Introduction: The excitatory neurotransmitter glutamate is widely distributed throughout mammals' nervous system. Glutamatergic pathways have been associated with different kinds of pain. Analgesia induced by exercise is a phenomenon observed in preclinical and clinical studies. However, the analgesia associated with this neurobiological mechanism has not been fully elucidated. Goal: To evaluate the effect of high-intensity swimming in the somatic hyperalgesia and nociception induced by glutamate analyzing the role of different receptors and pathways involved in it. Methodology: This study was approved by the Ethics Committee on Animal Use (CEUA /Unisul in the Portuguese acronym) under the number 13.020.4.06.IV. Swiss male mice were submited to one or two weeks of high-intensity swimming and compared to mice that did not exercise. The somatic nociception and hyperalgesia was induced by intraplantar injection (i.pl.) of glutamate (GLU). The paw response (licking time or removal frequency) against noxious stimuli was tested. Because paw (peripheral) and spinal cord (central) modulation are important sites for pain control, the involment of receptors of the main endogenous systems (opioid adenosinergic and cannabinoid) and the expression of protein kinase A (PKA) phosphorylated were evaluated by, respectively, specific antagonists of these systems and by Western blotting. Results: It was concluded that central and peripheral opioid and adenosinergic (A1) receptors are part of the spinal analgesia induced by high intensity swimming. In addition, spinal but not peripheral cannabinoid receptors CB1 are involved in this effect. An increased expression of phosphorylated PKA was induced by i.pl. injection of GLU, that was significantly reduced by swimming, was also found on both sites (paw and spinal cord). Coclusion: High intensity swimming reduces somatic nociception and hyperalgesia induced by glutamate. Inhibition of PKA phosphorylation by the activation of opioid, cannabinoid and adenosinergic receptors seem at least partially, to mediate this effect. / Submitted by Rogele Pinheiro (rogele.pinheiro@unisul.br) on 2017-10-23T17:17:35Z No. of bitstreams: 1 111846_Aline.pdf: 1636563 bytes, checksum: e2c62638d6b11fb08a152914d2c00176 (MD5) / Approved for entry into archive by Caroline Correa da Cruz (caroline.cruz@unisul.br) on 2017-10-23T20:16:12Z (GMT) No. of bitstreams: 1 111846_Aline.pdf: 1636563 bytes, checksum: e2c62638d6b11fb08a152914d2c00176 (MD5) / Made available in DSpace on 2017-10-23T20:16:12Z (GMT). No. of bitstreams: 1 111846_Aline.pdf: 1636563 bytes, checksum: e2c62638d6b11fb08a152914d2c00176 (MD5) Previous issue date: 2015-08 / Introdução: O neurotransmissor excitatório glutamato é amplamente distribuído pelo sistema nervoso de mamíferos. Vias glutamatérgicas tem sido associadas a diferentes tipos de dor. Analgesia induzida pelo exercício físico é um fenômeno observado em estudos pré-clínicos e clínicos. No entanto, o mecanismo neurobiológico adjacente a esta analgesia ainda não foi totalmente elucidado. Objetivo: Avaliar o efeito da natação de alta intensidade na nocicepção somática e hiperalgesia induzida pelo glutamato, analisando o papel de diferentes receptores e vias envolvidas neste efeito. Métodos: O presente estudo foi aprovado pela Comissão de Ética no Uso de Animais (CEUA/Unisul) sob o número 13.020.4.06.IV. Foram utilizados camundongos Swiss machos. Animais foram submetidos à uma ou duas semanas de natação de alta intensidade e comparados à camundongos não exercitados. A nocicepção e hiperalgesia mecânica somática foram induzida por uma injeção intraplantar (i.pl.) de glutamato (GLU). Testou-se a resposta da pata (tempo de licking) ou (frequência de retirada) frente a estímulos nocivos. Pelo fato da pata (periférico) e da medula espinal (central) serem sítios de modulação importantes no controle da dor, a participação dos receptores dos principais sistemas endógenos (opióde, adenosinérgico e canabinóide) e a expressão da proteína cinase A (PKA) fosforilada foram avaliados nestes locais, por meio de antagonistas específicos dos referidos sistemas e por Western Blotting, respectivamente. Resultados: Observou-se que receptores opióides e adenosinérgicos (A1) periféricos e espinais participam da analgesia induzida pela natação de alta intensidade. Além disso, receptores canabinóides CB1 espinais, mas não periféricos, também estão envolvidos neste efeito. Paralelo a estes resultados, também foi encontrado aumento na expressão de PKA fosforilada induzido pela injeção i.pl. de GLU, o qual foi reduzido significativamente pela natação, em ambos sítios, pata e medula espinal. Conclusão: Natação de alta intensidade reduz nocicepção somática e hiperalgesia induzida pelo glutamato. A inibição da fosforilação de PKA pela ativação de receptores opióides, adenosinérgicos e canabinóides parece, pelo menos em parte, mediar este efeito.

Submitted by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-04-15T14:35:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Giovanna Christinne Rocha.pdf: 5106952 bytes, checksum: fe0c347e66ce2a186ae1e34561ca40cb (MD5) / Made available in DSpace on 2015-04-15T14:35:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Giovanna Christinne Rocha.pdf: 5106952 bytes, checksum: fe0c347e66ce2a186ae1e34561ca40cb (MD5) Previous issue date: 2013-07-25 / FACEPE; Emerging Leaders in the Americas Program (ELAP) (Governo do Canadá) / A nova entidade química LPSF/FZ4 (4-cloro-benzil)-5-(4-nitro-benzilideno)-imidazolidina-2,4-diona) foi sintetizada pela Universidade Federal de Pernambuco (Brasil, Recife-PE) como uma alternativa para o tratamento da esquistossomose, uma doença negligenciada. Por se tratar de um protótipo, várias propriedades físico-químicas são praticamente desconhecidas, como, por exemplo, o seu pKa. O conhecimento desta propriedade é de fundamental importância para o desenvolvimento de medicamentos, uma vez que esta pode afetar a solubilidade e a permeabilidade do protótipo, aspectos importantes para a sua absorção, distribuição, metabolismo e excreção no organismo. Além disso, o LPSF/FZ4 possui limitada solubilidade em água (S<1μg/mL), sendo bem reconhecido que a baixa solubilidade de um fármaco muitas vezes limita sua taxa de dissolução e sua biodisponibilidade. Uma alternativa para esse entrave é a utilização das dispersões sólidas (DS). A fim de conhecer melhor o protótipo, o presente estudo objetivou a determinação do pKa do LPSF/FZ4 e, visando melhorar sua dissolução, investigou a técnica de DS. Utilizou-se o método espectrofotométrico com auxílio do equipamento SiriusT3 para determinação do pKa. Uma análise teórica da estrutura da molécula também foi realizada. Dessa forma, descobriu-se que o LPSF/FZ4 é uma base fraca monoprótica (BH+) com um grupo carbonila na posição 4 do anel imidazolidínico de pKa 8,47. Com um estudo de dissolução in vitro foi verificado que, com valores de pH superiores a 8,47, o LPSF/FZ4 apresenta-se predominantemente na sua forma neutra (B). À medida que o pH diminui, a base começa a se protonar (BH+), aumentando acentuadamente sua solubilidade. Para o desenvolvimento de DS com o protótipo, estudos teórico e prático preliminares permitiram selecionar o HPMC K4M, o PVP K-90, o PVPVA 64 e o SOLUPLUS. As DS com esses carreadores foram preparadas pelo método do solvente. A análise de Difração de raios-X (DRX) mostrou a concentração máxima de LPSF/FZ4 capaz de se manter amorfa em cada carreador, logo, indicou qual seria o último sistema de DS obtido com cada carreador. Através dos estudos de dissolução in vitro em condição non-sink utilizando o equipamento SiriusT3, e de estabilidade acelerada, pôde-se selecionar as dispersões sólidas mais promissoras para compor um futuro medicamento com o LPSF/FZ4. Percebeu-se que o sistema DS SOLUPLUS - LPSF/FZ4 10% pode aumentar em até 3000 vezes o desempenho do protótipo no estudo de dissolução, além de se mostrar estável. Por esse motivo, esse sistema revelou-se o mais promissor. O sistema DS PVP K-90 – LPSF/FZ4 10% obteve o segundo melhor resultado no estudo de dissolução in vitro e, assim, também foi selecionado. Prosseguiu-se com a caracterização física e química das DS com PVP K-90 e SOLUPLUS através das técnicas de Calorimetria Exploratória Diferencial, Microscopia de Polarização de Estágio à Quente, Microscopia de Polarização e Espectrofotometria de Absorção na Região do Infravermelho, permitindo verificar, por exemplo, que as DS com o SOLUPLUS formaram ligações de hidrogênio mais fortes com o protótipo do que o as com PVP K-90. Dessa forma, o presente estudo foi de grande importância, pois forneceu informações relevantes para a concepção racional de medicamentos com a nova entidade química, e obteve uma formulação adequada para superar as limitações de solubilidade do LPSF/FZ4.

Hyperactivation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) by dopamine D₁ receptor (D₁R) in the striatum is a characteristic feature of several neuropsychiatric conditions, including drug addiction and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) within individuals suffering from Parkinson’s disease. However, the current mechanistic gap in understanding of D₁R-mediated regulation of ERK1/2, both in physiology and disease, hampers effective treatment of these conditions. One important factor that is underexplored in this regard is the role played by proteins that physically interact with the intracellular regions of D₁R. Using yeast two-hybrid screens and co-transfected human embryonic kidney 293 (HEK293) cells, our laboratory has recently characterized an interaction between the third intracellular loop of D₁R and synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase family. Moreover, our lab identified endogenous D₁R-SAP102 complex within rat striatum and hippocampus. Interestingly, SAP102 regulates ERK signaling pathway within the hippocampus, and modulates adenosine A2A receptor-mediated ERK1/2 activation within transfected HEK293 cells. Capitalizing on the above findings, I hypothesized a role for SAP102 in controlling D₁R-mediated ERK1/2 activation. Herein, I demonstrate using co-transfected HEK293 cells that SAP102 alters the temporal activation of ERK1/2 by D₁R. Intriguingly, experiments using the protein kinase A (PKA) inhibitors H89 and protein kinase inhibitor 14-22 amide myristoylated show that SAP102 also facilitates a switch in D₁R-mediated ERK1/2 activation from a PKA-independent to PKA-dependent pathway. Furthermore, SAP102 reduces basal ERK1/2 activation in HEK293 cells, reminiscent of a previously documented role of hippocampal SAP102. To the best of my knowledge, my findings are the first to demonstrate scaffolding protein-mediated switching of a G protein-coupled receptor (GPCR) signaling pathway. Future studies aimed at uncovering the details of this process should provide valuable insight on the mechanisms contributing to D₁R-mediated ERK1/2 activation, and may also offer clues to the existence of similar phenomena for other GPCRs. These studies would also aid development of improved pharmacological treatment options for conditions with dysfunctional D₁R-dependent ERK1/2 activation.

Indiana University-Purdue University Indianapolis (IUPUI) / A histological marker of Parkinson’s disease (PD) is the loss of synapses located on striatal medium spiny neurons (MSNs) as a result of dopaminergic nigral cell depletion. The dendritic spines that give MSNs their name have a well-characterized structure and are the main regions of post-synaptic input. It has been shown that spines have altered functionality and morphology in many neurodegenerative diseases. Spine morphology, and potentially function, is dictated by an array of structural proteins and their associations with other proteins in a region dubbed the post-synaptic density (PSD). Spinophilin and neurofilament medium (NF-M) are two proteins that are enriched in the PSD and have potential implications in PD. Interestingly, preliminary data show that there is a decrease in the NF-M-spinophilin interaction in animal models of PD. Here it is shown that these two proteins interact in brain tissue and when overexpressed in a mammalian cell system. Moreover, we have begun to determine mechanisms that regulate this interaction. It is known that there is a misregulation of protein phosphatases and kinases in many neurodegenerative diseases. Moreover, the phosphorylation state of a protein can regulate its association with other proteins. Therefore, we hypothesize that the phosphorylation state of either protein affects the interaction between spinophilin and NF-M. Furthermore, we have conducted experiments utilizing protein phosphatases and kinases that are known to modulate the phosphorylation state of NF-M and/or spinophilin. Data show that both kinase and phosphatase activity and/or expression modulates the NF-M-spinophilin interaction in heterologous cell lines. Through the use of MS/MS analysis, we have begun to map specific phosphorylation sites that may play a role in regulating this interaction. Currently, we are elucidating the specific effects of these post-translational modifications on regulating the spinophilin-NF-M interaction. These data will enhance our knowledge of spinophilin’s interactions and how these interactions are altered in neurological disorders such as PD.

Thiols are one of the most important classes of chemicals used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. This study is an internally-referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and di-thiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature relative to an internal reference peak as a function of solution pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curveitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, first and second thiol pKa values for 1,2-benzenedithol in water were determined for the first time. This method is convenient to implement and its underlying theory is easy to follow.

No / Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K+ current (Ito) and inward rectifying K+ current (IK1) in rat cardiac muscle. Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 ¿mol l¿1. A similar degree of inhibition was observed when Ito and IK1 were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. The IC50 was 35.8 ¿mol l¿1 for Ito and 27.8 ¿mol l¿1 for IK1 compared to 5.4 ¿mol l¿1 for L-type Ca2+ current (ICa). The Hill coefficients for Ito, IK1 and ICa were ¿1.97, ¿1.60 and ¿1.21, respectively. In addition to inhibiting Ito amplitude, H-89 also accelerated the time to peak and the rate of voltage-dependent inactivation so that the time course of Ito was abbreviated. Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentration-dependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 ¿mol l¿1) used. In experiments investigating the effects of elevated cyclic AMP, the ß-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on Ito or IK1. Data suggest that the effects of H-89 on K+ currents are more complex than simple inhibition of PKA-mediated phosphorylation.

No description available.

Les parasites Theileria (Theileria. annulata and T. parva) sont des protozoaires intracellulaires qui font partie du phylum des Apicomplexa. Theileria infecte les leucocytes bovins et les transforment en cellules cancéreuses, induisant un genre de leucémie chez le bovin et conduisant à la mort de l’animal. Les cellules infectées par Theileria démontrent certaines caractéristiques de cellules cancéreuses telles qu’une importante capacité d’invasion et de migration cellulaire. Cependant, le traitement de cellules infectées avec une drogue Theiléricide spécifique (buparvaquone) permet l'élimination du parasite et la réversion du phénotype transformé. De plus, la virulence peut être atténuée par passages répétés sur culture cellulaire. La similitude entre les cellules transformées par Theileria et la leucémie humaine fait de Theileria un modèle très important permettant l’étude des mécanismes cellulaires induits par le parasite au cours de la transformation de la cellule hôte. Mon laboratoire d’accueil a publié une augmentation significative de TGF-β2 dans les cellules virulentes et a constaté que parmi les 1158 cibles de TGF-β, 68 gènes ont été reconnus d'avoir modifié leurs niveaux de transcription concomitante avec l'atténuation. Dans ce travail de thèse, nous avons étudié les voies de signalisations impliquées dans la régulation de l’adhésion et l’invasion des cellules infectées par Theileria. Nous nous sommes particulièrement intéressés à l’étude de la voie de signalisation TGF-β2 et ses effecteurs. Nos résultats montrent que l’activation de la voie de signalisation de TGF-β2 par Theileria entraîne une augmentation de l’invasion et de l’adhérence des cellules transformées par deux mécanismes différents, soit en activant la voie de signalisation PGE2/EP4/cAMP/PKA/EPAC/CREB, soit en stimulant la voie GRB2/PI3-K/AP-1. Les macrophages atténués infectés par Theileria sont plus stressés oxydativement ce qui diminue leur adhérence et leur invasion cellulaire. Ceci nous a amené à étudier en collaboration avec un autre doctorant (Mehdi Metheni) le rôle de TGF-β2 dans la régulation du stress oxydatif dans les macrophages infectés par Theileria. Nos données montrent que les niveaux élevés de TGF-β2 stimule l’expression de la catalase, une enzyme anti-oxydante qui convertit le H2O2 en H2O et la baisse de H2O2 favorise la virulence en augmentant l’invasion et l’adhésion des cellules infectées par Theileria (résultats supplémentaires). De plus, nous avons examiné le statut de stress oxydatif et le type de glycolyse utilisé par les cellules infectées par Theileria. Les cellules transformées par Theileria agissent comme des cellules cancéreuses, elles consomment énormément de glucose. La protéine BAD joue un rôle important dans l’apoptose ainsi que dans la voie de glycolyse. Son activité est régulée par phosphorylation en réponse à des facteurs de croissance et de survie. BAD peut être phosphorylée par la PKA sur le résidu sérine 155. Durant ma thèse, nous avons examiné le rôle de la phosphorylation de BAD par la PKA dans la régulation du métabolisme cellulaire des macrophages infectés par Theileria. Nos résultats montrent que l’abolition de la phosphorylation de BAD par la PKA dissocie le complexe mitochondrial formé entre BAD et HK2, ce qui induit l’ubiquitynation et la dégradation de HK2 par le protéasome. La baisse de HK2 stimule la voie de phosphorylation oxydative en faveur de l’effet Warburg dans les cellules infectées par Theileria. / Theileria parasites (Theileria. annulata and T. parva) are intracellular protozoa and members of the phylum Apicomplexa. Theileria parasites are the only eukaryotes that possess the property of being able to transform another eukaryote, their leukocyte host cells. Transformed leukocytes show many characteristics of tumour cells such as heightened invasive capacity; however the tumour-like phenotype can be totally reversed upon drug induced parasite death and attenuated by multiple in vitro passages. Such multiple-passaged attenuated lines are used as live vaccines against tropical theileriosis. The similarities in tumour hyper-invasiveness between Theileria-transformed leukcocytes and human lymphomas imply that observations on Theileria-induced leukocyte transformation have the potential to give generally applicable insights into the mechanisms underpinning tumour virulence. My host laboratory described higher TGF-β2 levels in virulent infected macrophages and following microarray analysis of virulent compared to attenuated macrophages found that among the 1158 TGF-β-targets, 68 genes had altered transcript levels concomitant with attenuation. In this study, we investigate the signalling pathways involved in the regulation of cellular adhesion and invasiveness of Theileria-infected cells. We were especially interested in the study of TGF-β2 signalling in Theileria-transformed virulent versus attenuated macrophages. My results indicate that following Theileria infection of macrophages, the TGF-β2 signalling pathway is activated and induces an increase in adhesion of virulent transformed macrophages through two different mechanisms: either by activating a PGE2 / EP4 / cAMP / PKA / EPAC / CREB signaling pathway, or by stimulating a GRB2 / PI3-K / AP-1 pathway. As attenuated macrophages display heightened oxidative stress, which underpins their loss of adhesion and invasiveness, in collaboration with another PhD student (Mehdi Metheni) we investigated the role of TGF-β2 in the regulation of the oxidative stress status of Theileria-infected macrophages. Our data show that high levels of TGF-β2 increase the expression of catalase, an anti-oxidant enzyme that converts H2O2 into H2O and the drop in H2O2 output results in regain of the virulence trait heightened adhesion of Theileria-transformed macrophages to fibronectin. Theileria-transformed macrophages display many features of cancer cells such as their consumption of larger quantities of glucose. The BCL-2 family protein BAD has an alternative function in glucose metabolism separate from its role in apoptosis. The activity of BAD is regulated by phosphorylation in response to growth/survival factors. BAD can be phosphorylated on Ser155 by PKA. So during my thesis studies I examined the role of PKA mediated phosphorylation of BAD in the regulation of the cellular metabolism of Theileria-transformed macrophages. My results showed that ablation of BAD S155 phosphorylation dissociates the mitochondrial complex of BAD and HK2 and cytosolic HK2 becomes ubiquitinated and degraded by the proteasome. Loss of HK2 switches the metabolism of Theileria-transformed leukocytes from Warburg-like to OXPHOS-like glycolysis.

Les parasites Theileria (Theileria. annulata and T. parva) sont des protozoaires intracellulaires qui font partie du phylum des Apicomplexa. Theileria infecte les leucocytes bovins et les transforment en cellules cancéreuses, induisant un genre de leucémie chez le bovin et conduisant à la mort de l’animal. Les cellules infectées par Theileria démontrent certaines caractéristiques de cellules cancéreuses telles qu’une importante capacité d’invasion et de migration cellulaire. Cependant, le traitement de cellules infectées avec une drogue Theiléricide spécifique (buparvaquone) permet l'élimination du parasite et la réversion du phénotype transformé. De plus, la virulence peut être atténuée par passages répétés sur culture cellulaire. La similitude entre les cellules transformées par Theileria et la leucémie humaine fait de Theileria un modèle très important permettant l’étude des mécanismes cellulaires induits par le parasite au cours de la transformation de la cellule hôte. Mon laboratoire d’accueil a publié une augmentation significative de TGF-β2 dans les cellules virulentes et a constaté que parmi les 1158 cibles de TGF-β, 68 gènes ont été reconnus d'avoir modifié leurs niveaux de transcription concomitante avec l'atténuation. Dans ce travail de thèse, nous avons étudié les voies de signalisations impliquées dans la régulation de l’adhésion et l’invasion des cellules infectées par Theileria. Nous nous sommes particulièrement intéressés à l’étude de la voie de signalisation TGF-β2 et ses effecteurs. Nos résultats montrent que l’activation de la voie de signalisation de TGF-β2 par Theileria entraîne une augmentation de l’invasion et de l’adhérence des cellules transformées par deux mécanismes différents, soit en activant la voie de signalisation PGE2/EP4/cAMP/PKA/EPAC/CREB, soit en stimulant la voie GRB2/PI3-K/AP-1. Les macrophages atténués infectés par Theileria sont plus stressés oxydativement ce qui diminue leur adhérence et leur invasion cellulaire. Ceci nous a amené à étudier en collaboration avec un autre doctorant (Mehdi Metheni) le rôle de TGF-β2 dans la régulation du stress oxydatif dans les macrophages infectés par Theileria. Nos données montrent que les niveaux élevés de TGF-β2 stimule l’expression de la catalase, une enzyme anti-oxydante qui convertit le H2O2 en H2O et la baisse de H2O2 favorise la virulence en augmentant l’invasion et l’adhésion des cellules infectées par Theileria (résultats supplémentaires). De plus, nous avons examiné le statut de stress oxydatif et le type de glycolyse utilisé par les cellules infectées par Theileria. Les cellules transformées par Theileria agissent comme des cellules cancéreuses, elles consomment énormément de glucose. La protéine BAD joue un rôle important dans l’apoptose ainsi que dans la voie de glycolyse. Son activité est régulée par phosphorylation en réponse à des facteurs de croissance et de survie. BAD peut être phosphorylée par la PKA sur le résidu sérine 155. Durant ma thèse, nous avons examiné le rôle de la phosphorylation de BAD par la PKA dans la régulation du métabolisme cellulaire des macrophages infectés par Theileria. Nos résultats montrent que l’abolition de la phosphorylation de BAD par la PKA dissocie le complexe mitochondrial formé entre BAD et HK2, ce qui induit l’ubiquitynation et la dégradation de HK2 par le protéasome. La baisse de HK2 stimule la voie de phosphorylation oxydative en faveur de l’effet Warburg dans les cellules infectées par Theileria. / Theileria parasites (Theileria. annulata and T. parva) are intracellular protozoa and members of the phylum Apicomplexa. Theileria parasites are the only eukaryotes that possess the property of being able to transform another eukaryote, their leukocyte host cells. Transformed leukocytes show many characteristics of tumour cells such as heightened invasive capacity; however the tumour-like phenotype can be totally reversed upon drug induced parasite death and attenuated by multiple in vitro passages. Such multiple-passaged attenuated lines are used as live vaccines against tropical theileriosis. The similarities in tumour hyper-invasiveness between Theileria-transformed leukcocytes and human lymphomas imply that observations on Theileria-induced leukocyte transformation have the potential to give generally applicable insights into the mechanisms underpinning tumour virulence. My host laboratory described higher TGF-β2 levels in virulent infected macrophages and following microarray analysis of virulent compared to attenuated macrophages found that among the 1158 TGF-β-targets, 68 genes had altered transcript levels concomitant with attenuation. In this study, we investigate the signalling pathways involved in the regulation of cellular adhesion and invasiveness of Theileria-infected cells. We were especially interested in the study of TGF-β2 signalling in Theileria-transformed virulent versus attenuated macrophages. My results indicate that following Theileria infection of macrophages, the TGF-β2 signalling pathway is activated and induces an increase in adhesion of virulent transformed macrophages through two different mechanisms: either by activating a PGE2 / EP4 / cAMP / PKA / EPAC / CREB signaling pathway, or by stimulating a GRB2 / PI3-K / AP-1 pathway. As attenuated macrophages display heightened oxidative stress, which underpins their loss of adhesion and invasiveness, in collaboration with another PhD student (Mehdi Metheni) we investigated the role of TGF-β2 in the regulation of the oxidative stress status of Theileria-infected macrophages. Our data show that high levels of TGF-β2 increase the expression of catalase, an anti-oxidant enzyme that converts H2O2 into H2O and the drop in H2O2 output results in regain of the virulence trait heightened adhesion of Theileria-transformed macrophages to fibronectin. Theileria-transformed macrophages display many features of cancer cells such as their consumption of larger quantities of glucose. The BCL-2 family protein BAD has an alternative function in glucose metabolism separate from its role in apoptosis. The activity of BAD is regulated by phosphorylation in response to growth/survival factors. BAD can be phosphorylated on Ser155 by PKA. So during my thesis studies I examined the role of PKA mediated phosphorylation of BAD in the regulation of the cellular metabolism of Theileria-transformed macrophages. My results showed that ablation of BAD S155 phosphorylation dissociates the mitochondrial complex of BAD and HK2 and cytosolic HK2 becomes ubiquitinated and degraded by the proteasome. Loss of HK2 switches the metabolism of Theileria-transformed leukocytes from Warburg-like to OXPHOS-like glycolysis.

Les étapes précoces du cycle réplicatif du VIH-1 sont assistées par des cofacteurs cellulaires dont la nature et la fonction restent mal connues. Nos travaux ont caractérisé la contribution de la protéine kinase dépendante de l'AMP cyclique (PKA), dans la cellule cible ou incorporée dans la particule virale VIH-1, dans les étapes post-entrée du cycle réplicatif. Les virus dépourvus d'activité PKA se caractérisent par un défaut de la synthèse de l'ADN proviral. En absence de Nef, la perte d'activité PKA associée aux particules VIH-1 induit une baisse plus modérée du pouvoir infectieux. En outre, le contournement des voies d'entrée classiques, par l'utilisation de particules pseudotypées, rend l'infectiosité indépendante de l'activité PKA. L'action de PKA s'exercerait donc au sein de la particule VIH-1 assemblée et impliquerait à la fois la protéine Nef et les voies de transport des complexes de transcription inverse au cours des étapes post-entrée. De plus, l'inhibition de l'activité PKA dans les cellules cibles entraîne également un défaut de synthèse de l'ADN proviral. Nos résultats indiquent que PKA agit comme un cofacteur de la transcription inverse. / The nature and function of cellular factors involved in post-entry steps of the HIV-1 life cycle are still poorly understood. We highlighted the role of cAMP-dependent protein kinase (PKA) in the early step of viral cycle, either as a host cellular protein in infected cells or as an incorporated protein into HIV-1 particles. PKA-deficient viruses failed to synthesize proviral DNA. In the absence of Nef, the loss of PKA activity associated to HIV-1 particles induces a minor diminution of infectiousness. Accordingly, VSV-G pseudotyped viruses, that use alternate entry pathway, exhibit full infectivity regardless of PKA deficiency. PKA action could therefore take place in the assembled HIV-1 particles, implying Nef protein and intracellular pathways of reverse transcription complexes. Moreover, the inhibition of PKA activity in target cells engenders a defective proviral DNA synthesis. Taken together, our data suggest that PKA may act as a cofactor required for HIV-1 reverse transcription.