Regulation of single exocytotic events by the cAMP signaling pathway

Permanece por esclarecer como a via de sinalização do cAMP modula a exocitose regulada. Os principais objetivos deste trabalho foram: i) avaliar o efeito do cAMP nos eventos exocitóticos, nas propriedades dos poros de fusão e na secreção hormonal; ii) perceber o impacto da sinalização por cAMP-HCN na exocitose e nas propriedades do poro de fusão; e iii) estudar as propriedades do poro de fusão na presença de um agente neurotóxico comum, como o alumínio. Lactotrofos, isolados a partir da hipófise anterior de ratos Wistar machos, foram usados como modelo celular. Os eventos unitários de fusão exocitótica e a prolactina (PRL) libertada foram avaliados, respetivamente, em ensaios eletrofisiológicos efectuados segundo a técnica de contacto hermético no modo sobre a célula aderida à pipeta porta-elétrodo e com recurso a métodos imunológicos de deteção. Os níveis intracelulares de cAMP foram aumentados por 3-isobutil-1-metilxantina (IMBX), forscolina e N6,2'-O-dibutiril adenosina- 3',5'-monofosfato cíclico (dbcAMP). A expressão dos canais HCN foi determinada por Western-blot, qRT-PCR e imunocitoquímica em combinação com microscopia confocal. Culturas primárias de lactotrofos foram também transfetadas com DNA plasmídico que codifica HCN2 juntamente com a proteína-verde-fluorescente e um agente farmacológico foi usado para avaliar o efeito de cAMP-HCN na exocitose. Observou-se que os lactotrofos responderam à forscolina e ao dbcAMP libertando PRL de um modo bifásico e dependente da concentração, uma vez que a secreção aumentou e diminuiu, respectivamente, na gama de baixas e altas concentrações. Os compostos que elevaram os níveis de cAMP aumentaram os eventos transientes e impediram a fusão completa. Além disso, o dbcAMP promoveu o aparecimento de eventos exocitóticos transientes de elevada periodicidade, cujos poros de fusão, de maior diâmetro, se mativeram abertos durante mais tempo. A expressão das quatro isoformas de HCN foi confirmada nos lactotrofos ao nível do mRNA e, tal como no coração, rim e hipófise, o mais abundante codifica a isoforma HCN2. Nos lactotrofos com sobre-expressão desta isoforma, o dbcAMP não só aumentou a frequência dos eventos transientes e a condutância dos poros, mas também a frequência dos eventos de fusão completa. Enquanto o bloqueador dos canais HCN, ZD7288, reduziu a frequência dos eventos transientes e de fusão completa desencadeados por dbcAMP e diminuiu o diâmetro dos poros de fusão. A simultânea diminuição da libertação de PRL, da frequência dos eventos transientes e do diâmetro dos poros de fusão representaram as principais alterações observados após pré-tratamento dos lactotrofos com concentração micromolar de alumínio. Em conclusão, os resultados demonstram que elevados níveis de cAMP reduzem a secreção de PRL devido à estabilização dos poros de fusão no estado de maior abertura. Além disso, a via de sinalização cAMP-HCN afecta a actividade exocitótica e modifica as propriedades dos poros de fusão, que parecem ser igualmente importantes na citotoxicidade induzida por alumínio.

Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.

Study of predictive biomarkers of manganese toxicity : control and prevention of neurotoxicity associated with parenteral nutrition

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

Selection and combination of biomarkers to control and prevent the risk of occupational exposure to the mixture of lead, arsenic and manganese

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

Topical bacteriophage therapy of the infected diabetic foot

Tese de doutoramento, Medicina (Medicina Interna), Universidade de Lisboa, Faculdade de Medicina, 2014

Nanotecnologia aplicada a formulações tópicas de fármacos anti-inflamatórios não esteróides

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Production of laminar extrudates containing particles of a model drug processed by supercritical fluids

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2015

Effets directs et aigus de médicaments insulinosensibilisateurs sur la cellule bêta des îlots pancréatiques : de l’outil de recherche à l’identification de la décélération métabolique comme mode d’action

Le diabète de type 2 (DT2) apparaît lorsque la sécrétion d’insuline par les cellules β des îlots du pancréas ne parvient plus à compenser la résistance à l’insuline des organes cibles. Parmi les médicaments disponibles pour traiter le DT2, deux classes agissent en améliorant la sensibilité à l’insuline : les biguanides (metformine) et les thiazolidinediones (pioglitazone et rosiglitazone). Des études suggèrent que ces médicaments protègent également la fonction des cellules β. Dans le but d’identifier des mécanismes par lesquels les médicaments insulinosensibilisateurs protègent les cellules β, nous avons étudié les effets aigus de la metformine et de la pioglitazone sur le métabolisme et la fonction des cellules INS 832/13, sécrétrices d’insuline et des îlots pancréatiques isolés de rats. Nous avons aussi validé in vivo avec des rats Wistar les principales observations obtenues en présence de pioglitazone grâce à des clamps glucidiques et par calorimétrie indirecte. Le traitement aigu des cellules β avec de la pioglitazone ou de la metformine inhibe la sécrétion d’insuline induite par le glucose en diminuant la sensibilité des cellules au glucose (inhibition en présence de concentrations intermédiaires de glucose seulement). Dans les mêmes conditions, les traitements inhibent aussi plusieurs paramètres du métabolisme mitochondrial des nutriments et, pour la pioglitazone, du métabolisme des lipides. Les composés affectent le métabolisme en suivant un patron d’inhibition similaire à celui observé pour la sécrétion d’insuline, que nous avons nommé « décélération métabolique ». La capacité de la pioglitazone à inhiber la sécrétion d’insuline et à ralentir le métabolisme mitochondrial de façon aigüe se confirme in vivo. En conclusion, nous avons identifié la décélération métabolique de la cellule β comme nouveau mode d’action pour les médicaments insulinosensibilisateurs. La décélération métabolique causée par les agents insulinosensibilisateurs les plus utilisés semble provenir d’une inhibition du métabolisme mitochondrial et pourrait être impliquée dans les bienfaits de ceux-ci dans un contexte de stress métabolique. Le fait que les deux agents insulinosensibilisateurs étudiés agissent à la fois sur la sensibilité à l’insuline et sur la sécrétion d’insuline, les deux composantes majeures du DT2, pourrait expliquer pourquoi ils sont parmi les agents antidiabétiques les plus efficaces. La décélération métabolique est une approche thérapeutique à considérer pour le traitement du DT2 et d’autres maladies métaboliques.

Gaba Receptor Gene Expression during Rat Liver Cell Proliferation and Its Function in Hepatocyte Cultures

The present thesis is an attempt to understand the role of GABA, GABAA and GABAB receptors in the regulation of liver cell proliferation using in vivo and in vitro models. The work also focuses on the brain GABAergic changes associated with normal and neoplastic cell growth in liver and to delineate its regulatory function. The investigation of mechanisms involving mitogenic models without cell necrosis may contribute our knowledge about both on cell growth, carcinogenesis, liver pathology and treatment. Objectives of the present study are, to induce controlled liver cell proliferation by partial hepatectomy and lead nitrate administration and uncontrolled cell proliferation by N-nitrosodiethylamine treatment in male Wistar rats, the changes in the content of GABA, GABAA,GABAB in various rat brain regions. To study the GABAA and GABAB receptor changes in brain stem, hypothalamus, cerebellum and cerebral cortex during the active cortex during the period of active DNA synthesis in liver of different experimental groups. The changes in GABAA and GABAB receptor function of the brain stem, hypothalamus and cerebellum play an important role sympathetic regulation of cell proliferation and neoplastic growth in liver. The decrease in GABA content in brain stem, hypothalamus and cerebellum during regeneration and neoplasia in liver. The time course of brain GABAergic changes was closely correlated with that of heptic DNA synthesis. The functional significance of these changes was further explored by studying the changes in GABAA and GABAB receptors in brain.

Prevalencia de obesidad y factores de riesgo cardiovascular en estudiantes de ciencias de la salud - universidad del Quindío - 2013

ANTECEDENTES: En Colombia, reportes del año 2010 de la Encuesta Nacional de la Situación en Nutrición ENSIN 2010(2), muestran uno de cada dos colombianos, presentan un índice de masa corporal mayor al esperado (3) METODO: El presente estudio de corte transversal, determino la prevalencia de obesidad y otros factores de riesgo cardiovascular en una población de estudiantes de Ciencias de la Salud de una Universidad regional en el primer periodo académico del año 2013. El tamaño de muestra fue n=113 sujetos que corresponden 60,5% a la carrera de medicina y 39,95% a enfermería. Con el fin de conocer su comportamiento con respecto a hábitos y estilos de vida específicos como el consumo de alcohol, el consumo de tabaco y el sedentarismo, así como su asociación a eventos inflamatorios relacionados con la fisiopatología de los procesos de salud asociados al peso, por medio de instrumentos de medición clínica, antropométrica y sérica, determino un modelo estadístico propicio para entender el comportamiento de la obesidad y la enfermedad Cardiovascular RESULTADOS: La prevalencia estimada de sobrepeso y obesidad por Índice de Masa Corporal (IMC), fue del 27,7% (IC 95%: 19.9%,37.2%); por el perímetro abdominal (OBPABD) se encontró una prevalencia estimada del 27,4% (IC 95%: 19,9% – 36,4%), y la prevalencia con el Índice Cintura Cadera (OBICC) fue de 3,5% (IC 95%:1,3% – 9,3%). CONCLUSIONES: La presencia de hábitos no saludables y la presencia de sobrepeso y obesidad se considera que es necesario en primera instancia una valoración general de estado nutricional de los universitarios de las diferentes facultados y plantear estrategias preventivas ya que la literatura documenta los efectos de los hábitos no saludables sino además documenta los efectos de la prevención de la misma ya que en si se ha encontrado asociación para enfermedades cardiovasculares. Se propone que para obtener mayor información del comportamiento de los factores de riesgo cardiovasculares se deberían realizar estudios retrospectivos en el que intervengan las demás carreras de la universidad y poder evaluar la totalidad de población universitaria

Modelo tipo de dermatite atópica em rato

A dermatite atópica (DA) é um tema importante na dermatologia clínica. Na verdade, a patogénese dessa doença inflamatória crónica da pele, caracterizada principalmente por pele seca e prurido, ainda está longe de ser totalmente compreendida. A fim de saber mais acerca desta complexa doença, ratos Wistar machos e adultos (n = 10) foram utilizados como modelo animal, nos quais o tratamento com acetona (AA) foi comparado com o tratamento com água por 3 dias (AW). No dia 3, uma hora após o último tratamento, a AA mostrou maior perda transepidérmica de água (TEWL), fluxo sanguíneo capilar e reduzida hidratação quando comparada com AW. A análise comportamental mostrou que a acção de coçar foi marcadamente mais frequente no grupo AA (n = 5) quando comparado ao grupo AW (n = 5). Estes resultados justificam a implementação deste modelo animal como uma ferramenta experimental para investigação da AD.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Preliminary study of the inflammatory response to subcutaneous implantation of three root canal sealers

P>Aim To evaluate the kinetics of the inflammatory tissue response to three root canal sealers using a physicochemical method for quantification of the enhanced vascular permeability and histopathological analysis. Methodology Twenty-eight male Wistar rats randomly assigned to four groups according to the evaluation periods (1, 3, 7 and 14 days) were used to assess the vascular permeability and histopathological reaction to RoekoSeal, AH Plus and Sealapex (new formulation) sealers, using saline and Chloropercha as negative and positive controls, respectively. Seven rats were sacrificed per period. The biocompatibility of the sealers was evaluated spectrophotometrically and histopathologically. Results At day 14, Sealapex produced significantly more inflammatory exudate than AH Plus and RoekoSeal (P < 0.05); however, there was no significant difference between AH Plus and RoekoSeal (P > 0.05). Sealapex (new formulation) was the most irritating sealer, producing severe inflammation with the presence of multinucleated giant cells. RoekoSeal was the most biocompatible sealer, producing the least amount of inflammatory exudate. Conclusions RoekoSeal root canal sealer was biocompatible when implanted in connective tissue.

Trigeminal nitric oxide synthase expression correlates with new bone formation during distraction osteogenesis

Nitric oxide synthase (NOS) has been reported to be involved with both bone healing and bone metabolism. The aim of this study was to test the null hypothesis that there is no correlation between new bone formation during mandibular distraction osteogenesis and NOS expression in the trigeminal ganglion of rats. Newly formed tissue during distraction osteogenesis and trigeminal NOS expression measured by the NADPH-diaphorase (NADPH-d) reaction were evaluated in 72 male Wistar rats by histomorphometric and histochemical methods. In animals submitted to 0.5 mm/day distraction osteogenesis, the percentage of bone tissue was higher in the basal area of the mandibles compared with the center and significantly increased through the experimental periods (P < 0.05). At the sixth postoperative week, the difference in bone formation between the continuous and acute distraction osteogenesis groups was the highest. Significant correlation between new bone formation by distraction osteogenesis and NADPH-d-reactive neurons was found, varying according to neuronal cell size (r = -0.6, P = 0.005, small cells strongly stained; r = 0.5, P = 0.018, large cells moderately stained). The results suggest that NOS may play a role in the bone healing process via neurogenic pathways, and the phenomenon seems to be neuronal cell morphotype-dependent. Further studies are now warranted to investigate the mechanistic link between the expression of trigeminal NOS and mandibular new bone formation by distraction osteogenesis.