Le purinorécepteur P2X7 joue un rôle majeur dans les phénomènes de dégénérescences (Alzheimer, DMLA) et mort cellulaires. Récemment des études ont montré que l’activation basale de ce récepteur est indispensable dans le processus de cicatrisation et de prolifération cellulaire. Nous avons essayé de mieux comprendre le paradoxe de cette activation du récepteur P2X7 qui oriente vers la prolifération et même les métastases tumorales, mais aussi vers les mécanismes de dégénérescence cellulaire. L'influence du microenvironnement (Lipides, matrice extracellulaire, oxygène) apparait essentielle pour comprendre ces différents effets. Notre premier objectif a été d’étudier l’impact de la modulation du microenvironnement du récepteur P2X7 par des composants de la matrice extracellulaire. Ainsi, sur un modèle de cicatrisation cutanée, nous avons mis en évidence l’impact de la taille des fragments du hyaluronate de sodium (composant prédominant dans la matrice extracellulaire). Nos résultats ont montré que l’activation du récepteur P2X7 dépend du poids moléculaire du hyaluronate de sodium. Notre deuxième objectif a été de moduler le microenvironnement lipidique du récepteur P2X7. Nous avons sélectionné une huile riche en acides gras insaturés et avons ainsi étudié son effet sur l’activation du récepteur P2X7. Sur nos modèles de cicatrisation, nous avons mis en évidence qu’une modulation du microenvironnement lipidique du récepteur P2X7 influence son activation. D’autre part la modulation du microenvironnement lipidique sur des cellules tumorale de mélanome active les voies de dégénérescence ouvrant la perspective de nouvelles stratégies thérapeutiques. / The purinoceptor P2X7 plays a role in cytotoxic degenerative processus (Alzheimer, AMD) and cell death. Recent studies have shown that basal activation of this receptor is essential in the healing process and cell proliferation. We tried to understand the paradox that activation of P2X7 receptor that directs to the same proliferation and tumor metastasis, but also to the mechanisms of cell degeneration. The influence of the microenvironment (lipids, extracellular matrix, oxygen) appears essential to understand these effects. Our first objective was to study the impact of the modulation of P2X7 receptor microenvironment by extracellular matrix components. On cell monolayer model of wound healing we have highlighted the impact hyaluronan molecular weight (predominant component in the extracellular matrix). Our results showed that activation of P2X7 receptor is dependent on hyaluronan molecular weight. Our second objective was to modulate lipid microenvironment P2X7 receptor. We selected an oil rich in unsaturated fatty acids and thus have studied its effect on the activation of P2X7 receptor. In our models of healing and melanoma cell, we have demonstrated that modulation of lipid microenvironment affects P2X7 activation.

Der ATP-getriggerte Ionenkanal P2X7 ist als purinerger Oberflächenrezeptor besonders auf Zellen des Immunsystems und auf Gliazellen im Nervensystem exprimiert. Seine Aktivierung führt zur Freisetzung proinflammatorischer Zytokine, zur Bildung reaktiver Sauerstoffspezies sowie zu einer Beeinflussung des Zellzyklus. Zwar konnte eine Beteiligung des Rezeptors an verschiedenen entzündlichen und degenerativen Erkrankungen nachgewiesen werden, allerdings bestehen nach wie vor viele Unstimmigkeiten darüber, ob P2X7 im Einzelfall protektiv oder schadend wirkt. Eine therapeutische Modulation des Rezeptors gestaltet sich daher bis heute schwierig. Weiterhin wurde trotz intensiver Bemühungen um selektive, potente P2X7-Modulatoren bisher kein Wirkstoff über Phase-II-Studien hinaus entwickelt. Der erste Teil der Arbeit beschreibt eine Studie zur Identifikation neuer P2X7-Modulatoren und deren Charakterisierung hinsichtlich Potenz, Bindeverhalten und Speziesspezifität. Ziel dieser Studie war es, die Basis für die Entwicklung möglicher neuer Therapeutika zu legen, für die ein hoher Bedarf besteht. Im zweiten Teil der Arbeit wurde die Beteiligung des P2X7-Rezeptors an den pathophysiologischen Vorgängen nach einem Hirninfarkt untersucht. Besondere Aufmerksamkeit lag dabei auf dem Einfluss, den der Rezeptor auf die Bildung eines begleitenden, oftmals fatalen Hirnödems ausübt. In einer Wirkstoffbibliothek enthaltene zugelassene Pharmaka und Naturstoffe wurden auf ihre Wirksamkeit am rekombinant exprimierten humanen P2X7-Rezeptor (hP2X7) getestet. Dazu wurde gemessen, inwiefern diese Wirkstoffe den P2X7-vermittelten Ca2+-Einstrom modulieren können. Für potenziell selektive Substanzen wurden Konzentrations-Wirkungs-Kurven erstellt. Für den potenten Inhibitor Tanshinon II A-Sulfonat (TIIAS) und den chemisch verwandten Wirkstoff Tanshinon II A (TIIA) erfolgte diese Untersuchung auch an den rekombinant exprimierten P2X7-Rezeptoren von Maus (mP2X7) und Ratte (rP2X7). Weiterhin erfolgte eine detaillierte, auf elektrophysiologischen Untersuchungen basierende Darstellung der pharmakodynamischen Eigenschaften von TIIAS. Die Selektivität der Wirkung gegenüber P2X2 und P2X4 wurde mithilfe entsprechender Zelllinien geprüft. Die Wirkung modulierender Pharmaka am nativen Rezeptor wurde in humanen, aus peripheren Blutmonozyten gereiften Makrophagen überprüft, wozu neben der Darstellung des Ca2+-Einstroms auch ein IL-1β-ELISA eingesetzt wurde. In allen Experimenten wurde die Beteiligung von P2X7 über bekannte Antagonisten verifiziert. Um zu klären, inwiefern P2X7 die pathophysiologischen Abläufe nach Hirninfarkt beeinflusst, wurde bei 20 P2X7-defizienten Mäusen (P2X7-/-) und bei 22 zugehörigen Wildtyp-Mäusen (WT) eine zerebrale Ischämie induziert, indem die mittlere Zerebralarterie mit einem dünnen Faden für 60 Minuten transient verschlossen wurde (middle cerebral artery occlusion, MCAO). In den folgenden 72 Stunden wurde über klinische Methoden und Magnetresonanzuntersuchungen die Entwicklung neurologischer Defizite, der Infarktgröße und des begleitenden Hirnödems evaluiert. Nach schmerzloser Tötung und Hirnentnahme wurden immunhistologisch die Aktivierung und Verteilung von Mikroglia und Astrozyten sowie der Zustand des Gefäßendothels untersucht. Sham-operierte Tiere dienten in allen Experimenten als Kontrollen. TIIAS hemmte hP2X7 mit einer IC50 von 4.3 μM, während die Potenz an mP2X7 geringer war und rP2X7 kaum geblockt wurde. TIIA modulierte P2X7 nicht. TIIAS hemmte als allosterischer Antagonist die Öffnung des Ionenkanals und band vermutlich an eine intrazelluläre Bindestelle. Die Wirkung von TIIAS wurde in humanen Makrophagen bestätigt, in denen der Wirkstoff den Ioneneinstrom und die IL-1β-Freisetzung hemmte. Obwohl die neurologische Untersuchung von P2X7-/-- und WT-Mäusen nach MCAO keine signifikanten Unterschiede ergab, zeigte sich in der bildgebenden Diagnostik, dass P2X7-/--Mäuse binnen 24 Stunden nach der OP ein signifikant stärkeres Hirnödem entwickelten, welches nicht durch Unterschiede in der Infarktgröße bedingt war. Der Infarkt führte in beiden Gruppen zu einer Gliaaktivierung, die im Fall der Mikroglia in Abwesenheit von P2X7 allerdings reduziert war. Differenzen hinsichtlich der Aktivierung von Astrozyten und der Expression von Laminin im Kapillarendothel wurden nicht festgestellt. Im Gegensatz zu TIIA, das häufig als gleichwertiger Wirkstoff eingesetzt wird, blockt TIIAS hP2X7 speziesspezifisch mit einer hohen Potenz. Maus und Ratte scheiden aufgrund der geringen Wirkung von TIIAS leider als Tiermodelle aus, um die Wirkung von TIIAS in vivo zu prüfen. Weitere Arbeiten sind notwendig, um die Potenz von TIIAS in anderen Spezies zu evaluieren oder Alternativen zum Tierversuch zu finden und eine mögliche therapeutische Anwendung bei Erkrankungen mit P2X7-Beteiligung zu testen. P2X7 beeinflusst die pathophysiologischen Vorgänge nach einem Hirninfarkt und begrenzt die Entwicklung eines zytotoxischen Hirnödems, nicht aber die des vasogenen Hirnödems, das sich zeitversetzt einstellt. Eine mögliche Erklärung für diesen Sachverhalt bieten die unterschiedlichen Funktionen, die Gliazellen zu verschiedenen Zeitpunkten nach zerebraler Ischämie übernehmen. Unsere Ergebnisse deuten auch darauf hin, dass verschiedene Tiermodelle des zerebralen Infarkts nicht in allen Punkten vergleichbar sind.:Inhaltsverzeichnis 1 Einleitung........................................................................................................... 1 1.1 Purinerge Signaltransduktion.......................................................................... 1 1.2 P2X-Rezeptoren.............................................................................................. 2 1.3 Pharmakologie des P2X7-Rezeptors............................................................... 3 1.4 Physiologische und pathophysiologische Bedeutung des P2X7-Rezeptor...... 5 1.5 Gegenstand dieser Arbeit............................................................................... 7 2 Veröffentlichungen............................................................................................. 9 2.1 Erste Publikation............................................................................................. 9 2.1.1 Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7................................. 9 2.1.2 Ergänzende Materialien zur ersten Publikation.......................................... 22 2.2 Zweite Publikation......................................................................................... 30 2.2.1 Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery................................................................................. 30 2.2.2 Ergänzende Materialien zur zweiten Publikation......................................... 42 2.2.3 Erratum to: Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery occlusion............................................ 46 3 Diskussion........................................................................................................ 49 4 Zusammenfassung........................................................................................... 55 5 Summary.......................................................................................................... 57 6 Literaturverzeichnis.......................................................................................... 59 7 Danksagung..................................................................................................... 66 / ATP-gated ion channel P2X7 is a purinergic cell surface receptor which is mainly expressed on immune and glia cell. Upon activation of P2X7, proinflammatory cytokines are released, reactive oxygen species are generated and the cell cycle may be altered. In this regard, it has been shown that P2X7 plays a role in diseases such as rheumatoid arthritis, Alzheimer’s disease and multiple sclerosis. However, results regarding protective or detrimental effects mediated by P2X7 under particular conditions are often inconsistent. Thus, up to now, any therapeutic modulation of the receptor remains a challenge. Although intensive research has been conducted to find selective, potent P2X7-modulators, no active compound has been developed beyond phase II clinical trials. The first part of this work describes a study realized to identify new P2X7 modulators and to characterize them in terms of pharmacodynamic properties like potency and species specificity. This study was aimed at providing a basis for the development of new therapeutic agents, which are urgently needed. During the second part of this work, the involvement of P2X7 in pathophysiological processes after cerebral infarction was examined. Particular attention was paid to the influence of the receptor on the development of an accompanying and often fatal brain edema. A compound library containing approved drugs and natural compounds was screened for modulators of the recombinantly expressed human P2X7 receptor (hP2X7). Therefor, their effect on P2X7-mediated Ca2+ influx was evaluated. Concentration-response-curves were established for potentially selective compounds. Tanshinone II A sulfonate (TIIAS) turned out to be a potent inhibitor of P2X7. Both TIIAS and tanshinone II A (TIIA), the natural compound TIIAS has been derived from, were also tested on recombinantly expressed mouse and rat P2X7 (mP2X7 and rP2X7, respectively). Furthermore, electrophysiological assays were conducted for a detailed characterization of mechanisms of P2X7 inhibition. Antagonist selectivity was revised using cell lines expressing purinergic receptors P2X2 and P2X4. Human monocyte-derived macrophages were used in fluorometric calcium and dye-uptake assays as well as an IL-1ß ELISA to evaluate the effects of modulating compounds on native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. In order to evaluate whether modulation of P2X7 may affect the outcome after cerebral infarction, cerebral ischemia was induced in 20 P2X7-deficient mice (P2X7-/-) and 22 mice of their corresponding wild type (WT) by transiently occluding their middle cerebral artery for 60 minutes with a thin filament (middle cerebral artery occlusion, MCAO). During 72 hours following surgery, neurological deficits, infarct size and edema development were monitored, applying clinical examinations and magnetic resonance measurements. After humane killing and brain removal, different antibodies were used in order to evaluate the distribution and activation state of microglia and astrocytes as well as the condition of the vascular endothelium. Sham-operated animals were used as negative controls in all experiments. TIIAS blocked hP2X7 with an IC50 of 4.3 μM, whereas it proved to be less potent at mP2X7 and poorly modulated rP2X7. TIIA did not modulate P2X7. TIIAS acted as an allosteric antagonist and reduced the opening of the ion channel; it presumably bound to an intracellular binding site. The effect of TIIAS could be confirmed in human macrophages. In these cells, TIIAS inhibited the ATP-induced Ca2+ entry, dye-uptake and release of IL-1β. Although neurological examinations did not reveal significant differences between P2X7-/- and WT mice that underwent MCAO, diagnostic imaging revealed that P2X7-/- mice developed significantly more severe brain edema within 24 hours after surgery, a development that was not due to differences in infarct sizes. Both groups displayed clear signs of activation of glia cells, but only microglia activation was attenuated in the absence of P2X7. Differences regarding the activation state of astrocytes or the expression of laminin by capillary endothelial cells could not be detected. TIIAS species specifically blocks hP2X7 with a high potency. TIIA does not convey this effect although both compounds are frequently used interchangeably. Due to the low potency TIIAS displays at mP2X7 and rP2X7, these species unfortunately cannot be used as animal models to evaluate the drug’s effect in vivo. Further research is necessary to evaluate the potency of TIIAS at other species’ P2X7 receptors or to find alternativespurinerge Signaltransduktion, P2X7, Tanshinon II A-Sulfonat, zerebrale Ischämie, Hirnödem to animal testing in order to study possible therapeutic applications of TIIAS in P2X7-related diseases. P2X7 does affect pathophysiological events following cerebral ischemia and restricts cytotoxic brain edema development, but does not limit vasogenic cerebral edema formation, which develops at a later stage of the disease. The different functions fulfilled by glia cells at distinct points in time after infarction may provide an explanation for this interesting fact. The results presented also imply that diverse animal models of cerebral ischemia may not be entirely comparable due to differences regarding the pathogenesis of brain edema.:Inhaltsverzeichnis 1 Einleitung........................................................................................................... 1 1.1 Purinerge Signaltransduktion.......................................................................... 1 1.2 P2X-Rezeptoren.............................................................................................. 2 1.3 Pharmakologie des P2X7-Rezeptors............................................................... 3 1.4 Physiologische und pathophysiologische Bedeutung des P2X7-Rezeptor...... 5 1.5 Gegenstand dieser Arbeit............................................................................... 7 2 Veröffentlichungen............................................................................................. 9 2.1 Erste Publikation............................................................................................. 9 2.1.1 Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7................................. 9 2.1.2 Ergänzende Materialien zur ersten Publikation.......................................... 22 2.2 Zweite Publikation......................................................................................... 30 2.2.1 Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery................................................................................. 30 2.2.2 Ergänzende Materialien zur zweiten Publikation......................................... 42 2.2.3 Erratum to: Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery occlusion............................................ 46 3 Diskussion........................................................................................................ 49 4 Zusammenfassung........................................................................................... 55 5 Summary.......................................................................................................... 57 6 Literaturverzeichnis.......................................................................................... 59 7 Danksagung..................................................................................................... 66

Estudos têm demonstrado que o microambiente tumoral é rico em ATP e adenosina, sugerindo o envolvimento da sinalização purinérgica no desenvolvimento e/ou manutenção do câncer. Ainda, o receptor purinérgico P2X7, conhecido pelo seu papel na indução de apoptose, encontra-se reduzido em alguns tecidos tumorais em comparação aos tecidos saudáveis, indicando que a sua redução possa ser um mecanismo de resistência celular à apoptose. Dessa forma, compreender o papel da sinalização purinérgica no contexto do câncer se torna indispensável e permite que novas abordagens terapêuticas sejam implementadas. Nesse trabalho, avaliamos a função dos nucleotídeos e nucleosídeos da adenina, bem como do receptor P2X7 na indução da morte celular em células de câncer cervical. Também verificamos o efeito do heat shock na potencialização da atividade do receptor P2X7 frente à curta exposição ao ATP em células de câncer de cólon. De acordo com os nossos resultados, o efeito citotóxico do ATP extracelular nas linhagens de câncer cervical é mediado principalmente pela ação do seu metabólito adenosina, que ao entrar no interior das células, promove o aumento dos níveis intracelulares de AMP, ativação de AMPK, aumento da p53 e indução de autofagia. O papel do receptor P2X7 nesse contexto parece ser apenas coadjuvante, visto que o seu bloqueio ou silenciamento impediu em apenas 20% a morte celular. Além disso, utilizando células de câncer de cólon, nós demonstramos que o heat shock aumenta a funcionalidade do receptor P2X7, independente da interação com heat shock proteins ou canais do tipo conexina/panexina, potencializando o efeito citotóxico do ATP. Esse efeito parece estar relacionado à mudanças na composição e arquitetura da membrana celular, visto que o uso do agente fluidizador de membrana benzil álcool foi capaz de mimetizar o efeito do heat shock na potencialização do receptor P2X7 a 37ºC. Este estudo fornece evidências adicionais sobre o papel da sinalização purinérgica no contexto da biologia celular tumoral e abre novas perspectivas para o uso dos nucleotídeos de adenina associados a hipertermia como agentes adjuvantes na terapia do câncer. / The tumor microenvironment is rich in ATP and adenosine, suggesting an involvement for purinergic signaling in cancer development and surveillance. The P2X7 receptor, among the P2 purinergic receptors, is broadly recognized as the “death receptor”, because it promotes cell apoptosis when exposed to high levels of extracellular ATP. Researches have been shown that P2X7 protein levels are decreased at the tumor site in comparison to adjacent healthy tissue, suggesting a mechanism of tumor escape to cell death. Thus, understanding purinergic signaling in a cancer context becomes urgent and opens a new field for therapeutic strategies. Here, we evaluated adenine nucleotides and nucleosides cytotoxicity, as well as P2X7 role in cell death induction using cervical cancer cell lines. Indeed, we investigated heat shock effect on P2X7 functionality through exposing colon cancer cell shortly to ATP at 40ºC. According to our data, adenosine uptake formed from ATP metabolism is the main responsible for the extracellular ATP cytotoxicity in cervical cancer cells. While inside of the cell, adenosine is converted to AMP, leading to AMPK activation, p53 increase and autophagy induction. ATP induced cell death per se through P2X7 in this context seems to be less important, since P2X7 blockage or knocking down reduced only 20% of cell death. In colon cancer cells, we found that heat shock stress was able to increase P2X7 pore formation independently of heat shock protein interaction or native pore-forming transporters association (e.g pannexin-or connexin-type channels), thus leading to an increase ATP cytotoxicity. The mechanism enrolled in this process seems to be related to changes in the lipid composition and architecture of membrane, as the membrane fluidizer benzyl alcohol could reproduce heat stress effect in potentiating P2X7 activation at 37ºC. In conclusion, our work provides further evidence for a purinergic signaling role in the cancer biology context and opens new perspectives for the utility of purine-based drugs associated to hypertermia as adjunctive agents in cancer therapy.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

<p>Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) V_H genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated V_H genes display superior survival compared to unmutated cases. Biased V_H gene usage has also been reported in CLL which may reflect antigen selection.</p><p>We performed V_H gene analysis in 265 CLL cases and confirmed the prognostic impact of the V_H mutation status. Preferential V_H gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular V_H gene, V_H3-21, displayed poor outcome despite that two-thirds showed mutated V_H genes. Many of the V_H3-21 cases expressed λ light chains, rearranged a V_λ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the V_H3-21 subset comprises an additional CLL entity.</p><p>To further explore the B-cell receptors in CLL, we analyzed the V_H gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted V_L gene usage as in the V_H3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL.</p><p>Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. <2%, 2-5% and >5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. </p><p>A 1513A/C polymorphism in the P2X₇ receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated V_H genes.</p><p> In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.</p>

Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) VH genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated VH genes display superior survival compared to unmutated cases. Biased VH gene usage has also been reported in CLL which may reflect antigen selection. We performed VH gene analysis in 265 CLL cases and confirmed the prognostic impact of the VH mutation status. Preferential VH gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular VH gene, VH3-21, displayed poor outcome despite that two-thirds showed mutated VH genes. Many of the VH3-21 cases expressed λ light chains, rearranged a Vλ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the VH3-21 subset comprises an additional CLL entity. To further explore the B-cell receptors in CLL, we analyzed the VH gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted VL gene usage as in the VH3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL. Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. &lt;2%, 2-5% and &gt;5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. A 1513A/C polymorphism in the P2X7 receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated VH genes. In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.

Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.

Le récepteur ionotropique P2X7 est impliqué dans diverses fonctions physiologiques telles que la prolifération, l’apoptose, la réponse inflammatoire et le trafic membranaire dans plusieurs types cellulaires. Cependant, peu est connu quant aux rôles physiologiques de P2X7 dans les cellules épithéliales intestinales (CEIs). Dernièrement, un collègue a démontré une nouvelle fonction pour P2X7 qui est de réguler l’internalisation du transporteur à glucose GLUT2. Comme l’un des rôles physiologiques majeurs des CEIs est l’absorption du glucose, la régulation de ce transporteur est d’une importance capitale. Par contre, nous ne savons pas ce qui permet de réguler l’expression du récepteur P2X7 dont la disponibilité pourrait être un des facteurs déterminants dans la régulation de l’absorption de glucose par le transporteur GLUT2. Ceci nous a menés à l’hypothèse suivante qui dit que le glucose lui-même pourrait stimuler certaines voies de signalisation menant à l’activation de facteurs de transcription, qui pourraient réguler l’expression du récepteur P2X7. Les objectifs de mes travaux de maîtrise étaient de déterminer si des variations dans la concentration de glucose affectaient l’expression de P2X7 dans les CEIs et d’identifier les facteurs de transcription impliqués dans ce processus. Les résultats obtenus démontrent que l’expression de P2X7 semble être affectée par la concentration de glucose. Cette régulation de l’expression de P2X7 en réponse au glucose semble impliquer les facteurs de transcription C/EBP? et ß. En fait, les essais d’immunoprécipitation de la chromatine (ChIP) effectués confirment que ces facteurs pouvaient lier le promoteur de P2X7 et que la liaison de C/EBPß au promoteur varie en fonction de la concentration de glucose. La capacité de liaison de C/EBPß au promoteur est comparable au niveau d’expression du gène du récepteur P2X7. In vivo, nous avons observé une diminution de l’expression du récepteur P2X7 dans les extraits de jéjunum de souris invalidées pour C/EBPß. Finalement, dans le modèle de souris diabétique NOD, il semble y avoir un dérèglement dans l’expression du récepteur P2X7, ce qui pourrait indiquer que P2X7 a bel et bien un rôle dans la régulation de l’homéostasie du glucose. [symboles non conformes]

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

Made available in DSpace on 2016-04-20T12:39:27Z (GMT). No. of bitstreams: 2 rafael_soares_ioc_mest_2015.pdf: 5479188 bytes, checksum: f6ce23593dd41a10564934ddfa4bd278 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O receptor P2X7, contido na família de receptores P2, é um receptor inotrópico purinérgico, cátion seletivo, ativado por nucleotídeos (ATP). Esses receptores são expressos em células do sistema nervoso central, periférico e células da linhagem hematopoiética além de estarem envolvidos em diversos processos biológicos como a transdução de sinais da dor, participação na inflamação granulomatosa, dentre outros. Dos seus sete subtipos (P2X1 \2013 P2X7) o P2X7 possui a característica de formar um poro cuja condutância é aumentada em aproximadamente 26 vezes em relação à condutância padrão de todos os receptores da família P2X (~15 pS). Este aumento na condutância se reflete em possibilitar a passagem de moléculas de até 900 Da. Este estudo propõe através da aplicação das técnicas de modelagem comparativa e dinâmica molecular analisar o comportamento dinâmico de um modelo do receptor P2X7 humano, desde seus movimentos de fechamento e abertura do canal de baixa condutância, até a passagem de íons catiônicos (Potássio) e aniônicos (cloreto) para avaliar a seletividade do canal. Isto é necessário pois ainda não se dispõe de um modelo experimental (Ressonância Magnética Nuclear ou Cristalografia de raio-X) e ainda existem dúvidas sobre o mecanismo de ativação que elucide o processo de transição entre o estado de menor condutância (um canal aberto) para o estado de maior condutância (formação de um poro) Foram construídos dois modelos do receptor P2X7 por meio de modelagem comparativa utilizando como molde o receptor P2X4 do organismo Danio rerio (único molde viável até o momento) em seus estados aberto e fechado. As simulações de dinâmica molecular revelaram o tempo de fechamento de canal (região transmembranar) e o rearranjo do sítio de ligação ao ATP em ~400ps e ~650ps, respectivamente. As dinâmicas de passagem dos íons confirmaram as características cátion seletivas do canal, visto que foi possível observar a passagem de todos os íons de potássio dispostos na região de estreitamento do canal e também foi observada a expulsão de uma das três dispostas na mesma região que os íons catiônicos avaliados. Estes resultados ressaltam que os sistemas simulados para avaliar o comportamento do receptor P2X7 descrevem comportamentos semelhantes aos descritos experimentalmente ressaltando a possibilidade de uso dos modelos construídos neste trabalho para aplicação em estudos piloto futuros (virtual screening, docking molecular) para avaliação de compostos promissores que atuem como agonistas ou antagonistas mais seletivos que os compostos existentes atualmente / The P2X7 is an inotropic, purinergic, cation-selective and activated by nucleotides (ATP) receptor that belongs to the P2 receptor family. These receptors are found in central and peripheral nervous system cells and in hematopoietic lineage cells. In addition, P2X7 receptors are involved in a vast number of biological processes such as pain signal transduction, participation in granulomatous inflammation, among others. From all seven subtypes (P2X1 \2013 P2X7), P2X7 receptor presents the characteristic of forming a pore whose conductance increases approximately 26 times more than the standard conductance from all others P2X receptors (~15 pS). This rise in the conductance allows the entrance of molecules up to 900 Da. The aim of this study is to employ comparative modeling and molecular dynamics methods to analyze the dynamic behavior of the human P2X7 receptor to assess the channel selectivity, regarding the opening and closing movements and the passage of cations (potassium) and anions (chloride) across the low conductance channel). The combination of these methods is necessary since there is no model determined by experimental methods such as Nuclear Magnetic Resonance or X-ray Crystallography, and doubts remain about the activation mechanism that drives the receptor form a state of low conductance (open channel) to a state of high conductance (pore formation) Two P2X7 receptor models were built by means of comparative modeling using as template the 3D structure of P2X4 receptor from Danio rerio organism in its open and close states. Molecular dynamics simulations revealed the channel closing time (transmembrane region) and the rearrangement of the ATP binding site took place, in ~400 ps and ~650 ps, respectively. The ion entry simulations confirmed the cation selectivity characteristics of the channel, since it was possible to observe the entrance of all potassium molecules present in the narrowed region of the channel. Furthermore, it was noted the expelling of the chloride molecules at the same regions. These results highlight that the P2X7model reproduce its experimental behavior. It is possible to describe similar mechanisms described experimentally; emphasizing the prospect of using these models in future studies (virtual screening, molecular docking) to evaluate lead compounds, capable of acting as more selective agonists or antagonist than the current available ones