No description available.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Multicellular organisms typically depend on O₂ for energy production to maintain normal cellular function, and even brief periods of O₂ deprivation may have fatal consequences. The aqueous environment is prone to changes in ambient water O₂ tension (PO₂) and thus the ability of fish to sense changes in water PO₂ and to elicit appropriate physiological responses is essential for their survival. Studies on fish O₂ chemoreception have identified neuroepithelial cells (NECs), which are characterized as having dense-cored vesicles containing serotonin (5-HT), as peripheral O₂ chemoreceptors. Upon exposure to hypoxia, isolated and cultured NECs in vitro depolarize, likely resulting in neurotransmitter release. However, to date there is no evidence that NECs are activated by hypoxia in vivo to initiate physiological responses such as the hypoxic ventilatory response (HVR), which is the focus of this thesis. Initial findings demonstrated that larval zebrafish fine-tune the HVR as early as 4 days post fertilization (dpf) and by 7 dpf, the HVR aids in O₂ uptake under hypoxic conditions. In addition, the HVR is multiphasic, with an initiation phase followed by a decline phase that gradually stabilizes above normoxic baseline values (Chapter 2). In the absence of tools to probe the hypoxia sensitivity of NECs in vivo, research focused on Merkel-like cells (MLCs), a newly proposed O₂ chemoreceptor in larval zebrafish. Using in vivo calcium imaging it was shown that MLCs are stimulated by hypoxia. Data suggest that MLCs are responsible for the initiation phase of the HVR, while peripheral sensory neurons (PSNs)/peripheral sensory ganglia (PSG) that innervate MLCs play a more important role in reducing ventilation during the decline phase of the HVR (Chapter 4). Attempts at identifying the putative neurotransmitter(s) involved in the O₂ signal transduction pathway revealed that adrenaline (AD), serotonin (5-HT), and dopamine (DA) are probable candidates (Chapter 4), though the presence of AD and DA within MLCs is yet to be confirmed. In addition, 5-HT likely plays a role in the central nervous system (CNS), integrating peripheral signals resulting in the final HVR (Chapter 3). Taken together, this thesis provides the first evidence of putative O₂ chemoreceptors responding to hypoxia in vivo and thus significantly advances models for O₂ signal transduction in larval zebrafish.

No description available.

Les gliomes sont les tumeurs cérébrales primitives les plus fréquentes de l’adulte. Le glioblastome (grade IV) en est la forme la plus agressive, caractérisé par sa résistance aux traitements actuels (chirurgie, chimiothérapie et radiothérapie). La mortalité de cette pathologie est quasi constante (survie médiane de 15 mois), ce qui justifie l’importance de découvrir de nouvelles cibles thérapeutiques. Le challenge est d'arriver à identifier des marqueurs spécifiques pour proposer un schéma thérapeutique alignant des stratégies de thérapies ciblées qui vont améliorer la prise en charge clinique, la survie globale et la survie sans progression des patients atteints de ces pathologies. Deux axes sont au centre des recherches fondamentales, translationnelles et cliniques. Le premier axe se définit autour du développement de molécules inhibitrices des voies de signalisation et le second autour du concept de cellules souches tumorales (CST) de glioblastomes (GBM) découvertes récemment dans le cerveau et qui révolutionnent la conception de la transformation tumorale.ASPM (Abnormal Spindle Like Microcéphaly Associated) est une cible candidate pertinente susceptible de participer au développement des gliomes (Horvath et al., 2007 ; Hagmann et al., 2008). Cette protéine régule la prolifération des neuroblastes, elle est fortement exprimée au stade embryonnaire, mais, reste faiblement exprimée dans le cerveau adulte. Par ailleurs, ASPM est impliquée dans divers processus de cancérisation (surexprimée dans les cancers du sein, du foie et du cerveau…), toute fois, le mécanisme responsable de cette dérégulation n’est pas encore bien caractérisé.Nos études menées sur une série de 169 gliomes humains, sélectionnés à partir de notre cohorte de patients, montrent que le gène ASPM est un marqueur de la progression vers la malignité, les grades les plus élevés exprimant le plus fortement ASPM. En outre, nous avons également montré que le niveau des transcrits d’ASPM est augmenté dans les récidives de gliomes et qu’en in vitro, ASPM contrôle la formation des gliomasphères (CST de GBM) avec une augmentation de l’expression de ses transcrits dans les cultures in vitro au fil des passages. En continuité de ces observations, nous avons alors développé un sh-miR-RNA spécifique d’ASPM permettant l’extinction post-transcriptionnelle de ce gène. Les résultats obtenus in vitro montrent que la perte d’expression d’ASPM conduit à un arrêt de la prolifération et aboutit à une mort cellulaire massive.Actuellement, des modèles de greffe de gliomasphères chez la souris (orthotopique) sont en cours de développement pour confirmer les effets observés in vitro et vérifier in vivo la validité de notre approche thérapeutique. En perspective, nous tenterons d’étudier les effets du silencing d’ASPM sur la voie de signalisation la plus dérégulée (pRB / E2F ou PI3K / AKT). Enfin, nous étudierons le rôle potentiel de cette protéine dans le contrôle du cycle cellulaire, et, in fine la mise en évidence de ses partenaires… / Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumors in adults; it is characterized by its resistance to current treatments (surgery, chemotherapy and radiotherapy). The prognosis is grim with a median survival of only 15 months underlining the importance to develop new therapeutic strategies. The recent development of the “tumor stem cell” (TSC) concept in hemopathies has been secondarily applied to gliomas with the identification of subpopulations of GBM cells which express neural stem cell markers and fulfill the criteria for stemness. Some evidences also suggest that this subpopulation could play a primary role in resistance to radio- and chemotherapy.ASPM (Abnormal Spindle Like Microcephaly Associated) is a protein regulating the proliferation of neuroblasts, highly expressed in the embryonic stage but weakly expressed in the adult brain. Preliminary reports suggesting that it could be involved in the development of gliomas (Horvath et al., 2007, Hagemann et al., 2008) prompted us to analyze further the role of this protein, focusing on its potential as a relevant candidate therapeutic target. In a series of 175 gliomas samples of various grades, we found that ASPM mRNA expression was strongly correlated with increasing tumor grade. We also found that ASPM expression increased at recurrence when compared to the initial lesion. Subsequently, we could demonstrate in vitro and in vivo that ASPM expression also increased over serial passages in gliomaspheres and in a mouse glioma xenograft model. In a therapeutic perspective, the effect of lentivirus-mediated shRNA post-transcriptional silencing of ASPM was evaluated in two different gliomasphere models and a dramatic proliferation arrest and cell death was observed. Taken together, these data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and could be an attractive therapeutic target in glioblastoma multiforme.Another potential candidate tumor stem cell target in glioma is the sonic hedgehog pathway (hedgehog-Gli) which is required for GBM growth and stem cell expansion. In a collaborative study, it was found that NANOG, a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells, modulates gliomasphere clonogenicity, CD133+ stem cell behavior and proliferation. NANOG was regulated by hedgehog-Gli signalling and was essential for GBM tumourigenicity in orthotopic xenografts suggesting that it could also be a useful potential therapeutic target.Conclusions: Accumulating evidences suggest that tumor stem cells play an important role in the oncogenesis of gliomas and in their resistance to treatment. Our data support this concept and suggest that specific stemness markers may become useful targets to improve treatment of this devastating disease.

In mammals, the trophoblast lineage of the embryo is specified before implantation. It is restricted to become the fetal portion of the placenta. We have isolated and cultured trophoblast-derived cells from day 10 and day 13 porcine embryos. These cells demonstrate morphological and biological characteristics that make them unique. We have demonstrated that these cells can grow in vitro in a defined, serum-replacement medium for over a year without showing any signs of senescence. Trophoblast-derived cells placed into serum-containing medium, however, rapidly senesce and fail to proliferate. Gene expression analysis by RT-PCR and Fluidigm analysis of cells in culture from 0-30 days confirmed expression of genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). These experiments revealed changes in gene expression over time and in response to serum-containing medium. We have demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. Also, immunofluorescence analysis results demonstrated that these cells do not only demonstrate epithelial characteristics by the expression of KRT18, but also they show expression of VIMENTIN which is a protein found in mesenchymal cells. These findings contradict studies done by Ramsoondar in 1993 and Flechon in 1995 which reported the negative expression of VIMENTIN in similar cells. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics which have taken us to the conclusion that they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

In mammals, the trophoblast lineage of the embryo is specified before implantation. It is restricted to become the fetal portion of the placenta. We have isolated and cultured trophoblast-derived cells from day 10 and day 13 porcine embryos. These cells demonstrate morphological and biological characteristics that make them unique. We have demonstrated that these cells can grow in vitro in a defined, serum-replacement medium for over a year without showing any signs of senescence. Trophoblast-derived cells placed into serum-containing medium, however, rapidly senesce and fail to proliferate. Gene expression analysis by RT-PCR and Fluidigm analysis of cells in culture from 0-30 days confirmed expression of genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). These experiments revealed changes in gene expression over time and in response to serum-containing medium. We have demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. Also, immunofluorescence analysis results demonstrated that these cells do not only demonstrate epithelial characteristics by the expression of KRT18, but also they show expression of VIMENTIN which is a protein found in mesenchymal cells. These findings contradict studies done by Ramsoondar in 1993 and Flechon in 1995 which reported the negative expression of VIMENTIN in similar cells. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics which have taken us to the conclusion that they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

Made available in DSpace on 2014-06-11T19:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-01Bitstream added on 2014-06-13T20:17:13Z : No. of bitstreams: 1 oliveira_cf_me_arafo.pdf: 444703 bytes, checksum: 9ede9f0590ee9d328107f7e49cda8549 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O laser de baixa intensidade, também conhecido como laser terapêutico, tem sido recomendado para uma variedade de procedimentos clínicos, dentre eles o tratamento da hipersensibilidade dentinária. Pesquisas in vivo, onde dentes foram tratados com laser, demonstraram aumento na síntese de matriz de dentina e menor intensidade na reação inflamatória pulpar. Entretanto, o mecanismo que rege este processo permanece desconhecido. Mediante ao exposto, a presente pesquisa teve como objetivo avaliar, in vitro, o metabolismo (MTT assay), expressão de fosfatase alcalina e síntese de proteínas quando células de linhagem odontoblástica MDPC-23 foram submetidas à irradiação com laser de baixa intensidade. A expressão dos genes que codificam para colágeno tipo-1 (Col-I) e fibronectina (FN) foi analisada por meio de RT-PCR. Para isto, células foram previamente cultivadas em placas de Petri (15.000 células/cm2) e submetidas a condições de estresse pelo período de 12 horas. Subseqüentemente, 6 aplicações do laser em parâmetros específicos foram realizadas em intervalos de 12 horas. Como grupo controle foi utilizado células não irradiadas. Tanto os valores do MTT quanto os níveis de proteína total não apresentaram valores estatisticamente diferentes daqueles observados para o grupo controle. Em contrapartida, as células irradiadas reduziram sua atividade de fosfatase alcalina. Os resultados de RT-PCR demonstraram haver uma tendência à redução específica, porém não estatística, na expressão dos genes avaliados após irradiação das células. Desta maneira, foi possível concluir, dentro das condições experimentais, que os parâmetros do laser de baixa intensidade utilizados na presente pesquisa não influenciaram o metabolismo celular, porém reduziram discretamente a expressão de algumas proteínas específicas. / Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. In vivo studies in which teeth were treated with LLLT have demonstrated an increase in dentin matrix synthesis and lower intensity of pulp inflammatory reaction. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-I) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, odontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15,000 cells/cm2) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Non-irradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p>0.05). On the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p<0.05). RT-PCR results demonstrated a trend to a specific reduction in gene expression after cell irradiation, though not significant statistically (Mann- Whitney test; p>0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins.

Orientador: Carlos Alberto de Souza Costa / Banca: Rosane Lizarelli / Banca: Cláudio Miguel da Costa Neto / Resumo: O laser de baixa intensidade, também conhecido como laser terapêutico, tem sido recomendado para uma variedade de procedimentos clínicos, dentre eles o tratamento da hipersensibilidade dentinária. Pesquisas in vivo, onde dentes foram tratados com laser, demonstraram aumento na síntese de matriz de dentina e menor intensidade na reação inflamatória pulpar. Entretanto, o mecanismo que rege este processo permanece desconhecido. Mediante ao exposto, a presente pesquisa teve como objetivo avaliar, in vitro, o metabolismo (MTT assay), expressão de fosfatase alcalina e síntese de proteínas quando células de linhagem odontoblástica MDPC-23 foram submetidas à irradiação com laser de baixa intensidade. A expressão dos genes que codificam para colágeno tipo-1 (Col-I) e fibronectina (FN) foi analisada por meio de RT-PCR. Para isto, células foram previamente cultivadas em placas de Petri (15.000 células/cm2) e submetidas a condições de estresse pelo período de 12 horas. Subseqüentemente, 6 aplicações do laser em parâmetros específicos foram realizadas em intervalos de 12 horas. Como grupo controle foi utilizado células não irradiadas. Tanto os valores do MTT quanto os níveis de proteína total não apresentaram valores estatisticamente diferentes daqueles observados para o grupo controle. Em contrapartida, as células irradiadas reduziram sua atividade de fosfatase alcalina. Os resultados de RT-PCR demonstraram haver uma tendência à redução específica, porém não estatística, na expressão dos genes avaliados após irradiação das células. Desta maneira, foi possível concluir, dentro das condições experimentais, que os parâmetros do laser de baixa intensidade utilizados na presente pesquisa não influenciaram o metabolismo celular, porém reduziram discretamente a expressão de algumas proteínas específicas. / Abstract: Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. In vivo studies in which teeth were treated with LLLT have demonstrated an increase in dentin matrix synthesis and lower intensity of pulp inflammatory reaction. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-I) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, odontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15,000 cells/cm2) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Non-irradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p>0.05). On the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p<0.05). RT-PCR results demonstrated a trend to a specific reduction in gene expression after cell irradiation, though not significant statistically (Mann- Whitney test; p>0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins. / Mestre