Dissertationen zum Thema „Adenosine pathway“

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.
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A leishmaniose cutânea (CL), causada por L. amazonensis, é caracterizada por uma intensa imuno- supressão e multiplicação descontrolada do parasito em modelos experimentais e é geralmente grave em humanos, variando desde a forma cutânea até a cutâneo-difusa. Não existem mecanismos precisos conhecidos sobre como L. amazonensis modula a resposta imunológica para que os macrófagos (MФ) infectados com L. amazonensis se tornem refratários à ativação por células T efetoras. Aqui, nós investigamos o possível mecanismo regulador que Leishmania provavelmente pode induzir em MФ residentes durante a interação precoce, de modo a impedir ativação das células. Neste estudo, analisou-se a expressão de CD39 e CD73, por citometria de fluxo, em MФ peritoneais murinos infectados com promastigotas metacíclicas de L. amazonensis e também a porcentagem dessas células que expressam a CD39 e CD73 foi avaliada. Nossos resultados mostraram que em 72hrs inativos os MФ tiveram baixa expressão de CD73. Curiosamente, no entanto, ao contrário de MФ tratados com LPS os infectados com L. amazonensis expressaram altos níveis de CD73. Esta informação foi posteriormente validada pelos resultados de estudos no contexto ex-vivo que mostrou igualmente que MФ infectados são predominantemente CD73+. Quando as atividades enzimáticas de CD39 e CD73 foram bloqueadas, tal como pelo uso de DIDS e MAD αβ, tanto a infecção quanto o número de amastigotas diminuiu significativamente após 48 horas de incubação. Da mesma forma, a inibição dos receptores de adenosina A2a e A2b de ZM241385 e MRS1754 também apresentou os mesmos efeitos sobre a sobrevivência do parasito e infectividade. Em estudo posterior, em busca de um possível papel da HIF- 1α na infecção por Leishmania, investigamos os efeitos da FM19G11, inibidor do HIF- 1α, na expressão de CD39 e CD73, bem como na infecção parasitária . Observou-se que, apesar de HIF - 1α poder influenciar na sobrevivência do parasito, os seus efeitos sobre a expressão de CD39 e CD73 não eram visíveis. Também foi avaliada, por PCR em tempo real, a expressão de receptores de adenosina em populações infectadas, nas quais não se observou nenhuma mudança significativa na expressão após 24 horas de infecção. Além disso, também foi avaliada a produção de citocinas, tais como TNF- α e IL-10 a partir da produção de NO nos grupos tratados. Surpreendentemente, não houve variação nos níveis destes mediadores, sugerindo a existência de outros mecanismos independentes da mediação por citocina para produção de Óxido Nítrico, tais como a produção de ROS ou efeitos leishmanacidas independentes do triptofano. Concluindo, nossos dados mostram que a infecção por L. amazonensis regula a expressão CD73 durante 24 horas de infecção e sua sobrevivência depende de atividades enzimáticas, bem como de receptores A2a e A2b. __________________________________________________________________________________________
ABSTRACT:Cutaneous leishmaniasis (CL) caused by L. amazonensis is characterized by intense immune-suppression and uncontrolled parasite multiplication in experimental models and is usually severe in humans ranging from cutaneous to diffuse cutaneous leishmaniasis. There are no precise mechanisms known how L. amazonensis modulates immune response so that macrophages (MФ) infected with L. amazonensis are refractory to activation by effector T cells. Here, we investigated the possible regulatory mechanism that Leishmania can likely induce in host MФ during early interaction so as to prevent their host cells from activation. In this study, we analyzed the expression of CD39 and CD73, by flow cytometry, in murine peritoneal MФ infected with metacyclic promastigotes of L. amazonensis and percentage of those cells expressing CD39 and CD73 was evaluated. Our results showed that 72hrs rested MФ down regulated CD73 expression. Interestingly, however, unlike LPS treated MФ, L. amazonensis infected MФ up regulated CD73 expression. This data was further validated by the findings from in ex-vivo studies which equally support that infected MФ are predominantly CD73 positive. When CD39 and CD73 enzymatic activities were blocked such as by the use of DIDS and αβ MAD, both infection and amastigote number decreased significantly within 48hrs of incubation. Similarly, inhibition of adenosine receptors A2a and A2b by ZM241385 and MRS1754 also had the same effects on the parasite survival and infection. In another study, in search of a possible role of HIF-1α in Leishmania infection, we investigated the effects of FM19G11, inhibitor of HIF-1α, on expression of CD39 and CD73 as well as parasitic infection. We observed that although HIF-1α can influence in the parasite survival, their effects on CD39 and CD73 expression were not visible. We also evaluated the expression of adenosine receptors in infected population by real time PCR in which we observed no significant change in the expression after 24hrs of infection. Moreover, we also evaluated cytokine production such as TNF-alpha, IL-10 and NO production from the treated groups. Surprisingly, there was no alternation in the levels of these mediators suggesting other mechanisms, independent of cytokine mediated nitric oxide production such as ROS production or tryptophan independent oxygen anti-leishmanacidal effects, involved in it. In conclusion, our data show that L. amazonensis infected up regulates CD73 expression during 24hrs of infection and its survival is dependent on enzyme activities as well as A2a and A2b receptors.

Malignant pleural mesothelioma is an aggressive cancer and, for this disease, chemotherapy, surgery and radiotherapy are not curative. During the last years, numerous of studies have focused on immune therapies exploiting the use of immune checkpoint inhibitors. So far, these treatments are based on the use of molecules that inhibit proteins and receptors such as PD-1, PD-L1 and CTLA-4 [1]. Thus, there is the urgent need to find other immune targets. The purinergic pathway is a field of great interest as in fact there is evidence that the hypoxic environment of tumors induces increased expression of CD73 and CD39 (enzymes that produce adenosine starting from ATP) which promote the increase in extracellular adenosine [2]. High levels of adenosine are characteristic of the tumor microenvironment and induce immunosuppressive signals promoting growth and progression of tumors. For this reason [3], inhibitors of the purinergic pathway drawing attention to restore immune response to cancer cells. Our study is aimed at the identification of adenosine pathway members in MPM tissue and if this same pathway is active in this tumor. We have detected high expression of the Adenosine receptors and CD73 in MPM cells. Accordingly, treatment with the A2Br antagonist (MRS1754) provided the evidence that adenosine signaling is active in MPM cells and is a potential novel druggable target against MPM.

Since the early twentieth century, biological responses to extracellular ATP have been documented in Virtually every major organ and tissue system. Our laboratory has focussed on the ability of extracellular ATP to induce elevations in the intracellular cAMP content of acute myelocytic leukaemia HL-60 cells, which subsequently results in their differentiation towards a more mature neutrophil-like cell type.

[ES] Las modificaciones químicas post-transcripcionales implican un nuevo nivel de modulación de la expresión génica. Al comienzo de esta Tesis, algunos componentes del complejo de metilación del nitrógeno en posición 6 de la adenosina (m6A) habían sido caracterizados en plantas. Sin embargo, a diferencia de mamíferos y levadura, ninguno de los 13 homólogos de AlkB (atALKBH1-10B) - potenciales desmetilasas (o erasers) - y las 13 proteínas de la familia YTH (ECT1- 11, AT4G11970 y CPSF30) - potenciales proteínas de reconocimiento de m6A (o readers) - identificadas en el genoma de Arabidopsis se habían caracterizado funcionalmente. Además, varios estudios describen la presencia de m6A en RNAs de virus de mamíferos y las diferentes funciones que desempeña esta modificación en la regulación de esas infecciones. No obstante, no se ha estudiado la posible implicación de este mecanismo molecular en las infecciones virales de plantas. El descubrimiento de la interacción entre la CP del virus del mosaico de la alfalfa (AMV) y una proteína de Arabidopsis (atALKBH9B) con homología a una eraser humana fue el punto de partida de esta Tesis. En este trabajo se confirma esta interacción, y se demuestra que atALKBH9B también puede reconocer los RNAs virales. Los resultados revelan que atALKBH9B tiene la capacidad de desmetilar m6A a partir de moléculas de RNA monocatenario in vitro. Esta proteína se acumula en gránulos citoplasmáticos que se colocalizan con siRNA bodies y se asocian a P-bodies, lo que sugiere que su actividad podría estar relacionada con el silenciamiento y/o degradación de mRNA. Por otro lado, ensayos preliminares muestran que los RNAs del AMV, el virus del mosaico del pepino (CMV), el virus de la arruga del nabo (TCV) y el virus del mosaico de la coliflor (CaMV) se metilan durante la infección en Arabidopsis. Además, para AMV y CMV, los resultados fueron corroborados por UPLC-PDA-Tof-MS y los sitios m6A a lo largo de los RNAs del AMV fueron identificados mediante MeRIP-seq. Los resultados presentados confirman que la relación m6A/A a lo largo de los RNAs virales aumenta en plantas atalkbh9b en comparación con las silvestres, mientras que la traducción y/o replicación se ven afectadas y el movimiento sistémico a los tallos florales está prácticamente bloqueado. A diferencia de la CP de AMV, la de CMV no interacciona con atALKBH9B por Y2H y, como ocurre con el resto de virus analizados (CMV, TCV y CaMV), su ciclo de infección no se ve afectado en plantas atalkbh9b. Además, la secuenciación de mRNA realizada en este trabajo revela que la infección por AMV induce algunos genes de Arabidopsis pertenecientes a la maquinaria m6A, MTA, MTB, VIR y ECT5. De acuerdo con el efecto antiviral dependiente de m6A para el AMV y teniendo en cuenta que ECT2, ECT3 y ECT4 fueron recientemente caracterizadas como readers citoplasmáticas, la supresión del módulo ECT2/ECT3/ECT5 aumenta significativamente los títulos sistémicos de AMV y CMV. El efecto antiviral de ECT2 sobre AMV parece estar modulado por su unión directa a los residuos de m6A presentes en los RNAs virales, ya que un mutante de ECT2 defectuoso en el reconocimiento de m6A pierde la actividad antiviral que sí presenta la proteína original y no es capaz de arrastrar RNAs virales in vivo. Por otro lado, acorde a la localización previamente descrita para ECT2 y ECT4 y la capacidad de ECT2 para experimentar una fase similar al gel in vitro, la expresión transitoria de ECT5 muestra un patrón citoplasmático con formación de agregados. Se propone que, como se ha descrito para las proteínas YTH de mamíferos, la interacción entre las ECTs y el RNA polimetilado (en este caso, RNA viral) promovería la formación de gránulos de estrés y, en consecuencia, reduciría las tasas de traducción y replicación viral. En resumen, en este trabajo se caracteriza la primera m6A eraser de plantas, atALKBH9B, y, por primera vez, se describe la influencia del mecanismo de metilación m6A en las infecciones virales de plantas.
[EN] Post-transcriptional chemical modifications entail a new level of gene expression modulation. At the beginning of this Thesis, some components of the N6-adenosine methylation (m6A) complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) - putative demethylases (or erasers) - and 13 proteins of the YTH family (ECT1-11, AT4G11970 and CPSF30) had been identified in the Arabidopsis genome. However, unlike mammals and yeast, no functional roles had been described for any of these proteins. Besides, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses and the critical roles that this modification plays regulating viral infections. However, the potential relevance of this molecular mechanism on plant viral infections remained fully unexplored. The discovery of the interaction between the AMV CP and an Arabidopsis protein (atALKBH9B) with similarity to a human eraser was the starting point of this Thesis. Here, this interaction is confirmed and it is demonstrated that atALKBH9B can also recognize the viral RNAs. Furthermore, the obtained results prove that atALKBH9B has the capability of demethylating m6A from single-stranded RNA molecules in vitro. This protein was observed to accumulate in cytoplasmic granules that colocalize with siRNA-bodies and associate to P-bodies, suggesting that atALKBH9B activity could be related to mRNA silencing and/or decay processes. On the other hand, preliminary assays show that viral RNAs of AMV, Cucumber mosaic virus (CMV), Turnip crinkle virus (TCV) and Cauliflower mosaic virus (CaMV) become methylated during infection in Arabidopsis. Besides, for AMV and CMV, the results were corroborated by UPLC-PDA-Tof-MS and m6A sites along the RNAs of AMV were identified through MeRIP-seq approach. The results presented here confirm that m6A/A ratio along viral RNAs is increased in atalkbh9b plants compared to wild type, whereas translation and/or replication are impaired and systemic movement to the floral stems is practically blocked. In contrast to AMV, CMV CP does not interact with atALKBH9B by Y2H and, as it occurs with the rest of the assayed viruses (CMV, TCV and CaMV), its infection cycle is not affected in atalkbh9b plants. Furthermore, the mRNA-seq analysis performed in this Thesis reveals that some Arabidopsis factors belonging to the m6A machinery, MTA, MTB, VIR and ECT5 genes, are upregulated upon AMV infection. Consistent with the m6A-dependent antiviral effect for AMV and considering that ECT2, ECT3 and ECT4 were recently characterized as cytoplasmic m6A readers, mutations of ECT2/ECT3/ECT5 Arabidopsis module significantly increase AMV and CMV systemic titers. The antiviral effect of ECT2 on AMV seems to be modulated via its direct binding to the m6A residues presented in the viral RNAs, since an ECT2 mutant defective in m6A recognition loses wild type antiviral activity and is not able to pull down viral RNAs in vivo. On the other hand, according to the previous subcellular localization described for ECT2 and ECT4 and the ability of ECT2 to undergo gel-like phase in vitro, the transitory expression of ECT5 displays a cytoplasmic pattern with the formation of some aggregates. As found for mammal YTH proteins, the interaction between ECTs and poly-methylated RNA (in this case viral RNA) is proposed to promote the formation of stress granules and, consequently, reduce viral translation and replication rates. In summary, in this work, atALKBH9B is reported as the first m6A eraser identified in plants and, for the first time, it is described the influence of m6A methylation mechanism in plant viral infections.
[CA] Les modificacions químiques post-transcripcionals impliquen un nou nivell de modulació de l'expressió gènica. Al començament d'esta Tesi, s'havien caracteritzat alguns components del complex de metilació del nitrogen en posició 6 de la adenosina (m6A) en plantes. No obstant això, a diferència de mamífers i llevat, cap dels 13 homòlegs d'AlkB (atALKBH1-10B) - potencials desmetilases (o erasers) - i les 13 proteïnes de la família YTH (ECT1- 11, AT4G11970 i CPSF30) - potencials proteïnes de reconeixement de m6A (o readers) - identificades en el genoma d'Arabidopsis s'havien caracteritzat funcionalment. A més, diversos estudis han descrit la presència de residus m6A en RNAs de virus de mamífers i les diferents funcions que exercix esta modificació en la regulació de les infeccions virals. No obstant això, no s'ha estudiat la possible implicació d'este mecanisme molecular en les infeccions virals de plantes. El descobriment de la interacció entre la CP del virus del mosaic de l'alfals (AMV) i una proteïna d'Arabidopsis (atALKBH9B) amb homologia a una eraser humana va ser el punt de partida d'esta Tesi. En este treball es confirma esta interacció, i es demostra que atALKBH9B també pot reconéixer els RNAs virals. Els resultats revelen que atALKBH9B té la capacitat de desmetilar m6A a partir de molècules de RNA monocatenari in vitro. Esta proteïna s'acumula en grànuls citoplasmàtics que es colocalitzen amb siRNA bodies i s'associen a P-bodies, la qual cosa suggerix que l'activitat atALKBH9B podria estar relacionada amb els processos de silenciament i/o degradació de mRNA. D'altra banda, assajos preliminars mostren que els RNAs virals de l'AMV, el virus del mosaic del cogombre (CMV), el virus de l'arruga del nap (TCV) i el virus del mosaic de la coliflor (CaMV) es metilen durant la infecció en Arabidopsis. A més, per AMV i CMV els resultats van ser confirmats per UPLC-PDA-Tof-MS i els llocs m6A al llarg dels RNAs d'AMV s'identificaren mitjançant MeRIP-seq. Els resultats presentats confirmen que la relació m6A/A al llarg dels RNAs virals augmenta en les plantes atalkbh9b en comparació amb les silvestres, mentre que la traducció i/o replicació es veuen afectades i el moviment sistèmic a les tiges florals està pràcticament bloquejat. A diferència de la CP d'AMV, la de CMV no interacciona amb atALKBH9B per Y2H i, com ocorre amb la resta dels virus analitzats (CMV, TCV i CaMV), el seu cicle d'infecció no es veu afectat en plantes atalkbh9b. A més, la seqüenciació de mRNA realitzada en este treball revela que la infecció per AMV indueix alguns gens d'Arabidopsis pertanyents a la maquinària m6A, MTA, MTB, VIR i ECT5. D'acord amb l'efecte antiviral dependent de m6A per a l'AMV i tenint en compte que ECT2, ECT3 i ECT4 van ser recentment caracteritzades com readers citoplasmàtiques, la supressió del mòdul ECT2/ECT3/ECT5 augmenta significativament els títols sistèmics d'AMV i CMV. L'efecte antiviral d'ECT2 sobre AMV sembla estar modulat a través de la seua unió directa als nucleòtids m6A presents en els RNAs virals, ja que un mutant de la proteïna ECT2 defectuós en el reconeixement de m6A perd l'activitat antiviral que sí que presenta la proteïna original i no és capaç d'arrossegar RNAs virals in vivo. D'altra banda, d'acord amb la localització subcel·lular descrita prèviament per a ECT2 i ECT4 i la capacitat d'ECT2 per a experimentar una fase similar al gel in vitro, l'expressió transitòria d'ECT5 mostra un patró citoplasmàtic amb la formació d'agregats. Es proposa que, com s'ha descrit per a les proteïnes YTH de mamífers, la interacció entre les ECTs i el RNA polimetilat (en aquest cas, RNA viral) promouria la formació de grànuls d'estrès i, en conseqüència, reduiria les taxes de traducció i replicació viral. En resum, en este treball es caracteritza la primera m6A eraser de plantes, atALKBH9B, i, per primera vegada, es descriu la influència de l'mecanisme de metilació M6A en les infeccions virals de plantes.
Martínez Pérez, M. (2020). Involvement of the host RNA N6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155976
TESIS

Adenosine blood levels and complications of prematurity in a selected population of very low birth weight infants. We have recently reported high blood adenosine levels in extremely premature infants, positively correlating to their prematurity in function of the body weight classes. Acting via purinergic receptors, adenosine can affect different vulnerable organs including the developing brain.

Les chondrosarcomes, tumeurs rares des os, sont considérés chimio- et radio- résistants pouvant métastaser au niveau pulmonaire. À l’heure actuelle, seule la chirurgie est le traitement de référence, cependant, elle peut être très délabrante et augmente la morbidité associée. Le but de ce travail de recherche vise à identifier de nouvelles stratégies chimiothérapeutiques pouvant être applicables aux chondrosarcomes et permettant de mieux comprendre les mécanismes tumoraux mis en place dans ces tumeurs. L’identification de ces nouvelles stratégies thérapeutiques nous a amenés à nous intéresser à la voie de l’adénosine, mais également à la méthylation des histones et notamment la méthylation de la lysine 36 de l’histone H3 (H3K36). Dans un premier temps, nous avons montré que l’utilisation d’analogues de l’adénosine, déjà utilisés en clinique, la cladribine et la clofarabine pouvaient avoir des effets antitumoraux in vitro et in vivo sur les chondrosarcomes. Ces résultats confirment ceux déjà obtenus avec un autre analogue de l’adénosine, le 3-deazaneplanocine A (DZNep). Dans un second temps, nous avons voulu comprendre par quels mécanismes d’action pouvaient fonctionner les analogues, notamment le DZNep. Nous avons donc identifié la triméthylation de H3K36 (H3K36me3) comme cible potentielle. Cependant, l’inhibition pharmacologique de la méthyltransférase responsable de H3K36me3, SETD2, par l’EZM0414 n’a montré que peu d’effets sur les chondrosarcomes. Ainsi, cette étude souligne un lien entre le métabolisme, la méthylation et la voie de l’adénosine dans les chondrosarcomes et donc un possible intérêt pour associer plusieurs molécules thérapeutiques pour traiter les chondrosarcomes
Chondrosarcomas, rare bone tumors, are considered chemo and radio-resistant and can metastasize to the lung. At present, surgery is the standard treatment, but it can be damaging and increases the associated morbidity. The aim of this research work is to identify new chemotherapeutic strategies that could be applied to chondrosarcomas and provide a better understanding of the tumor mechanisms involved in these tumors. The identification of these new therapeutic strategies led us to investigate both the adenosine pathway and histone modification, in particular the methylation of lysine 36 of histone H3 (H3K36). Firstly, we demonstrated that the use of the adenosine analogs already in clinical use, cladribine and clofarabine, could have antitumoral effects in vitro and in vivo on chondrosarcoma. These results confirm those already obtained with another adenosine analog, 3-deazaneplanocine A (DZNep). Secondly, we wanted to understand the mechanisms by which analogs, and DZNep in particular, might work. We therefore identified H3K36 trimethylation (H3K36me3) as a potential target. However, pharmacological inhibition of methyltransferase responsible for H3K36me3, SETD2, by EZM0414 showed limited effects on chondrosarcomas. Thus, this study highlights a link between metabolism, methylation and adenosine pathways in chondrosarcomas, and therefore a possible interest in combining multiple therapeutic molecules to treat chondrosarcomas

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

Thesis (Ph. D.)--Georgia State University, 2007.
Vincent Rehder, committee chair; Sarah Pallas, Walter William Walthall, committee members. Electronic text (248 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 28, 2008; title from file title page. Includes bibliographical references (p. 218-248).

L’adénosine extracellulaire appartient à la voie de signalisation purinergique et réguledivers processus physiologiques à travers l’activation de ses récepteurs spécifiques (adora).La disponibilité de cette purine dans l’espace extracellulaire est régulée par plusieurs ectoenzymesassurant sa production ou sa dégradation, mais également par des transporteurs denucléosides permettant son passage à travers la membrane. Chez l’adulte, le rôle del’adénosine est assez bien connu. Cependant, l’implication de cette purine au cours del’embryogenèse reste très peu étudiée. Pourtant, un excès d’adénosine dans les phasesprécoces du développement est létal chez la souris et l’oursin, démontrant l’importance de larégulation des concentrations de cette molécule de signalisation lors de l’embryogénèse. Lebut de ma thèse est de comprendre le rôle de l’adénosine au cours de l’embryogenèse enutilisant l’amphibien xénope. En effet, ce modèle a permis de mettre en évidence in vivol’implication de l’ADP au cours du développement de l’oeil chez les vertébrés. La premièrepartie de ce projet a permis de caractériser les acteurs de la voie de signalisation del’adénosine chez le xénope afin d’établir la première carte comparative de leur profild’expression embryonnaire. Cette partie a également permis de mettre en avant laphosphatase alcaline alpl pour son profil d’expression particulier, dans le rein et la rétine. Laseconde partie s’est focalisée sur l’étude fonctionnelle de cette enzyme. Les expériences deperte de fonction montrent son implication lors de la formation de ces deux tissus
Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development

Adenosine is an ubiquitous autacoid that modulates a variety of cellular functions through occupancy of four cell surface G-protein-coupled receptors, named A1, A2A, A2B and A3. In particular, adenosine was found to exert its effects on cell proliferation, clone formation ability, UV resistance, and cell death mainly through the A3 subtype, which is highly expressed in tumor cells. Adenosine also plays a role in the promotion of angiogenesis. In this thesis I have characterized the signal transduction pathways modulated in hypoxia by A3 adenosine receptors in two different human tumor models: the colon cancer HT29 and the melanoma A375 cell lines. I have performed the study in hypoxia, present in most solid tumors, which shifts the cellular phenotype toward an increase in adenosine. Furthermore, hypoxic tumor cells are resistant to conventional chemiotherapy and radiotherapy. I have investigated the modulation of hypoxia-inducible factor-1 (HIF-1) through adenosine via its receptor subtypes. HIF-1 is a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1 is a heterodimer composed of an inducibly expressed HIF-1α subunit and a constitutively expressed HIF-1β one. In normoxia, HIF-1α is rapidly degraded by the ubiquitin proteasome system, whereas exposure to hypoxic conditions prevents its degradation. I have studied the signaling pathways modulated by A3 receptors which involved Akt, MEK, and p38 MAPK. I have demonstrated that adenosine increased in hypoxic colon carcinoma cells HIF-1α and vascular endothelial growth factor (VEGF) through the A3 receptor stimulation. Furthermore, the stimulation of A2B receptor increased interleukin-8 (IL-8) expression. Pretreatment of cells with caffeine, which is a methylxanthine antagonist of adenosine receptors, significantly reduced adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The kinases have a key role in A3 receptor ability to enhance HIF-1α and VEGF protein expression. Moreover, Akt, ERK 1/2, and p38 MAPK are required for the IL-8 expression increase induced by A2B receptor activation. Then, I have examined the modulation of IL-8, VEGF and HIF-1 by the DNA-damaging agents etoposide and doxorubicin, and I have analyzed the influence of the adenosinergic signaling on the chemotherapeutic drug effects. I have demonstrated that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with etoposide and doxorubicin. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. Exposure of melanoma cells to the chemotherapeutic drugs resulted in the increase of p38, Akt and ERK1/2 phosphorylation levels. Moreover, etoposide and doxorubicin strongly inhibited HIF-1α protein expression. The A2B receptor antagonist MRE 2029F20 attenuated the increase in p38, Akt and ERK1/2 phosphorylation levels induced by the chemotherapeutic drugs, and when used alone it reduced p38, Akt and ERK1/2 phosphorylation basal levels. The A3 receptor antagonist MRE 3008F20 alone reduced HIF-1α protein, and in combination with the chemotherapeutic drugs further decreased HIF-1α protein accumulation. Therefore, the results of this thesis provide evidence of how human tumor growth may be influenced through the adenosinergic system and how the adenosine receptors modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.

O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos.
Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.

Au cours de l’ischémie cardiaque, la lésion est initiée au niveau de l’endothélium, et progresse aux cardiomyocytes environnants. La signalisation purinergique joue un rôle important au cours d’épisodes l’ischémie/reperfusion (I/R). De multiples travaux ont portés sur l’étude des mécanismes de protection des nucléotides et nucléosides, sans pour autant étudier leurs rôles spécifiques sur l’endothélium. Dans ce travail, nous mettons en évidence une augmentation des concentrations extracellulaires en ATP et adénosine provenant de l’endothélium soumis à une hypoxie. Au niveau endothélial, nous mettons en évidence un effet protecteur de l’ATP extracellulaire ainsi qu’un rôle complémentaire des récepteurs P2 et P1. Les récepteurs P2 impliquent les voies de signalisation PI3K, ERK1/2, le canal mKATP et mettent également en jeu la NOS. La protection médiée par les récepteurs P1 implique les voies MEK/ERK1/2, PKA et NOS. Dans un second temps, nous rapportons un nouveau mécanisme cytoprotecteur du ticagrelor, indépendant des éléments figurés du sang et de son effet antiagrégant plaquettaire. Ce mécanisme est initié par l’augmentation de la biodisponibilité de l’adénosine extracellulaire qui déclenche les effets protecteurs via ses récepteurs A3 et A2A. Ceci peut expliquer, en partie, les effets cardioprotecteurs du ticagrelor décrit chez l’homme. L’ensemble de nos données conforte le rôle protecteur de l’ATP et de l’adénosine vis-à-vis de l’hypoxie au niveau endothélial et suggèrent un rôle bénéfique de ces médiateurs dans diverses ischémies notamment cardiaque, rénale ou cérébrale
During cardiac ischemia, the lesion is triggered in the endothelium and progresses to the surrounding cardiomyocytes. Purinergic signalling plays an important role during ischemia/reperfusion (I/R) events. Many studies have been carried out to study the mechanisms of protection of nucleotides and nucleosides, without studying their specific roles on the endothelium. In this work, we report an increase in extracellular concentrations of ATP and adenosine from the endothelium exposed to hypoxia. We report a protective effect of extracellular ATP and a complementary role of the P2 and P1 receptors. P2 receptors protective effects involve the PI3K, ERK1/2, mKATP channel signalling and also involve NOS. The protection mediated by the P1 receptors involves the MEK/ERK1/2, PKA and NOS. In a second step, we describe a new cytoprotective mechanism of ticagrelor, independent of the blood element and its antiplatelet anti-aggregating effect. This mechanism is initiated by the increased of extracellular adenosine bioavailability, which triggers protective effects via its A3 and A2A receptors. This may explain, in part, the reported cardioprotective effects of the ticagrelor in clinical studies. Together, our data support the protective role of ATP and adenosine against deleterious effects ofendothelium hypoxia and suggest a beneficial role for these mediators in different ischemia, including cardiac, renal or cerebral ischemia

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
Introdução: A hipóxia é uma característica comum dos tumores sólidos e desempenha um papel crítico nas neoplasias malignas, incluindo o cancro da bexiga (CB). O factor induzível por hipóxia-1α (HIF-1α) desempenha um papel importante na regulação da resposta das células tumorais sujeitas ao stress hipóxico que resulta em alterações metabólicas e na ativação de mecanismos de sobrevivência. As células hipóxicas sobreexpressam as ecto-nucleotidases CD39 e CD73 que estão envolvidas na geração de adenosina extracelular. Este nucleosídeo atua como um importante regulador de processos inflamatórios em condições fisiológicas mas no cancro, inibe o sistema imunitário permitindo que as células cancerígenas escapem ao controlo do sistema imunitário. Além disso, existem evidências que associam a ativação da via adenosinérgica em resposta à hipóxia com a agressividade tumoral. No entanto, os mecanismos que estão subjacentes a este processo não estão completamente clarificados.Objetivo: Explorar o papel patofisiológico da via adenosinérgica mediada por hipóxia nas características malignas do CB e avaliar a contribuição do HIF-1α e da adenosina nesses processos biológicos.Métodos: Duas linhas celulares humanas de CB, UM-UC3 e HT-1376, foram expostas a hipóxia usando um sistema anaeróbio GasPakTM EZ na ausência e na presença do inibidor do HIF-1α (Digoxina). A expressão do HIF-1α, CD39, CD73, dos receptores de adenosina A2A e A2B e do PD-L1 foi avaliada por Western blot. Os níveis extracelulares de adenosina no sobrenadante foram medidos usando um kit comercial de medição de adenosina. A proliferação celular e a quimiossensibilidade à cisplatina foram avaliadas pelo ensaio de WST-1. A migração celular foi determinada pelo ensaio de scratch. Os marcadores de superfície e os fatores de transcrição da transição epitelial-mesenquimal foram analisados por qPCR. Estas experiências foram realizadas em condições de normóxia na presença de adenosina.Resultados: Ambas as linhas celulares em condições de hipóxia estabilizaram o HIF-1α e ativaram a via adenosinérgica como demonstrado pela sobreexpressão das ecto-nucleotidases CD39 e CD73, produção de adenosina extracelular e sobreexpressão do receptor A2B. A hipóxia diminuiu a susceptibilidade das células de CB à cisplatina e aumentou a expressão do PD-L1, antecipando o desenvolvimento de mecanismos de evasão do sistema imunológico. A hipóxia induziu a transição epitelial-mesenquimal em ambas as linhas celulares e aumentou a capacidade de migração das células HT-1376, mas não das UM-UC3. O tratamento com adenosina exacerbou as características malignas das células de CB, semelhante às induzidas pelas condições de hipóxia. A Digoxina atenuou a expressão de HIF-1α nas células de CB em condições de hipóxia sem contudo prevenir a produção de adenosina extracelular e os seus efeitos pro-tumorais.Conclusão: A hipóxia ativou a via adenosinérgica nas células de CB e promoveu a agressividade tumoral ao interferir com a proliferação celular, quimiorresistência e capacidade de invasão. A adenosina desempenha um papel importante no efeito pro-tumoral da hipóxia. Assim sendo, estratégias terapêuticas incorporando inibidores da via hipóxia-CD39-CD73-A2BR podem ser benéficos no controlo da progressão tumoral e na resposta à terapia.
Introduction: Hypoxia is a common feature of solid tumors and a critical hallmark of malignant disease, including bladder cancer (BC). The hypoxia-inducible factor-1α (HIF-1α) is a crucial regulator of cancer cells response to hypoxia stress and results in metabolic changes and activation of survival mechanisms. Hypoxic cells upregulate the ecto-nucleotidases CD39 and CD73 that are involved in the generation of extracellular adenosine. This nucleoside in physiological conditions acts as an important regulator of inflammatory processes but in cancer, dampen the immune system allowing cancer cells to escape the immune control. Moreover, accumulating evidences suggest a link between the adenosinergic response to hypoxia and tumor aggressiveness. However, the mechanisms behind this process are not completely clarified.Objectives: To explore the pathophysiological role of the hypoxia-driven adenosinergic pathway on the malignant features of BC and to unravel the contribution of HIF-1α and of adenosine in those biological processes.Methods: Two human BC cell lines, UM-UC3 and HT-1376, were exposed to hypoxia using the GasPak ez anaerobe system in the absence and in the presence of a HIF-1α inhibitor (Digoxin). The expression of HIF-1α, CD39, CD73, adenosine receptors A2A and A2B and of PD-L1 were measured by Western blot. Extracellular levels of adenosine in supernatants were measured using an adenosine assay kit. Cell proliferation and chemosensitivity to cisplatin were evaluated using the WST-1 assay. Cell migration was assessed via a wound-healing assay. Epithelial-to-mesenchymal transition surface markers and transcription factors were detected by qPCR. A parallel set of experiments were conducted under normoxic conditions in the presence of adenosine.Results: Both cell lines under hypoxic conditions stabilized HIF-1α and activated the adenosinergic pathway as shown by the upregulation of CD39, CD73 and extracellular generation of adenosine, accompanied by an upregulation of the A2B receptor. Hypoxia impaired the susceptibility of BC cells to cisplatin and upregulates the expression of PD-L1, anticipating the development of an immune escape mechanism. Hypoxic stress induced epithelial-to-mesenchymal transition in both cell lines and increased the migratory rate of HT-1376 cells, but not of UM-UC3 cells. Treatment with adenosine exacerbated the malignant features of BC cells in a fashion similar to those induced by hypoxic conditions. Digoxin attenuated the protein expression of HIF-1α in BC cells under hypoxic conditions without preventing the extracellular production of adenosine and its pro-tumoral effects.Conclusion: Hypoxia activated the adenosinergic pathway in BC cells and promoted tumor aggressiveness by interfering with cell proliferation, chemoresistance and invasiveness. Adenosine has a high contribution to the tumor-promoting effects of hypoxia. Therefore, therapeutic strategies incorporating inhibitors of the hypoxia-CD39-CD73-A2BR pathway might be beneficial in controlling tumor growth and response to therapy.
Outro - This work was funded by National Funds via FCT (Foundation for Science and Technology) through the Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB)

Adenosine is an endogenous nucleoside that has been recognised to be a molecule with autocrine/paracrine functions, acting as a signal molecule to preserve host defence and tissue integrity during inflammation and trauma in addition to its important role as homeostatic regulator. The physiological activities of adenosine involve its interactions with four types of receptors, designed as A1, A2A, A2B and A3. Adenosine mediates its anti-inflammatory activity primarily through the A2A receptor (A2AAR). The ecto-5’nucleotidase/CD73 degrades AMP to adenosine and represents a key enzyme for adenosine accumulation at the site of injury. The aim of this research work was to explore different aspects of adenosine signalling pathway in inflammation. Several findings implicate the adenosine signalling pathway as an innate mechanism to attenuate excessive tissue damage and identify CD73 as critical control points for endogenous adenosine generation. It has been shown that CD73 plays an important role in regulating vascular permeability and leukocyte trafficking in inflammatory disease; and a crucial role in the regulation of immune/inflammatory cell function. A better understanding of the role of CD73 enzyme in the development of inflammatory processes can help to identify new therapeutic strategies aimed at strengthening the endogenous anti-inflammatory mechanisms. For this reason we sought to investigate on the role of CD73, the key enzyme in “switching on” adenosine signalling, in the development of inflammation through its pharmacological blockade by using the selective inhibitor, adenosine 5'-(α,β-methylene) diphosphate (APCP; 400 μg/site), in an in vivo model of acute inflammation represented by carrageenan-induced pleurisy in rats. We found that local inhibition of CD73 significantly increased cell accumulation, exudate formation and pro-inflammatory cytokine content into the pleural cavity in the acute phase of inflammation with no differences in the sub-acute phase. The in vivo treatment with APCP induced cells recruited into the pleural cavity to change in a phenotype with increased ability to migrate in vitro either in the presence or in the absence of a chemotactic stimulus. In parallel, these cells showed a reduced CD73 expression and activity compared to cells collected from control group. In addition, APCP, in vitro, strongly increased the ability of cells from control groups to migrate in the presence of a chemotactic stimulus. Local inhibition of CD73 increased also the infiltration of the lung with polymorphonuclear leukocytes (PMNs) and the degree of lung injury 4 hours following carrageenan injection. The interest to explore the role of adenosine signalling pathway in the control of inflammation has been growing further following evidence that adenosine signalling is also involved in the mechanism of action of some well-known anti-inflammatory drugs. With regard to this, we focused our interest on the possible involvement of adenosine signalling in the anti-inflammatory mechanism of nimesulide, in vivo (rat paw oedema) and in vitro (J774A.1cell line); indeed, there is evidence that nimesulide anti-inflammatory effect is the consequence of regulation of the production and actions of a wide range of inflammatory mediators, independently from the sole cyclooxygenase-2 (COX-2) enzyme inhibition. To date, the molecular mechanisms at the basis of nimesulide peculiar pharmacological effects are still unclear. In vivo, in the model of carrageenan-induced rat paw oedema, we found that the anti-inflammatory effect of nimesulide (5 mg/kg i.p.) was inhibited by pre-treatment with the adenosine A2A receptor antagonist, ZM 241385 (3 mg/kg i.p.), and by local administration of the CD73 inhibitor, APCP (400 μg/paw). Furthermore, we observed increased activity of 5'-nucleotidase/CD73 in plasma and paws of nimesulide-treated rats, 4 h following oedema induction that represented the inflammatory peaking point. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin (PG)E2 (PGE2) production by lipopolysaccharide (LPS)-activated J774 macrophage cell line was again reverted by ZM 241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by LPS-activated small interfering RNA (SiRNA) CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide is, in part, mediated by CD73-derived adenosine acting on A2A receptors. There is evidence that A2AAR activation beside anti-inflammatory effects promotes wound healing and extracellular matrix production; given that extracellular matrix and fibroblasts take an active part in the modulation of inflammation beside wound healing, we investigated whether and how extracellular matrix was involved in the anti-inflammatory effect of A2A receptor. Specifically, we evaluated changes in tissue fibroblast growth factor-2 (FGF-2), an important growth factor for fibroblasts that has been shown to facilitate not only tissue regeneration but also to dampen inflammation, following systemic administration of the A2A agonist, CGS 21680, in a rat model of acute inflammation (paw oedema). We observed that CGS 21680 prevented oedema development and inflammation, confirming an anti-inflammatory effect of A2AR. The effect of CGS 21680 was specific, through A2A adenosine receptor stimulation, as revealed by co-administration with ZM 241385 that reverted CGS 21680 inhibitory effect. On the basis of histological analysis showing an increased matrix deposition following rat treatment with CGS 21680, we evaluated whether the beneficial effect of A2A agonist, CGS 21680, was paralleled by changes in FGF-2 expression. Interestingly, we found that the expression of FGF-2 in rat paws, evaluated at each hour following carrageenan injection, was increased following rat treatment with CGS 21680. Immunofluorescence analysis confirmed data obtained by western blot and also showed spots of co-localization between A2AR and FGF-2. In conclusion, in this research work we demonstrate that CD73 regulates cell migration in the acute phase of inflammation and that the anti-inflammatory effects mediated by A2AR activation are paralleled by changes in extracellular matrix morphology. These findings suggest the important role of CD73/adenosine/A2A signalling in the control of the acute phase of inflammation, characterised by PMNs infiltration, but also in the control of a late phase, characterised by re-arrangement of extracellular matrix. In addition, we also demonstrate that CD73/adenosine/A2A axis is involved in the mode of action of nimesulide. Our study may open a path to re-evaluate the mechanism of action of nimesulide and to identify new therapeutic opportunities in COX-2 inhibitors which display a more potent activity on adenosine signalling. Furthermore, these results may give the cue to project an innovative anti-inflammatory strategy based on the manipulation of endogenous anti-inflammatory pathways.

This thesis presents a study of possible role of the extracellular adenosine signaling pathway in the regulation of metabolism of energetic reserves in Drosophila melanogaster. I focus on a study of metabolism of carbohydrate reserves and show a connection of adenosine signaling pathway to regulation of glucose homeostasis. Results of hemolymph and whole larval and pupal carbohydrates concentration measurements are shown together with concentration of other metabolites. They suggest that glucose homeostasis is impaired in the adgf-a mutant, which is accompanied by impaired metabolism of energetic reserves. The work is based on genetic screening for dominant suppressors of larval lethality caused by a loss of function mutation in the main larval adenosine deaminase, ADGF-A. Possible interactions of adenosine signaling pathway and regulation of immune response are disscussed.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Adenosine triphosphate (ATP) is best known as the key molecule in bioenergetics and in multiple cellular processes, but it is now well-recognized as an extracellular signalling molecule as well. ATP can signal directly through the activation of P2Rs or indirectly through P1Rs activated by adenosine formed upon the extracellular catabolism of ATP by ecto-nucleotidases. In the adult brain, purinergic receptors display a widespread regional and cellular distribution, fulfilling a neurotransmitter and neuromodulatory role, controlling astrocytic function or formatting microgliaresponsiveness. Purinergic signaling also plays a role in brain development ranging from neurogenesis to neuronal migration, neuritogenesis and synaptogenesis. Particularly regarding neuronal development, it has been shown that P2Rs bidirectionally regulates axonal growth, promoting axonal elongation through P2Y1R and inhibiting it through P2Y13R and P2X7R. A2ARs also promotes axonal growth and dendritic branching in ratcortical neurons. Unpublished data from our group showed that A2ARs is not only involved in axonal growth but also in axon formation. This was observed both in vitro inmice cortical neurons and in vivo, since it is required for cortical principal neurons migration in the transition from the intermediate zone (IZ) to the cortical plate (CP).The bidirectional regulation of axonal growth by P2Rs was shown to be mediated by a convergent but differential regulation of adenylate cyclase 5 and PI3K-Akt-GSK3βpathway. The A2AR-driven axonal outgrowth in rat cortical neurons was also shown to involve PI3K. Yet, how the purinergic receptors are able to control both dendritic and axonal development remains ill-defined. One protein that might be targeted and mediating the effects of purinergic receptors in neuronal development is collapsin response mediator protein 2 (CRMP2), a microtubule-associated protein shown to contribute to dendritogenesis and axon specification and outgrowth, and critical in cortical principal neurons migration, precisely in the IZ-CP transition. CRMP2 is negatively regulated by phosphorylation by two independent pathways, one mediated by cyclin-dependent kinase (Cdk5) that phosphorylates S522, priming the phosphorylation of T514 by GSK3β, and the other mediated by Rho kinase at T555, also a target of PKC. Thus, to start addressing the putative involvement of CRMP2 regulation in the effects induced by purinergic receptors, it was necessary to evaluate the involvement of each of these kinases namely PKC, GSK3 and Rho kinase.E18-derived rat hippocampal neurons cultured in the presence of the selective A2AR agonist, CGS21680 (30 nM), from day in vitro 0 (DIV0) to DIV3 displayed an increase in the number of axons per neuron and longer axons, an effect not observed in cellselectroporated with shRNA-A2AR. Thus, in contrast to the observed in mice cortical neurons, where A2AR is tonically necessary for the formation of the normal axon, in rathippocampal neurons A2ARs are not involved in the formation of the normal axon.Instead, the exogenous activation of A2AR induces the formation of secondary axons. Moreover, A2ARs activation decreased dendritic length. Preliminary data indicate that all these effects depend on BDNF activity. Furthermore, we observed that P2Y1R does notcontrol axon formation, but it promotes axon outgrowth. The agonist of P2Y1, P2Y12 and P2Y13 receptors, ADPβS (5 μM), and the P2Y1R selective agonist, MRS2365 (100 nM), induced an increase in axon length, while the P2Y1R antagonist, MRS2179 (10 μM), caused a decrease in axon length, showing a tonic action of P2Y1R controlling axon outgrowth. The pharmacological blockade of P2X7R with BBG (100 nM) increased axonal length, indicating that P2X7R is tonically inhibiting axonal growth also in rat hippocampalneurons whilst not modifying dendritic morphology, as previously reported.All these effects were then evaluated in the presence of the kinase inhibitors. Itwas found that A2AR-driven axon elongation involves GSK3, whereas axon formation induced by A2AR was prevented in the presence of PKC and Rho kinase inhibitors. The inhibition of dendritic growth by A2ARs activation depends on PKC and Rho kinase. P2Y1Rpromotes axonal outgrowth through a PKC- and GSK3-dependent mechanism, whereas the inhibition of dendritic growth involves PKC- and Rho kinase.The identification of the enzymes involved in the control of dendritic and axonal development by A2AR- and P2Y1R - support and set the grounds to characterize the involvement of CRMP2 and hence fully identify the signaling pathways recruited by purinergic signaling in the control of neuronal development. Besides, this study also revealed region-specificity in the control of neuronal development by purinergic signalling that is now mandatory to be fully elucidated.
Adenosina trifosfato (ATP) tem um papel central como molécula energética e em múltiplos processos celulares, mas também desempenha um papel na sinalização extracelular. O ATP pode sinalizar ou pela ativação direta dos recetores P2 (P2Rs) ou através dos recetores P1 (P1Rs) ativados pela adenosina formada a partir do catabolismo extracelular de ATP pelas ecto-nucleotidases. No cérebro adulto, os recetores purinérgicos apresentam uma distribuição generalizada, contribuindo para afisiologia sináptica astrocítica e da microglia. A sinalização purinérgica também tem um papel no desenvolvimento do cérebro, nomeadamente na neurogénese, migração neuronal, neuritogénese e sinaptogénese. Em relação ao desenvolvimento neuronal, foi mostrado que os P2Rs podem regular bidireccionalmente o crescimento axonial, aumentando através dos P2Y1R e inibindo através dos P2Y13R e P2X7R. Os A2AR também promovem o crescimento axonial e a ramificação das dendrites em neurónios corticais de rato. Resultados não publicados do nosso grupo mostram que os A2AR estão também envolvidos na formação do axónio. Isto foi observado quer in vitro em neurónios corticais de ratinho, quer in vivo, sendo um processo necessário para a migração dos neurónios corticais principais na transição da zona intermédia (IZ) para a placa cortical (CP).Relativamente às vias de sinalização envolvidas, foi mostrado que a regulação bidirecional do crescimento axonial pelos P2Rs é mediada pela regulação convergente mas diferencial da adenilato ciclase 5 e da via PI3K-Akt-GSK3β. Foi mostrado também que o crescimento promovido pelos A2AR envolve PI3K. Contudo ainda não se sabe qual o(s) mecanismo(s) a jusante que medeia(m) o controlo por parte dos recetores purinérgicos do desenvolvimento das dendrites e axónios. Uma proteína que pode estar envolvida é a proteína collapsin response mediator protein 2 (CRMP2), uma proteínaassociada aos microtúbulos envolvida na dendritogénese e na especificação e crescimento axonial, e essencial na migração dos neurónios corticais principais, precisamente na transição IZ-CP. A CRMP2 é regulada negativamente por fosforilação através de duas vias independentes, uma mediada pela enzima cdk5 que fosforila o resíduo S522, que permite a fosforilação de T514 pela GSK3β, e a outra mediada pela Rho cinase no resíduo T555, que também pode ser fosforilada pela PKC. Para avaliar o possível envolvimento da CRMP2, foi neste estudo avaliado o envolvimento de cada um destas enzimas cinases, nomeadamente PKC, GSK3 e Rho cinase, nos efeitos induzidos pelos recetores purinérgicos.Em culturas primárias de neurónios de hipocampo de rato derivados de embriões E18, a exposição ao agonista seletivo dos A2AR, CGS21680 (30 nM), desde o dia in vitro 0 (DIV0) até DIV3 induziu um aumento no número de axónios e no seu comprimento, um efeito que não foi observado em neurónios eletroporados com shRNA-A2AR. Portanto, ao contrário do observado em neurónios corticais de ratinho, os A2AR não estão envolvidos na formação do axónio normal em neurónios de hipocampo de rato. Em vez disso, a ativação exógena dos A2AR promove a formação de axónios secundários. Em relação às dendrites, a ativação dos A2AR diminuiu o o seu comprimento. Resultados preliminares mostram que todos estes efeitos dependem de BDNF. Em relação ao P2Y1R, os resultados obtidos indicam que este recetor não controla a formação do axónio, mas promove o seu crescimento. O agonista dos recetores P2Y1,12,13, ADPβS (5 μM), e o agonista seletivo de P2Y1, MRS2365 (100 nM), induziram um aumento no comprimento do axónio, enquanto que o bloqueio farmacológico dos P2Y1R, com MRS2179 (10 μM), induziu uma diminuição no comprimento do axónio, demonstrando uma ação tónica dos P2Y1R no crescimento axonial. O bloqueio farmacológico do P2X7R com BBG (100 nM) aumentou o comprimento dos axónios, indicando uma inibição tónica do crescimento axonial, nãotendo modificado a morfologia das dendrites.Relativamente ao envolvimento das diferentes enzimas, os resultados demonstraram que o crescimento axonial induzido pela ativação dos A2AR envolve GSK3, enquanto que a formação de axónios secundários foi prevenida pela presença de inibidores da PKC e da Rho cinase. A inibição do crescimento das dendrites induzido pelo A2AR depende de PKC e Rho cinase. O P2Y1R promove o crescimento axonial por um mecanismo dependente de PKC e GSK3, enquanto que a inibição do crescimento das dendrites envolve PKC e Rho cinase.A identificação destas enzimas envolvidas no controlo do desenvolvimento de dendrites e axónios pelos A2AR e P2Y1R suportam a hipótese de haver um envolvimento de CRMP2 no controlo do desenvolvimento neuronal pelos recetores purinérgicos. Para além disso definem como poderá ser feita essa regulação, que terá que ser avaliadaexperimentalmente. Além disso, este estudo revelou que existem diferenças no controlo do desenvolvimento neuronal pelo sistema purinérgico em diferentes regiões que também será importante elucidar.
Outro - Esta dissertação foi financiada pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do Programa Operacional Regional Centro 2020 sob o projeto CENTRO-01-0145-FEDER-000008: BrainHealth2020, através do COMPETE 2020 - Programa Operacional Competitividade e Internacionalização e através de fundos nacionais via FCT - Fundação para a Ciência e a Tecnologia sob projetos POCI-01-0145-FEDER-028160 e UID/NEU/04539/2019.