introduction: Systemic sclerosis (SSc) is a generalized connective tissue disease affecting the skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. To date, none of the tested drugs have demonstrated convincing efficacy in the treatment of SSc. S100A4 is involved in the regulation of cell motility, proliferation, apoptosis, angiogenesis and remodeling of the extracellular matrix. It was originally described as a promoter of metastasis in tumors, however, its pro-inflammatory properties have recently been demonstrated in inflammatory rheumatic diseases. The aim of this study was to assess the role of S100A4 in pathological activation of fibroblasts in SSc and in experimental models of dermal fibrosis. Results: The expression of S100A4 was increased in the skin of SSc patients, in SSc fibroblasts and in experimental fibrosis in a TGF-β / Smad dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, inhibition of S100A4 or its complete deficit abrogated the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4 knock-out mice (S100A4-/- ) were protected from bleomycin-induced skin fibrosis with reduced dermal thickening,...

abstract: Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014