The studies that have been published since 1990 which are dealing with the relationship of the genetic polymorphism of the dopamine D2 receptor gene (DRD2) in relation to alcoholism or other disorders or diseases have brought contradictory results. These results have led the author of this thesis to closely investigate the relationship between the genetic polymorphism of the A1 allele gene for DRD2 and hazardous alcohol consumption in the Czech Republic with a focus on the artistic area. A total of 29 participants (15 artists and 14 "non-artists") were involved in the study. The criterion for selecting the examined sample was the type of professional focus (artistic, non-artistic). A genetic analysis of blood (PCR-RFLP method) was used to determine the genotype. Only in the "non-artistic" group the presence of the genetic polymorphism of the A1 allele for DRD2 was found (a predisposition to a risk behavior in relation to alcohol). Additional parameters were found using EEG, an AUDIT test and a temperament questionnaire. A statistical analysis confirmed an association between the appearance of the A1 allele for DRD2 and a higher amplitude of the P300 component (p = 0.0000421). Statistically proven riskier alcohol consumption was found among introverted artists (n = 9) in the AUDIT test (p = 0.02298).

Dans le cerveau, les fonctions de la phosphatase alcaline non spécifique des tissus (TNAP) ne sont pas clairement identifiées. La localisation et l'expression de cette enzyme au niveau neuronal suggère cependant, qu'elle joue un rôle important dans le développement et le fonctionnement du cerveau. Cela est supporté par la présence de graves crises d'épilepsie chez les humains porteurs d'une mutation de la TNAP. Ces crises d'épilepsie sont létales chez les souris KO pour la TNAP. Des études chez la souris montrent que la TNAP pourrait réguler l'inhibition postsynaptique médiée par le GABA et elle pourrait être impliquée dans l'inhibition présynaptique médiée par l'adénosine. L'adénosine est, en partie, synthétisée via la déphosphorylation successive de l'ATP en ADP puis en AMP par des ectonucléotidases. Parmi elles, la TNAP et l'ecto- 5'-nucléotidase (NT5E) catalysent l'hydrolyse de l'AMP en adénosine dans le cortex cérébral. L'adénosine agit principalement au niveau présynaptique par l'intermédiaire des récepteurs A1. Ainsi l'adénosine a une influence sur la transmission synaptique et sur la plasticité synaptique. Ceci pourrait expliquer, en partie, les crises d'épilepsie observées chez les souris KO pour la TNAP. Les deux objectifs principaux de ma thèse ont été : (1) évaluer la contribution de la TNAP dans la production d'adénosine dans le cerveau ; (2) étudier l'influence de l'adénosine sur la plasticité synaptique. Premièrement, l'étude de la contribution de la TNAP dans la production d'adénosine dans le cerveau a été réalisée au moyen de deux approches complémentaires. Une approche métabolomique (spectroscopie RMN du proton) sur des cerveaux entiers de souris KO pour la TNAP a permis de montrer que la TNAP participe, entre autre, à la synthèse d'adénosine dans le cerveau. Une deuxième approche, électrophysiologique sur tranches de cerveaux de souris in vitro, nous permet d'examiner les conséquences de l'inhibition des ectonucléotidases intervenant dans la synthèse de l'adénosine. Elle a révélé que l'inhibition des ectonucléotidases (TNAP et NT5E) ne supprime pas l'effet inhibiteur de l'AMP médiée par les récepteurs A1. Deuxièmement, nous avons étudié l'influence de l'adénosine sur la plasticité synaptique à courte terme. Nous avons enregistré des potentiels de champs dans la couche Ia du cortex piriforme en réponse à des stimulations électriques (3,125 à 100 Hz) présentée avec des fréquences recouvrant la gamme d'oscillations physiologiques. Nos résultats montrent qu'avec de fortes concentrations d'adénosine, la facilitation est accentuée par rapport à celle observée en situation contrôle. Cet effet est observé pour des fréquences supérieures ou égales à 25 Hz. De plus, cette accentuation est d'autant plus grande que la fréquence est élevée (maximum atteint à 100 Hz pour 100 µM). En bloquant l'action de l'adénosine endogène, l'effet contraire est observé : une facilitation déficitaire par rapport au contrôle et dont le défaut est croissant avec la fréquence de stimulation. Tous ces résultats convergent vers l'hypothèse qu'une déficience en TNAP, traduite par une absence d'adénosine, pourrait contribuer au maintien des processus épileptiques générés par un déséquilibre de l'inhibition et de l'excitation dû à une diminution de GABA. L'effet inhibiteur de l'AMP médié par les récepteurs A1 ne serait pas suffisant pour contrecarrer les crises d'épilepsie observées chez les sujets hypophosphatasiques et les souris KO pour la TNAP. / The functions of Tissue Nonspecific Alkaline Phosphatase (TNAP) in the brain are not clearly identified. The localization and expression of TNAP at the neuronal level, however, suggests that it plays a prominent role in the development and the function in the brain. This is supported by the presence of severe epileptic seizures in humans carrying TNAP mutation. These epileptic seizures are lethal in TNAP KO mice. Studies in mice show that TNAP could regulate GABA-mediated postsynaptic inhibition and may be involved in presynaptic inhibition mediated by adenosine. Adenosine is, partly, synthesized via the successive dephosphorylation of ATP to ADP and then to AMP by ectonucleotidases. Among them TNAP and ecto-5'-nucleotidase (NT5E) are able to hydrolyze AMP into adenosine. Adenosine acts mainly at the presynaptic level via A1 receptors activation. Adenosine has an influence on synaptic transmission and thus on synaptic plasticity. This could partly explain the epileptic seizures observed in TNAP knock-out mice. The two main purposes of my thesis were: (1) to evaluate the contribution of TNAP in adenosine production in the brain; (2) to study the influence of adenosine on synaptic plasticity. Firstly, the study of the contribution of TNAP in adenosine production in the brain was carried out using two complementary approaches. A metabolomic approach (proton NMR spectroscopy) on whole brains of TNAP KO mice showed that TNAP in involved in adenosine synthesis in the brain. In a second approach, in vitro electrophysiological recordings on mouse brain slices allowed us to examine the consequences of the inhibition of the ectonucleotidases involved in adenosine synthesis. This revealed that inhibition of ectonucleotidases (TNAP and NT5E) did not suppress the inhibitory effect of AMP mediated by A1 receptors. Secondly, we studied the influence of adenosine on short-term synaptic plasticity. Field potentials were recorded in response to electrical stimulations (3.125 to 100 Hz) applied with frequencies encompassing the range of physiological oscillation. Our results show that, with high adenosine concentrations, the facilitation is emphasized compared to that observed in the control situation. This effect is observed for frequencies greater than or equal to 25 Hz. In addition, the higher the frequency, the greater the facilitation. Finally, by blocking the action of endogenous adenosine, the opposite effect was observed: a deficient facilitation with respect to the control, whose defect was increasing with stimulation frequency. All these results converge towards the hypothesis that TNAP deficiency, expressed by absence of adenosine, could contribute to the maintenance of the epileptic processes generated by an imbalance of the neuronal inhibition and the excitation due to a decrease of GABA. AMP inhibitory effect mediated by A1 receptors, would not be sufficient to counteract epileptic seizures observed in hypophosphatasic patients and TNAP KO mice.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-18T15:03:59Z No. of bitstreams: 1 DISS_2014_Caroline Marques Caloi.pdf: 1667299 bytes, checksum: 818ef12f2884a1595534e6fe5023eb67 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-22T11:54:23Z (GMT) No. of bitstreams: 1 DISS_2014_Caroline Marques Caloi.pdf: 1667299 bytes, checksum: 818ef12f2884a1595534e6fe5023eb67 (MD5) / Made available in DSpace on 2017-09-22T11:54:23Z (GMT). No. of bitstreams: 1 DISS_2014_Caroline Marques Caloi.pdf: 1667299 bytes, checksum: 818ef12f2884a1595534e6fe5023eb67 (MD5) Previous issue date: 2014-09-22 / A hanseníase é uma doença infecciosa crônica, causada pelo Mycobacterium leprae (M. leprae), parasita intracelular obrigatório que afeta, preferencialmente, macrófagos no tecido conectivo e células de Schwann nos nervos periféricos. O objetivo desse trabalho foi identificar as células mnonucleares presentes na pele de pacientes com hanseníase, com as formas clínicas tuberculóide-tuberculóide (TT), tuberculóide-borderline (TB), borderline-borderline (BB), virchowiano-borderline (BV) e virchowiano-virchowiano (VV); e mensurar a expressão dos mediadores anti-inflamatórios anexina-A1 (ANXA1) e interleucina-10 (IL-10), investigando seu possível papel na patogênese da doença. Após o diagnóstico clínico os pacientes foram submetidos à biópsia da lesão para análise histopatológica. Os cortes histopatológicos foram submetidos à técnica de imunofluorescência para detecção dos mediadores ANXA1 e IL-10. Os resultados demonstraram uma correlação na diferença dos tipos de leucócitos presentes nas diferentes formas clínicas dos pacientes com hanseníase com os níveis dos mediadores ANXA1 e IL-10 produção diferencial de mediadores reguladores do processo infeccioso. Os dados demonstraram que os histiócitos, células T CD4+ e T CD8+ dos pacientes com as formas clínicas BV e VV apresentavam uma expressão aumentada da proteína ANXA1, assim como nos níveis da citocina IL-10. Além disso, pode-se verificar uma correlação positiva na expressão da ANXA1 e nos níveis de IL-10 nos pacientes BB, BV e VV, indicando possivelmente que essa proteína possa contribuir com a regulação da produção dessa citocina durante o processo infeccioso induzido por M. leprae. Em conclusão, a proteína ANXA1 foi caracterizada como um possível regulador do processo infeccioso induzido por M. leprae, indicando que, a presença dessa proteína poderia estar reduzindo o potencial pró-inflamatório nos pacientes BB, BV e VV, levando a disseminação do M. leprae. Do contrário, em pacientes TT e BT, a proteína ANXA1 apresenta-se reduzida, o que poderia contribuir com a ativação dos leucócitos e a eliminação das bactérias. / Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae), a intracellular obligate parasite that affects mainly macrophages and Schwann cells. This study’s goal was to identify monocytic cells present in the skin of leprosy patients with tuberculoid- tuberculoid clinical forms (TT), tuberculoid-borderline (TB), borderline-borderline (BB), borderline-lepromatous (BV) and lepromatous (LL); and evaluating the expression of annexin-A1 (ANXA1), and anti-inflammatory mediators interleukin-10 (IL-10), evaluating its potential role in pathogenesis disease. After the clinical diagnosis, the patients underwent biopsy for histopathological analysis. Histopathological cuts were submitted to immunofluorescence staining for detection of mediators ANXA1 and IL-10. The results showed a difference in the of leukocytes’s profile in patients with different clinical forms of leprosy, that is essential to allow the differential production of regulatory mediators of the infectious process. The data showed that the histiocytes, CD4 + and CD8 + T cells of patients with clinical forms BV and VV had a higher expression of ANXA1 protein as well as high levels of IL-10. Furthermore, it can be seen a positive correlation in expression of ANXA1 and IL-10 in BB, BV and VV patients, possibly indicating that this protein may contribute to the regulation of this cytokine production during the infectious process induced by M. leprae. In conclusion, ANXA1 protein was characterized as a possible regulator of the infectious process induced by M. leprae, indicating that the presence of this protein could be reducing the pro-inflammatory potential in BB, BV and VV patients taking the spread of the bacterium M. leprae. Otherwise, patients in TT and BT, ANXA1 protein comes in lower levels, which could contribute to more effective activation of pro-inflammatory action of the immune system, favoring the elimination of bacterias.

Submitted by Caroline de Freitas Zanon (carolfzanon@yahoo.com.br) on 2018-07-19T18:02:39Z No. of bitstreams: 1 TESE VERSAO FINAL.pdf: 2398136 bytes, checksum: a2ce0aa2c7a816d48d27d6f13d6a0fec (MD5) / Rejected by Elza Mitiko Sato null (elzasato@ibilce.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: Problema 01) As páginas pré-textuais não estão na ordem correta, a ordem correta das páginas pré-textuais (capa, folha de rosto, ficha catalográfica, folha de aprovação, dedicatória, agradecimentos, epígrafe, resumo na língua vernácula, resumo em língua estrangeira, listas de ilustrações, de tabelas, de abreviaturas, de siglas e de símbolos e sumário). Problema 02) Nos agradecimentos consta também a FAPESP, então deve constar o nome dela também na folha de rosto e de aprovação com o número de processo e nos agradecimentos precisa constar o número do processo também, é norma do convênio, coloque também os números do processo da CNPQ. Problema 03) No repositório você colocou o nome de Caroline de Freitas Zanon e na tese está Caroline de Freitas Zanon de Carvalho, se esse é o seu nome atual por favor coloque também este nome no repositório. Sua submissão será rejeitada para que você possa fazer as correções Lembramos que o arquivo depositado no repositório deve ser igual ao impresso, o rigor com o padrão da Universidade se deve ao fato de que o seu trabalho passará a ser visível mundialmente. Agradecemos a compreensão. on 2018-07-19T19:04:58Z (GMT) / Submitted by Caroline de Freitas Zanon de Carvalho (carolfzanon@yahoo.com.br) on 2018-07-21T14:52:58Z No. of bitstreams: 1 TESE VERSAO FINAL.pdf: 2394414 bytes, checksum: 6f54895bda93635e996f95a66bf389be (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2018-07-23T13:58:02Z (GMT) No. of bitstreams: 1 carvalho_cfz_dr_sjrp.pdf: 2370535 bytes, checksum: d8d49b7c960d1ac32653f9d268eda5df (MD5) / Made available in DSpace on 2018-07-23T13:58:02Z (GMT). No. of bitstreams: 1 carvalho_cfz_dr_sjrp.pdf: 2370535 bytes, checksum: d8d49b7c960d1ac32653f9d268eda5df (MD5) Previous issue date: 2018-06-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os avanços recentes nos mecanismos de inflamação e a descoberta de vários mediadores endógenos anti-inflamatórios levaram a novas investigações sobre as possibilidades terapêuticas. A demonstração, em estudos científicos, da ação anti-inflamatória da proteína anexina A1 (ANXA1) e do seu peptídeo mimético Ac2-26, têm sido fonte de sucesso para o desenvolvimento de novos fármacos. A descoberta da interação biofísica da piplartina (PL) com a região N-terminal da ANXA1 abriu um novo e instigante campo de investigação para o nosso grupo de pesquisa. Diante destas considerações, o objetivo do presente trabalho foi investigar a atuação da PL nas células endoteliais da veia umbilical humana (HUVEC), ativadas pelo lipopolissacarídeo (LPS). Ainda, se a interação Ac2-26/PL influência nas ações anti-inflamatórias da PL, favorecendo ou atenuando os seus efeitos nos processos inflamatórios. Inicialmente foi utilizada a Espectroscopia de Absorbância UV-Vis e de Fluorescência para investigar as interações entre o ligante PL e Ac2-26. In vitro, as células HUVEC foram ativadas pelo LPS (10 μg/mL), nos tempos de 8, 24, 48 e 72 horas, seguido pelos tratamentos com Ac2-26 (1µM) e/ou PL (10µM), demonstrando resultados expressivos em 24 e 72 horas. Os efeitos foram avaliados nos seguintes aspectos: proliferação celular, viabilidade celular, dosagens da quimiocina MCP-1 e das citocinas IL-8 e IL-1β e expressão da proteína α-tubulina. A presença de um anel trimetoxiaromático na estrutura da PL, o que pode favorecer uma interação com a tubulina, motivou as análises de Western blotting, as quais demonstraram que a PL inibiu a síntese da proteína endógena α-tubulina, em todas as condições experimentais. Nossos resultados mostraram efeitos pró-proliferativos do peptídeo Ac2-26 e, por outro lado, antiproliferativos e pró-apoptóticos da PL. Os níveis das citocinas pró-inflamatórias confirmaram o papel anti-inflamatório do Ac2-26 nas células ativadas pelo LPS, com redução dos níveis de MCP-1 e IL-8, em 24 horas. O tratamento PL reduziu os níveis de MCP-1 nas células ativadas pelo LPS, e aumentou IL-8, inclusive no tratamento associado Ac2-26/PL. Em conjunto, os resultados com as células HUVEC indicam que a PL inibe a proliferação por meio da ativação da apoptose celular, regulada pela inibição da polimerização da α-tubulina e dos níveis elevados da citocina IL-8. A PL e o Ac2-26 mostraram ações anti-inflamatórias, e a interação Ac2-26/PL parece ser neutra em relação às propriedades anti-inflamatórias da PL. / Recent advances in inflammation mechanisms and the discovery of several endogenous anti-inflammatory mediators have led to further investigations into the therapeutic possibilities. The demonstration, in scientific studies, of the annexin A1 protein (ANXA1) anti-inflammatory action and its peptide mimetic Ac2-26, have been a source of success for the development of new drugs. The discovery of the biophysical interaction of piplartin (PL) with the N-terminal region of ANXA1 opened a new and exciting field of investigation for our research group. In view of these considerations, the objective of the present study was to investigate the role of PL in lipopolysaccharide (LPS) activated human umbilical vein endothelial cells (HUVEC). Also, if the interaction Ac2-26/PL influences the anti-inflammatory actions of PL, favoring or attenuating its effects on inflammatory processes. Initially, UV-Vis Absorbance Spectroscopy and Fluorescence were used to investigate the interactions between the PL ligand and Ac2-26. In vitro, HUVEC cells were activated by LPS (10 μg/mL) at 8, 24, 48 and 72 hours, followed by treatments with Ac2-26 (1μM) and/or PL (10μM), demonstrating expressive results in 24 and 72 hours. The effects were evaluated in the following aspects: cell proliferation, cell viability, MCP-1 chemokine and IL-8 and IL-1β cytokines and α-tubulin protein expression. The presence of a trimethoxyaromatic ring in the PL structure, which may favor an interaction with tubulin, motivated Western blotting analyzes, which demonstrated that PL inhibited endogenous α-tubulin protein synthesis in all experimental conditions. Our results showed pro-proliferative effects of the peptide Ac2-26 and, on the other hand, antiproliferative and pro-apoptotic of PL. Proinflammatory cytokine levels confirmed the anti-inflammatory role of Ac2-26 in LPS-activated cells, reducing MCP-1 and IL-8 levels within 24 hours. PL treatment reduced MCP-1 levels in LPSactivated cells, and increased IL-8, including in the treatment associated with Ac226/PL.Taken together, the results with HUVEC cells indicate that PL inhibits proliferation through the activation of cellular apoptosis, regulated by the inhibition of α-tubulin polymerization and elevated IL-8 cytokine levels. PL and Ac2-26 showed anti-inflammatory actions, and the Ac2-26/PL interaction appears to be neutral in relation to the anti-inflammatory properties of PL. / CNPq: 142274/2014 / CNPq UNIVERSAL: 474596/2013-3

Orientador: Flávia Cristina Rodrigues-Lisoni / Coorientador: Sonia Maria Oliani / Banca: Eny Maria Goloni Bertollo / Banca: Cristiane Damas Gil / Resumo: O carcinoma epidermoide de cabeça e pescoço (CECP) representa o sexto câncer mais comum em todo o mundo, sendo o câncer na cavidade oral o mais comum entre as regiões anatômicas de abrangência do CECP. Há evidências de que o desenvolvimento e a progressão do câncer dependem não somente de suas características genéticas, mas também de interações de suas células com as células do microambiente tumoral. A carcinogênese também está relacionada com angiogênese e processos inflamatórios, que podem ser controlados pela ação de mediadores anti-inflamatórios, e entre esses, destacamos a proteína anti-inflamatória anexina A1 (ANXA1). Mas os mecanismos pelos quais a ANXA1 atua na tumorigênese e a sua relação com a interação entre células endoteliais e tumorigênicas não são bem conhecidos e, considerando seu papel na inflamação, compreendem um alvo importante para estudo. Desse modo, o presente trabalho teve como objetivo, analisar a interação de células endoteliais e tumorigênicas juntamente com a ação do peptídeo sintético Ac2-26 da proteína ANXA1(ANXA1Ac2-26) em linhagem de carcinoma de cabeça e pescoço, verificando como o peptídeo atua junto ao meio condicionado de células endoteliais e/ou de células tumorais, elucidando como os fatores excretados por ambas as células e o peptídeo atuam no processo tumorigênico. Para isso, a observação da morfologia foi realizada em microscópio invertido, o índice de proliferação pela curva de crescimento, a viabilidade celular foi determinada pelo... / Abstract: Head and Neck Squamous Cells Carcinoma (HNSCC) is the sixth most common cancer in the world, being cancer in the oral cavity the most common among the anatomical regions of HNSCC. There is evidence that the development and progression of cancer depends not only on its genetic characteristics, but also because of the interactions of cells, cancer and tumor microenvironment cells. Carcinogenesis is also related to angiogenesis and inflammatory processes which can be controlled by the action of anti-inflammatory mediators, among which we highlight the anti-inflammatory protein annexin A1 (ANXA1). But the mechanisms that ANXA1 acts in tumorigenesis and its relation to the interaction between endothelial and tumorigenic cells are not well known and considering their role in inflammation, comprise an important target for study. Therefore, the present study aimed to analyze the interaction of endothelial and tumorigenic cells with the action of the synthetic peptide Ac2-26 of the ANXA1 protein (ANXA1Ac2-26) in head and neck carcinoma cell line, verifying how the peptide acts with the conditioned medium of endothelial cells and/or tumor cells, elucidating how the factors excreted by both cells and the peptide acts in the tumorigenic process. For this, the morphology observation was performed under inverted microscope, proliferation index by growth curve, cell viability was determined by the colorimetric method MTS (IC50), cell migration by transwell bilayer method, genotoxicity by ... / Mestre

No description available.

Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d’água» retrouvée dans l'État du Mato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse. L’objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d’extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine (Huh-7). Nous avons également évalué l’effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. L' activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses et sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. L'étude phytochimique de la fraction ‘acétate d'éthyle’ du latex de C. urucurana bioguidé a permis d’obtenir un nouveau cyclopeptide que nous avons nommé [1-9-NαC]-crourorb A1. Le crourorb présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) à un niveau équivalent à celle de la doxorubicine (témoin positif). Ce composé est de plus pratiquement inactif (GI50 = >250 μg/mL) sur la lignée non cancéreuse NIH/3T3. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et temps-dépendant. Nous avons mesuré une forte activation des caspases 3 et 7 et une nette augmentation de l’expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée par le crourorb A1. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l’induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. Le crourob A1 induit un arrêt du cycle cellulaire en phase G2/M. En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C. Nous avons également confirmé, l’effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D. / Croton urucurana Baillon (Euphorbiaceae), plant species popularly known in Brazil as “sangra-d’água”, is commonly found in Mato Grosso do Sul State (midwestern Brazil), where its latex is popularly used to treat several types of cancer. Despite the ethnopharmacological significance of the species, no phytochemical or cytotoxic studies of its latex have been published. In this context, the aim of the present study was to assess the cytotoxic activity of crude extracts, fractions and isolated compound from the latex of C. urucurana and to investigate the effects of crourorb A1 on the viability, apoptosis, cell cycle and cell migration of human hepatocarcinoma cell lines (Huh-7). Also, we have evaluated the viability of Huh-7 cells treated with crourorb A1 in 3D collagen gels. The cytotoxicity was detected using the sulforhodamine B (SRB) assay against six human cancer cells lines and one nontumor NIH/3T3 cell line (murine fibroblast). Studies on the mechanism of cell death of crourorb A1 were conducted in different test systems in vitro. The bioactive ethyl acetate phase obtained from the latex of C. urucurana afforded a novel cyclic peptide, [1−9-NαC]-crourorb A1, that proved active against NCIADR/ RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells (GI50 = > 250 μg/mL). Crourorb A1 treatment decreases the cell viability of Huh-7 at dose and time dependent manner associated with induction of apoptosis by caspase 3/7 activation and increasing expression of pro-apoptotic proteins: Bak, Bid, Bax, Puma, Bim, and Bad and clived-caspase3 protein. The JNK/MAP pathway is involved in this induced cell death. Crourorb A1 effect is also associated with G2/M phase cell cycle arrest. In parallel, we observed that crourorb A1 treatment resulted in an increase of cyclin-dependent kinase (CDK1), cyclin B1 and cyclin D1 expressions. A significant reduction of Huh-7 cells migration by crourorb A1 can be assessed and we also show that cells grown on 3D collagen gels exhibited increased resistance to crourorb A1 when compared to 2D cell culture. / Croton urucurana Baillon (Euphorbiaceae) é uma espécie arbórea conhecida popularmente no Brasil como “sangra d´água”, comumente encontrada no estado de Mato Grosso do Sul, onde seu látex tem sido utilizado na medicina popular no tratamento de vários tipos de câncer. Apesar de sua importância etnofarmacológica, não há relatos de estudos relacionados à sua composição química ou atividades anticâncer na literatura. Nesse contexto, o objetivo do presente estudo foi avaliar a atividade citotóxica de extratos, fases, frações e da substância isolada do látex de C. ururucurana e investigar os mecanismos celulares e moleculares in vitro responsáveis pela citotoxicidade da substância isolada considerando seu potencial apoptótico e influência sobre a progressão do ciclo celular em uma linhagem de hepatocarcinoma celular humano (Huh-7), bem como, os efeitos desta substância sobre a migração e viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D. O ensaio de citotoxicidade in vitro foi realizado com o corante sulforrodamina B (SRB) em seis linhagens de células neoplásicas humanas e uma linhagem de células não neoplásicas NIH/3T3 (fibloblasto murinho). Os estudos de mecanismos de ação com crourorb A1 foram conduzidos em diferentes sistemas-teste in vitro. O estudo fitoquímico biomonitorado da fase bioativa acetato de etila do látex de C. urucurana resultou no isolamento de um novo ciclopeptídeo denominado [19-NαC]-crourorb A1, que apresentou potente atividade citotóxica frente à linhagem de células neoplásicas NCI-ADR/RES (ovário com fenótipo de resistência a múltiplos fármacos), com a mesma potência que a doxorrubicina (controle positivo), e foi inativa até a maior concentração testada (GI50 = >250 μg/mL) frente à linhagem de células não neoplásicas 3T3. A exposição de células Huh-7 ao crourorb A1 resultou em uma diminuição da viabilidade celular de maneira dose e tempo-dependentes. Nós observamos um aumento da atividade de caspases 3 e 7 e um aumento da expressão de proteínas próapoptóticas (Bak, Bid, Bax, Puma, Bim e Bad) e caspase 3 clivada após tratamento com crourorb A1. Além disso, observamos que a via de sinalização celular MAP/JNK foi necessária para induzir a morte celular em células Huh-7 tratadas com crourorb A1. Os efeitos de crourorb A1 foram também associados com o acúmulo de células na fase G2/M do ciclo celular. Em paralelo, as expressões da cinase dependente da ciclina (CDK1), ciclina B1 e ciclina D1 foram aumentadas após tratamento com crourorb A1. Foi demonstrado também o envolvimento de crourorb A1 na redução da migração de células Huh-7 cultivadas na presença e ausência de mitomicina C. A avaliação da viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D apontou uma maior resistência destas células ao crourorb A1 quando comparadas com a cultura de células em 2D.

Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required. Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment. Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results - Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring. Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5- methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with Ki values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively. A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > pfluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin- 4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor. The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log Ki (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 μM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities. Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy. Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Trypanosoma cruzi é o agente causador da Doença de Chagas, que segundo a OMS, atinge oito milhões de pessoas principalmente na América Latina, causando danos à saúde pública, juntamente com um impacto econômico negativo. Durante o processo de infecção, uma variedade de eventos celulares ocorre apenas pelo simples contato do parasito com a célula hospedeira, levando a modificações no metabolismo celular e alterações morfológicas. O parasita é capaz de modular respostas celulares e imunológicas da célula hospedeira para sua própria sobrevivência. Além do que, pode alterar compartimentos celulares como o número e tamanho de nucléolos, sugerindo que a presença do parasita poderia estar interferindo na maquinaria nuclear. Porém, pouco se conhece sobre a organização nuclear da célula hospedeira quando infectada por Trypanosoma cruzi. O objetivo deste estudo foi de investigar pela primeira vez os compartimentos nucleares das células hospedeiras durante o curso da infecção por T. cruzi. Células LLC-MK2 foram infectadas com T. cruzi e reações de imunofluorescência indireta foram realizadas utilizando anticorpos e marcadores específicos para proteínas nucleares. As análises das imagens de microscopia confocal e quantificação das fluorescências pelo ImageJ mostraram padrões distintos nos compartimentos nucleares quando comparadas com células não infectadas. Corpos de Cajal e Speckles sofrem alterações quando a célula está infectada e isso depende do ciclo celular do parasita. Neste trabalho também foi investigado através de quantificação de imagem e immunoblotting o comportamento das Ribonucleoproteínas A1 e A2B1 durante a parasitemia. Estas análises demonstram que o T. cruzi pode modular a célula hospedeira quando infectada a favor de sua sobrevivência, promovendo alterações na dinâmica dos compartimentos nucleares durante o seu ciclo celular. Esse estudo inédito poderá auxiliar a compreender a biologia do parasita e sua interação com a célula hospedeira e desta maneira contribuir na busca de possíveis alvos terapêuticos / Trypanosoma cruzi is the causal agente of Chagas disease, that affects about eight million people mostly in Latin America according to the WHO, causing damage to public health and a negative economic impact. During infection, a variety of signaling processes occur after contact of the parasite to the host cell, what can lead to metabolic modifications as well morphological alterations in both cells. The parasite can modulate host cell cellular and immunological responses for its own survival. In addiction, the presence of T. cruzi can modify the nuclear compartments such as nucleoli, suggesting that the presence of the parasite could be interfering with the nuclear machinery. However, little is know about the nuclear organization when the host cell is infected with Trypanosoma cruzi. This study aimed to investigate for the first time the nuclear compartment of host cells infected by T. cruzi using specific antibodies and fluorescent markers for nuclear compartments, in order to investigate the morphological and functional changes in the nucleus of the host cell. Using LLC-MK2 cells infected with T. cruzi, we performed indirect immunofluorescence using distinct nuclear antibodies. Confocal microscopy analysis of infected cells showed pattern variations in the nuclear compartments when compared to uninfected cells. Cajal bodies and Speckles suffer alterations when the cell is infected and it is related to the parasite life cycle. In this work we also investigated by image quantification and immunoblotting the behavior of Ribonucleoproteins A1 and A2B1 during infection. These evidences support the idea that T. cruzi can modulate host cell response to ensure its own survival during the infection, promoting changes in the dynamics of the nuclear compartments. This unpublished data may help to understand the biology of the parasite and its interaction with the host cell and thus contributing to seek for potential therapeutic targets

A inflamação é fundamental para a manutenção da homeostasia e para a resposta do organismo à injúria. A resposta inflamatória deve ser adequada aos estímulos agressores; no sistema nervoso central, sua inadequação conduz à gênese de diferentes doenças neurodegenerativas. A proteína anexina A1 (ANXA1) e os receptores ativados por proliferadores de peroxissomo (PPAR) controlam a inflamação, pois ambos inibem o desenvolvimento da inflamação e aceleram sua resolução. Nosso grupo de pesquisa tem mostrado que a ANXA1 modula a expressão de PPARγ em macrófagos. Assim, o presente trabalho investigou a modulação da expressão do PPARγ e das suas funções em células da microglia pela ANXA1. Foram empregadas células imortalizadas da linhagem BV2 (microglia murina), inalteradas ou transfectadas para redução da expressão de ANXA1, tratadas com ANXA1 exógena (recombinante - rANXA1) ou com agonista ou antagonista de PPARγ (pioglitazona e GW9662, respectivamente). Os resultados obtidos mostraram que: 1) tratamento com rANXA1 aumenta as expressões gênica (RT-PCR) e proteica (Western Blotting) de PPARγ, e ambas as expressões estão reduzidas em células com deficiência endógena de ANXA1, sendo que tal efeito foi revertido pela ação da rANXA1; 2) tratamento com rANXA1 não induz a expressão dos fatores de transcrição ligados a expressão de PPARγ: proteínas ligantes de elementos de resposta ao cAMP - CREB - e transdutores de sinais e ativadores de transcrição - STAT6 - (Western Blotting), mas os níveis de ambos os fatores estão reduzidos em células transfectadas, e tal efeito não foi revertido pelo tratamento com rANXA1; 3) tratamento com pioglitazona ou com rANXA1 individualmente aumenta a fagocitose de células PC12 apoptóticas (citometria de fluxo), mas o tratamento simultâneo não altera a fagocitose induzida por pioglitazona ou rANXA1; no entanto, tratamento com GW9662 inibiu a fagocitose induzida pelo tratamento com rANXA1; 4) o tratamento com rANXA1 aumenta a expressão de CD36 (citometria de fluxo); a expressão de CD36 está reduzida em células transfectadas e tal expressão não é revertida pelo tratamento com rANXA1. Em conjunto, os dados obtidos mostram a modulação da ANXA1 sobre PPARγ em células da micróglia, com possível ação sobre a fagocitose de células apoptóticas, e que a redução da expressão de ANXA1 reduz acentuadamente a expressão dos fatores de transcrição STAT6 e CREB, bem como a expressão de CD36. A elucidação dos efeitos resultantes destas alterações desencadeadas pela deficiência de ANXA1 endógena poderá contribuir para compreensão da fisiopatologia da neuroinflamação. / Inflammation is a key process in maintaining homeostasis and is essential for the body\'s response to injury. The inflammatory response must be proportional to the aggressor stimuli; in the central nervous system, a failed proper modulation leads to the development of different neurodegenerative diseases. Protein annexin A1 (ANXA1) and peroxisome proliferated-activated receptors (PPAR) control inflammation, as both inhibit development of inflammation and accelerate its resolution. Our research group has demonstrated that ANXA1 modulates PPARγ expression in macrophages. Thus, the present work investigated the modulation of PPARγ expression and its functions in microglia cells by ANXA1. In order to assess such, immortalized cells from cell line BV2 (murine microglia), either unadulterated or transfected for reduced expression of ANXA1, were treated with exogenous ANXA1 (recombinant protein - rANXA1) or either with PPARγ agonist or antagonist (pioglitazone and GW9662, respectively). The obtained results demonstrated that: 1) treatment with rANXA1 increases both gene (RT-PCR) and protein (Western Blotting) expressions of PPARγ, and also that both expressions are reduced in cells with endogenous deficiency of ANXA1, and such effect was reversed by the actions of rANXA1; 2) treatment with rANXA1 does not promote the expression of transcription factors associated with PPARγ expression: cAMP response element binding protein - CREB - and signal transductor and activator of transcription 6 - STAT6 (Western Blotting), but the expression levels of both factors are reduced in transfected cells, and such effect was not reversed by treatment with rANXA1; 3) individual treatment with pioglitazone or rANXA1 increases phagocytosis of apoptotic PC12 cells (flow cytometry), but simultaneous treatment does not affect pioglitazone/rANXA1-induced phagocytosis; however, treatment with GW9662 inhibited rANXA1-induced phagocytosis; 4) treatment with rANXA1 increases CD36 expression (flow cytometry); the expression of CD36 is reduced in transfected cells, and such expression is not reversed by treatment with rANXA1. The obtained data demonstrate the modulation ANXA1 exerts upon PPARγ in microglia cells, with a possible action upon phagocytosis of apoptotic cells, and that reduction of ANXA1 expression greatly reduces the expression of transcription factors STAT6 and CREB, as well as the expression of CD36. Elucidation of such effects that arise from a deficiency of endogenous ANXA1 will contribute to a better comprehension of the pathophysiology of neuroinflammation.