Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T00:41:43Z (GMT). No. of bitstreams: 1 Melo_EstevaoAzevedo_M.pdf: 1050744 bytes, checksum: b28d2d7f1bf1683ed75a9b3e47166ad2 (MD5) Previous issue date: 2015 / Resumo: A ácido graxo sintase (FASN) é a enzima responsável pela biossíntese endógena de ácidos graxos e apontada como uma oncoproteína metabólica, por favorecer a proliferação e sobrevivência das células tumorais nas quais sua expressão é elevada. Vários são os compostos capazes de inibir a atividade de FASN, dentre eles o orlistat (Xenical®), que possui efeitos antiproliferativos previamente mostrados em células de câncer de mama, próstata, boca e melanoma. O sistema imunológico apresenta um importante papel na prevenção e defesa do organismo contra neoplasias malignas. As células do sistema imune que se infiltram nos melanomas são produtoras de uma vasta gama de citocinas, dentre elas interleucina 12 (IL-12) e interferon gama (IFN-?) que favorecem uma resposta imune bem sucedida contra os tumores, porém, as células dos melanomas possuem capacidade de produzir interleucina 10 (IL-10) e fator de crescimento transformante beta (TGF-?), capazes de inibir as células imunocompetentes, favorecendo a progressão tumoral e disseminação metastática. O objetivo deste estudo foi avaliar a secreção das citocinas IL-10, IL-12, IFN-? e TGF-? pelas células de melanoma murino B16-F10 após tratamento com orlistat. Para isto, inicialmente determinou-se a dosagem de orlistat capaz de inibir a proliferação celular em 50% (IC50). Em seguida, as células foram tratadas por 24 e 48 horas, quando realizou-se a quantificação da secreção das citocinas por ELISA. Após 24 horas de tratamento, observou-se aumento da secreção de IL-10 e IL-12, no entanto, após 48 horas de tratamento não foi detectada diferenças estatisticamente significantes na secreção de ambas as citocinas, quando comparadas aos seus controles. IFN-? e TGF-? não foram detectáveis. Assim, os resultados desta pesquisa mostram que o tratamento com orlistat alterou a produção das citocinas IL-10 e IL-12, sugerindo que o tratamento promove um equilíbrio entre estas citocinas pró e anti-inflamatórias nas células estudadas / Abstract: Fatty acid synthase (FASN) is the enzyme responsible for the endogenous biosynthesis of fatty acids suggested as a metabolic oncoprotein by promoting proliferation and survival of cancer cells. Several compounds are known to inhibit FASN activity, including orlistat (Xenical®), which has antiproliferative effects in breast, prostate, and oral cancer as well as melanoma cells. Melanoma is an aggressive malignant tumor of melanocytes with high propensity for metastatic spread and resistant to chemotherapy. The immune system plays an important role in the prevention and defense against malignant neoplams. In fact, immune cells that infiltrate melanomas produce a wide range of cytokines, such as interleukin 12 (IL-12) and interferon gamma (IFN-?), which favor a successful immune response against the tumor. However, melanomas cells are able to produce interleukin 10 (IL-10) and transforming growth factor beta (TGF-?) and in turn inhibit immunocompetent cells, favoring tumor progression and metastatic spread. The aim of this research was to evaluate the effect of the FASN inhibitor orlistat on the secretion of the cytokines IL-10, IL-12, IFN-? and TGF-? by B16-F10 mouse melanoma cells. For this purpose, we first searched for the IC50 Of orlistat in B16-F10 cells. Then, cells were treated for 24 and 48 hours with the drug and the secretion of cytokines quantified by ELISA. After 24 hours of treatment the secretion of IL-10 and IL-12 was increased, however, after 48 hours there were no statistically significant changes in the secretion of both cytokines, compared to their controls. IFN-? and TGF-? were not detectable. Thus, the results of this study show that the treatment with orlistat change the production of IL-10 and IL-12, suggesting a balance between the secretion of pro- and anti-inflammatory cytokines / Mestrado / Estomatopatologia / Mestre em Estomatopatologia

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Pelargonium sp., appelé aussi « géranium » à odeur de rose ou « Géranium rosat » est l’une des plantes aromatiques et médicinales les plus cultivées au niveau international, essentiellement pour son huile essentielle (HE), utilisée par les industries des cosmétiques et de la parfumerie. Cette essence est extraite des feuilles par distillation vapeur et donne une HE riche de plusieurs centaines de molécules volatiles. Cette complexité est le résultat d’un long processus évolutif et de sélections variétales. Parmi ces composés volatils, les monoterpènes comme le géraniol, le citronellol et l’isomenthone, ou les sesquiterpènes comme le 10- γ-épi-eudesmol et le 6,9-guaiadiène, jouent un rôle prépondérant dans le parfum du Pelargonium. Les proportions relatives de ces différents composés sont d’ailleurs utilisées comme marqueurs de la qualité de l’HE et déterminent la typicité du parfum des différents cultivars et origines (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Égypte’ et P. cv. ‘rosat Grasse’). Malgré de très nombreux travaux portant sur la chimie de cette HE, il n’existe aucune information sur les voies de biosynthèse de ces molécules et aucun gène intervenant dans ces voies n’a été isolé. Durant cette thèse, nous avons cloné et caractérisé fonctionnellement par expression et purification des protéines recombinantes chez Escherichia coli des gènes codant les enzymes clés de ces voies de biosynthèse, les terpène synthases. Nous avons ainsi pu caractériser quatre terpène synthases, dont une géraniol synthase mono-produit. Nous avons isolé deux autres monoterpène synthases multi-produits, produisant pour l’une majoritairement du myrcène mais aussi trois autres monoterpènes, et pour l’autre majoritairement du 1,8-cinéole ainsi que 10 autres monoterpènes minoritaires. Enfin, une sesquiterpène multi-produit, la 10-γ-épi-eudesmol synthase, a été caractérisée. Nous avons ensuite analysé l’expression de la géraniol synthase et de la 10-γ-épi-eudesmol synthase dans différentes accessions de Pelargonium par RT-qPCR et nous avons montré la relation entre la capacité de production des différents composés volatils et le niveau d’expression dans les feuilles de ces deux terpène synthases. L’efficacité de la transformation génétique du Pelargonium par Agrobacterium tumefaciens étant élevée, des expériences de transgénèse ont aussi été réalisées afin de compléter la caractérisation fonctionnelle des gènes isolés. Dans une deuxième partie, nous avons réalisé l’analyse des essences produites par 64 espèces et cultivars de Pelargonium d’odeurs très diverses (citron, menthe, rose, abricot, pin, épices…). A l’aide d’analyses statistiques (ACP, analyse discriminante…), nous avons mis en évidence des relations entre la biochimie de ces cultivars, leurs odeurs et leurs proximités génétiques et cela afin de nous donner des pistes sur des croisements potentiellement intéressants. Enfin, un dernier chapitre est consacré à l’amélioration de la production d’HE en Égypte. Grâce à ce programme commencé il y a trois ans, nous améliorons chaque année la qualité et le rendement en HE de plus de 10 Ha de plantation de Pelargonium en Égypte. Un travail d’optimisation de la distillation ainsi que des améliorations des pratiques culturales, nous ont permis de produire une HE de qualité avec un rendement de plus de 60 kg.Ha-1 d’HE. D’autres expériences présentées dans ce chapitre soulignent l’influence de l’environnement et notamment de la température sur le ratio entre le citronellol et le géraniol ainsi que sur la biosynthèse de l’isomenthone, du 10-γ-épi-eudesmol et du 6,9-guaiadiène / Pelargonium sp, also named rose scented « geranium » or « Geranium rosat » is one of the the most cultivated aromatic and medicinal plant worldwide, especially for its essential oil (EO), which is used by cosmetic and perfumery industries. This essence is extracted from leaves by steam distillation and gives an EO containing several hundreds of organic volatile compounds (VOC). This complexity is the result of a long evolutive process and varietal selections. Among these VOC, the monoterpenes like geraniol, citronellol and isomenthone and the sesquiterpenes like 10-γ-epieudesmol and 6,9-guaiadiene, play an important role for the Pelargonium fragrance. The relative proportions of these compounds are used as EO quality markers and determine the different cultivars origins (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Egypt’ and P. cv. ‘rosat Grasse’). Despite the important researches on the chemistry of these EO, there is no information on the biosynthesis pathways for these molecules and no genes involved in the pathways have been isolated. During this PhD thesis, we have functionally characterized by recombinant proteins expression and purification in Escherichia coli, four genes, three monoterpene and one sesquiterpene synthases, coding for key enzymes in terpene biosynthesis pathway. The first enzyme is a mono-product geraniol synthase. The second enzyme is a multi-product enzyme with a major peak of myrcene and 3 minor peaks of other monoterpenes. The third enzymes is also a multi-product protein, producing 1,8-cineol as major product and 10 others monoterpenes. The last one is a multi-products sesquiterpene synthase producing mainly the 10-γ-epi-eudesmol and other sesquiterpenes. We have also analyzed the level of expression of the geraniol and 10 γ-epi-eudesmol synthases in several Pelargonium accessions by RT-qPCR and we have demonstrated the relationship between the level of expression of these two terpene synthases and the quantity of the related terpenes produced in leaves. Pelargonium transformation efficiency by Agrobacterium tumefaciens was tested in order to complete the functional characterization of the genes. In a second part, we have analyzed the essence of 64 species and cutivars of Pelargonium having very different fragrances like lemon, mint, rose, apricot, pine, spices… With different statistical tools (PCA, discriminant analysis…), we have highlighted the links between the biochemistry of these species and cultivars, their odors and their phylogenetic relationships. This worked gave us some interesting ideas for some new crossings. Finally, the last chapter concerns the EO production improvements in Egypt. Thanks to these researches, started 3 years ago, we are improving year after year our EO yield and quality in our 10 Ha R&D plantation. An important work was done to optimize the distillation process and improve the agricultural practices which abled us to reach a yield of 60 kg of EO per hectare. Some other experiments show the effect of the environmental factors such as the temperature on the biosynthesis of several important molecules like citronellol and geraniol, 6,9-guaiadiene and 10-γ-epi-eudesmol

Background. Myocardial hypertrophy is a risk factor for cardiovascular morbidity and mortality. Independent of underlying disease, the cardiac muscle strives in different ways to compensate for an increased workload. This remodelling of the heart includes changes in the extracellular matrix which will affect systolic and diastolic cardiac function. Furthermore, signal transduction, molecular diffusion and microcirculation will be affected in the hypertrophic process. One important extracellular component is the glycosaminoglycan hyaluronan. It has been shown to play a major role in other conditions that feature cellular growth and proliferation, such as wound healing and malignancies. The aim of this thesis was to investigate hyaluronan and its role in both an experimental rat model of cardiac hypertrophy as well as in cultured mouse cardiomyocytes and fibroblasts. Methods. Cardiac hypertrophy was induced in rats by aortic ligation. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were quantified after 1, 6 and 42 days after operation, in cardiac tissue from the left ventricular wall. Localization of hyaluronan and its receptor CD44 was studied histochemically. Hyaluronan synthesis was correlated to gene transcription using microarray gene expression analysis. Cultures of cardiomyocytes and fibroblasts were stimulated with growth factors. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were detected. Hyaluronan size was measured and crosstalk between cardiomyocytes and fibroblasts was investigated. Results. Increased concentration of hyaluronan in hypertrophied cardiac tissue was observed together with an up-regulation of two hyaluronan synthase genes. Hyaluronan was detected in the myocardium and in the adventitia of cardiac arteries whereas CD44 staining was mainly found in and around the adventitia. Hyaluronan synthesis correlated to the expression of genes, regulated by transcription factors known to initiate cardiac hypertrophy. Stimulation of cardiomyocytes by PDGF-BB induced synthesis of hyaluronan. Cardiomyocytes also secreted a factor into culture media that after transfer to fibroblasts initiated an increased synthesis of hyaluronan. When stimulated with hyaluronan of different sizes, a change in cardiomyocyte gene expression was observed. Different growth factors induced production of different sizes of hyaluronan in fibroblasts. The main synthase detected was hyaluronan synthase-2. Cardiomyocytes were also shown to secrete microvesicles containing both DNA and RNA. Isolated microvesicles incubated with fibroblasts were observed by confocal microscopy to be internalized into fibroblasts. Altered gene expression was observed in microvesicle stimulated fibroblasts. Conclusion. This study shows that increased hyaluronan synthesis in cardiac tissue during hypertrophic development is a part of the extracellular matrix remodelling. Cell cultures revealed the ability of cardiomyocytes to both synthesize hyaluronan and to convey signals to fibroblasts, causing them to increase hyaluronan synthesis. Cardiomyocytes are likely to express receptors for hyaluronan, which mediate intracellular signalling causing the observed altered gene expression in cardiomyocytes stimulated with hyaluronan. This demonstrates the extensive involvement of hyaluronan in cardiac hypertrophy.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is believed to play a role in human lung cancer induced by tobacco smoking. NNK biotransformation may involve the enzymes prostaglandin H synthase (PHS)-1, PHS-2 and cytochrome 450 (CYP) 2F. PHS activity is thought to be important in extrahepatic tissues, where CYP activity is low. The CYP2F subfamily contains a single functional enzyme in humans (CYP2F1) and goats (CYP2F3); these enzymes are preferentially expressed in the lung, with little or no expression in other organs. The role of these enzymes in the pulmonary biotransformation of NNK was investigated. 4.2 µM [5-3H]NNK was incubated with human lung microsomes under NADPH-dependent and arachidonic acid-dependent conditions. Metabolites reflective of NNK α-carbon hydroxylation, N-oxidation and carbonyl reduction were detected in the presence of NADPH, and metabolite levels for all three biotransformation pathways were lower in the presence of arachidonic acid compared with NADPH (p<0.05, N=4). Incubation of microsomes with the PHS-1 selective inhibitor SC-560 and the PHS-2 selective inhibitor NS-398 did not change NNK biotransformation either in the presence of NADPH or in the presence of arachidonic acid (p>0.05, N=4). Incubation of [5-3H]NNK with ovine PHS-1 or PHS-2 did not result in formation of α-carbon hydroxylation or N­-oxidation metabolites; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was measurable only in the presence of PHS-2. Incubation of goat recombinant CYP2F3 with [5-3H]NNK resulted in formation of keto acid, keto alcohol and NNK-N-oxide (65.0%, 17.5% and 30.0% (µmol enzyme)-1 minute-1, respectively). Metabolite formation was inhibited by 3-methylindole (3-MI), a mechanism-based inactivator of CYP2F3. Based on an N value of 3, incubation of human lung microsomes with 3-MI inhibited N-oxidation (p<0.05) but did not alter NNK bioactivation or carbonyl reduction (p>0.05). However, when metabolite formation was examined in lung microsomes from different individuals, decreases in NNK biotransformation (ranging from 19.6 to 68.5%) were observed and were more pronounced in some patients than others, suggesting inter-individual variability in CYP2F1 activity. These studies demonstrate the ability of CYP2F to biotransform NNK and suggest inter-individual variability in the importance of CYP2F1 for this activity in human lung. They also strongly argue against the involvement of PHS enzymes. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2007-12-30 16:12:58.228

Les mécanismes à l'origine de la Bronchopneumopathie Chronique Obstructive (BPCO) et de ses complications cardiovasculaires restent partiellement connus. Le NO est produit par les NO synthases en quantité importante dans le poumon des sujets emphysémateux, mais son rôle dans la maladie n'est pas connu. Une dysfonction endothéliale précoce liée à une diminution de la disponibilité en NO au niveau vasculaire a aussi été observée chez les patients BPCO. Dans un premier travail, nous avons montré que iNOS et eNOS étaient induites de manière diffuse dans le poumon de souris développant un emphysème après une instillation d'élastase. Le recours à des souris iNOS-/- et eNOS -/-, ainsi qu'à un inhibiteur pharmacologique d'iNOS (1400W) ont permis de montrer que l'induction d'iNOS dans le poumon était responsable d'une accumulation de protéines nitratées dans les pneumocytes de type 2 et d'une diminution de l'oxydation protéique. Cependant ni iNOS ni eNOS n'étaient nécessaires au développement de l'emphysème induit par l'élastase. Dans un deuxième travail, nous avons exploré l'effet de la polyglobulie, une complication de la BPCO hypoxique, sur la fonction endothéliale chez 15 patients polyglobuliques et 13 normoglobuliques atteints de BPCO de sévérité égale. La polyglobulie était associée de base à une viscosité sanguine plus élevée et un diamètre artérielbrachial plus important mais avec des forces de cisaillement calculées similaires. L'étude de la vasodilatation en réponse à l'hyperhémie et celle du flux sanguin de l'avant bras mesuré par plethysmographie d'occlusion veineuse en réponse à une perfusion d'acétylcholine (ACh), et de N-monomethyl-L-arginine (L-NMMA) ont permis de montrer que les artères systémiques despatients polyglobuliques ajustent leur diamètre aux forces de cisaillement aigues et chroniques de manière adaptée grâce à une libération adaptée de NO. De plus, nos résultats suggèrent que la polyglobulie modérée n'a pas d'effet délétère sur la fonctionvasculaire chez les patients BPCO.

Secondary metabolites are compounds that are not necessary for the survival of the organism, but which are to be related to the organism's interaction with its environment. This work studied compounds of secondary metabolism through the study of chemical and biological extracts and fractions Simaba Maiana Casar. and the involvement of candidate genes (TPS10, TPS14, and CYP71B31 CYP76C3) in the metabolism of monoterpenes in Arabidopsis thaliana. The extracts from roots and stems of Simaba Maiana were tested in antioxidant activity, molluscicide, inhibition of lymphocyte proliferation, inhibition of NO production, anti-Leishmania amazonensis and anti-Trypanosoma cruzi. The four candidate genes (CYP76C3, CYP71B31, TPS10 and TPS14) were selected with the CYPedia, which calculates the coexpression between genes based on the Arabidopsis Affymetrix ATH1 microarray. The extracts and fractions Simaba Maiana showed a lower antioxidant activity by DPPH method, low concentrations of total phenols measured by Folin-Ciocalteu, however, a good antioxidant activity by TBARS method, using three agents of oxidative damage (AAPH, FeSO4 and H2O2). The extract showed cytotoxic activity and molluscicidal concentration of 100 mg/mL. The crude extract of the stem was not active for leishmanicidal and trypanocidal. This extract did not inhibit NO production, but showed a high percentage of inhibition of lymphoproliferation. The skimmianine furoquinoline alkaloid and pellopterin furanocoumarin were isolated from chloroform fraction. The candidate genes showed a similar pattern of expression of the stamen, more specifically the top of the filaments. Heterologous expression in transiently expressed in leaves of Nicotiana benthamiana, in the volatiles of TPS10 TPS14 (alone) were found enantiomers R-(-)-linalool and S-(+)-linalool. In the extraction buffer leaf discs were found that CYP76C3 converts linalool to E-8-hydroxy-linalool and E-8-oxo-linalool, and CYP71B31 in 1,2-epoxy-linalool. The analysis of the methanol extract of the discs incubated in S-(+)-linalool showed the use of this substrate by P450s converting to lilac alcohol for both P450s. Analysis of flowers of Arabidopsis mutants showed minor differences, analyzes with extracts of fresh flowers in UPLC-MS/MS MRM mode, has been found a compound having the same signature as linalool, however with different retention time and may be is an indication of linalool bound. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os metabólitos secundários são compostos que não são necessários para a sobrevivência do organismo, mas que apresentam-se relacionados com a interação do organismo com o seu ambiente. O presente trabalho estudou compostos do metabolismo secundário através do estudo químico e biológico dos extratos e frações Simaba maiana Casar. e a envolvimento dos genes candidatos (TPS10, TPS14, CYP76C3 e CYP71B31) no metabolismo de monoterpenóides em Arabidopsis thaliana. Os extratos das raízes e caule de Simaba maiana foram testadas em ensaios de atividade antioxidante, moluscicida, inibição da linfoproliferação, inibição da produção de NO, anti-Leishmania amazonensis e anti-Trypanosoma cruzi. Os quatro genes candidatos (CYP76C3, CYP71B31, TPS10 e TPS14) foram selecionados com o CYPedia, que calcula a co-expressão entre os genes de Arabidopsis com base no Affymetrix ATH1 microarray. Os extratos e frações de Simaba maiana mostraram uma baixa atividade antioxidante pelo método do DPPH, baixas concentrações de fenóis totais avaliados pelo método de Folin-Ciocalteu, entretanto, uma boa atividade antioxidante pelo método de TBARS, usando três agentes de danos oxidativos (AAPH, FeSO4 e H2O2). Os extratos mostraram atividade moluscicida e citotóxica na concentração de 100 mg/mL. O extrato bruto do caule não foi ativo para as atividades anti-Leishmania e anti-Trypanosoma. Este extrato não inibiu a produção de NO, mas apresentou uma alta porcentagem da inibição da linfoproliferação. O alcalóide furoquinolínico esquiamina e a furanocumarina felopterina foram isolados da fração clorofórmica. Os genes candidatos mostraram um similar padrão de expressão nos estames, mais especificamente na parte superior dos filamentos. Na expressão heteróloga transitoriamente expressa em folhas de Nicotiana benthamiana, nos voláteis de TPS10 e TPS14 (sozinho) foram encontrados os enantiômeros R-(-)-linalol e S-(+)-linalol. No tampão de extração de discos de folhas, verificou-se que CYP76C3 converte linalol em E-8-hidroxi-linalol e E-8-oxo-linalol, e CYP71B31 em 1,2-epoxilinalol. A análise do extrato metanólico dos discos foliares incubados em S-(+)-linalol mostrou a utilização deste substrato por P450s convertendo para lilac álcool para ambos os P450s. Análises de flores em plantas mutantes de Arabidopsis thaliana mostraram pequenas diferenças, em que análises com extratos de flores frescas em UPLC-MS/MS no modo MRM, foi encontrado um composto com a mesma assinatura que linalol, no entanto, com tempo de retenção diferente e pode ser uma indicação de forma ligada do linalol.

Although survival rates are increasing, brain injury continues to be a leading cause of death after cardiac arrest (CA). Permanent brain damage after CA is determined by limited tolerance to ischemia from CA and cardiopulmonary resuscitation (CPR), as well as the unique cerebral response to reperfusion after return of spontaneous circulation (ROSC). A major pathway leading to neurotoxic cascade and neuronal injury after CA involves the increased presence of reactive oxygen and nitrogen species generated during ischemia and reperfusion. The magnitude of cerebral oxidative injury induced by free radicals increased with the duration of CA (Paper I). Nitric oxide (NO), a free radical responsible for the formation of reactive nitrogen species, is increased during global ischemia from CA and reperfusion (Paper IV). Hypothetically, the administration of a drug that counteracts the overproduction of NO and also acts as a scavenger of oxygen free radicals might be warranted in order to reduce the damage caused by nitrosative and oxidative stress. For these purposes we used methylene blue (MB), an old dye that has been used in medicine for almost half a century, and an experimental pig model of 20 min of ventricular fibrillation (VF) to reflect a clinical scenario of ischemia/reperfusion injury. Administration of MB added to a hypertonic-hyperoncotic solution (MBHSD) that was started during CPR and continued for 50 min after ROSC increased short-term survival by decreasing myocardial damage, as well as cerebral peroxidation and inflammatory injury (Paper II). Immunostaining of cerebral tissue collected at different time points after CA and ROSC (Paper IV) provided experimental evidence that cortical blood-brain barrier (BBB) disruption begins as early as  during the initial phase of untreated as well as treated CA. The results indicated that MB administration reduced the neurologic injury and BBB disruption considerably, but did not reverse the ongoing detrimental processes. The demonstrated positive effects of MB were related to a decrease of nitrite/nitrate tissue content, and thus to a decrease of excess NO due to the MB inhibitory effects on NOS isoforms. A mixture of MB in hypertonic sodium lactate (MBL) was investigated to facilitate administration of MB in “the field.” Based on findings that MBL cardio- and neuroprotective properties were similar to those of MBHSD, there is reason to believe that the use of MBL might be extended during ongoing CPR and after ROSC (Paper III). It would therefore make sense to try using MB as a pharmacological neuroprotectant during or after clinical CPR in order to expand the temporal therapeutic window before other measures for neuroprotection such as hypothermia are available.

Ο καρκίνος του παχέος εντέρου αποτελεί αναμφισβήτητα ένα παγκόσμιο πρόβλημα, όντας η δεύτερη αιτία θανάτου από καρκίνο. Τα γεγονότα που οδηγούν σε καρκίνο του παχέος εντέρου ακολουθούν στις περισσότερες περιπτώσεις συγκεκριμένη αλληλουχία. Έτσι, ξεκινώντας από υπερπλαστικό επιθήλιο, εξελίσσεται σε αδένωμα/δυσπλασία, σε ενδοεπιθηλιακή νεοπλασία και καταλήγει σε καρκίνο. Κατά την εξέλιξη σε καρκίνο σημαντικές αλλαγές λαμβάνουν χώρα στη σύσταση και οργάνωση του εξωκυττάριου χώρου του εντερικού τοιχώματος. Για το λόγο αυτό απαιτείται μελέτη του μεταβολισμού των εξωκυττάριων μακρομοριακών συστατικών, με στόχο την κατανόηση και διασαφήνιση της διηθητικής και μεταστατικής συμπεριφοράς των κακοήθων νεοπλασιών του παχέος εντέρου. Στην παρούσα εργασία κρίθηκε χρήσιμη η μελέτη των ενζύμων μεταβολισμού του υαλουρονικού οξέος (ΗΑ), συστατικό του εξωκυττάριου χώρου που φαίνεται να εμπλέκεται στο μηχανισμό της ανάπτυξης του όγκου, της διείσδυσης των καρκινικών κυττάρων και στη διάδοση των μεταστάσεων. Για τον παραπάνω λόγο μελετήθηκε η δραστικότητα των υαλουρονιδασών (Hyals) με ζυμογράφημα-ΗΑ, σε εκχυλίσματα ιστών και σε ορούς, και των συνθασών του ΗΑ (HAS-1, HAS-2) καθώς και του υποδοχέα του ΗΑ, CD44, με RT-PCR ανάλυση. Τα αποτελέσματα που προκύπτουν από το ζυμογράφημα-ΗΑ σε εκχυλίσματα ιστών συναινούν σε αυξημένη δραστικότητα των Hyals σε καρκινικά σε σύγκριση με τα μακροσκοπικώς φυσιολογικά δείγματα στην πλειονότητα των σταδίων, ενώ και οι δύο δραστικότητες είναι σημαντικά αυξημένες σε σχέση με τα δείγματα υγιών ιστών. Όσον αφορά τα δείγματα ορών, τα επίπεδα των Hyals φαίνεται να μειώνονται αισθητά 7 ημέρες μετεγχειρητικά σε σχέση με την ημέρα πριν την εγχείρηση, ενώ 1, 3 και 6 μήνες μετά εμφανίζεται και πάλι σταδιακή αύξηση των επιπέδων τους. Σε γονιδιακό επίπεδο, τα επίπεδα της HAS-1, HAS-2 και του CD44 εμφανίζονται αυξημένα στα καρκινικά δείγματα έναντι των μακροσκοπικώς φυσιολογικών. / Colon cancer is indisputably a great problem, being the second cause of death by cancer. The facts that lead to colon cancer have certain concatenation. Thereby, starting most of the times as hyperplastic epithelium, it develops to endoepithelium adenoma, and it finally leads to cancer. During this development important changes happen at the constitution and organization of the extracellular matrix of the intestinal tract. For this purpose, study of the metabolism of the extracellular matrix constituents is required, so as to understand the metastatic behaviour of the malignant tumors of colon cancer. The present study focuses on the metabolism enzymes of hyaluronic acid (HA), a constituent of the extracellular matrix that seems to be involved in the tumor growth mechanism, the infiltration of the cancerous cells and metastasis. Particularly, hyaluronidases (Hyals) activity (HA-zymography) and hyaluronan synthases (HAS-1, HAS-2) as well as hyaluronan receptors (CD44) expression (RT-PCR analysis) are studied at this study. The results of the study show increased Hyals levels in cancerous samples compared to the macroscopically normal ones, in all anatomic sites of colon examined, while both activities remain significantly increased compared to healthy samples.. as far as it concerns Hyals levels in sera, they seem to decrease perceptibly 7 days postoperatively, while 1, 3 and 6 months afterwards gradually increased to reach the amount preoperatively. In gene level, HAS-1, HAS-2 and CD44 expression levels were increased in cancerous samples compared to the macroscopically normal ones.

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