<div> <p>Fluorescent proteins and the biosensors created with them have been used extensively to monitor chemical species inside and outside of the cell. They have been used to increase our knowledge of cellular function in normal and diseased states. Fluorescent biosensors are advantageous because they can be genetically encoded, do not require exogenous reagents, and can be quantitative. Fluorescent biosensors are also able to measure analytes with high spatial and temporal resolutions, enabling measurements at the scale of physiological events. In this thesis efforts have made to increase the available fluorescent biosensor tools for imaging cellular events. This work includes creation of new sensors for two molecules not yet detectable via fluorescent protein biosensor, acetylcholine and adenosine diphosphate. Efforts were also made to improve the current available biosensors for adenosine triphosphate and cellular redox, to make them more compatible with multiplex and deep tissue imaging. Here I present my work to design, characterize and utilize these fluorescent biosensors.</p> </div> <br>

Vários estudos têm apontado a sinalização e os receptores purinérgicos, representados em mamíferos pelos receptores ionotrópicos (P2X1 P2X7) e metabotrópicos (P2Y1,2,4,6, 11,12,14), como um sistema primitivo, envolvido não somente na sinalização neuronal, mas também em muitos outros processos vitais incluindo resposta imune, inflamação, dor, agregação plaquetária e nos processos de diferenciação, proliferação e morte celular, que ocorrem no desenvolvimento e na regeneração tecidual. Condizente com as descrições da literatura, dados do nosso laboratório, baseados na farmacologia dos receptores purinérgicos, sugeriram o envolvimento do subtipo P2Y2 na proliferação e na neurogênese in vitro de células de carcinoma embrionário P19. Tendo em vista a ausência de agonistas e antagonistas específicos para a maioria dos subtipos de receptores purinérgicos, o que vem dificultando a elucidação das funções exatas desses receptores em processos fisiológicos e patológicos, optamos para o screening de uma biblioteca combinatória de oligonucleotídeos para a identificação de ligantes de alta afinidade e especificidade para o receptor P2Y2 (procedimento de SELEX, Evolução Sistemática de Ligantes por Enriquecimento Exponencial). Essa abordagem envolve passos reiterativos de seleção in vitro de moléculas de RNA, estabilizadas por substituição do grupo 2´OH das pirimidinas por um átomo de flúor, que possuem afinidade pelo receptor, até que a mistura de RNAs, originalmente de 1013 diferentes seqüências que adotam uma gama de estruturas secundárias e terciárias, esteja purificada para uma população homogênea de ligantes de alta afinidade pelo receptor P2Y2. O processo envolve a transcrição in vitro da biblioteca de DNA para RNA, a apresentação desta ao alvo, a eluição dos ligantes específicos, denominados aptâmeros, e a regeneração da biblioteca de DNA por RT-PCR, a qual, após uma reação de transcrição in vitro, gera a mistura de RNAs para o próximo ciclo de seleção. Neste trabalho, nós utilizamos como alvo o receptor P2Y2 recombinante humano expresso na linhagem de células de astroglioma humano 1321N1. Ao final de nove ciclos de SELEX, nós isolamos 46 sequências que foram agrupadas em três classes estruturais, de acordo com a presença de regiões consensos. A mistura destas moléculas se ligou ao receptor P2Y2 humano com uma constante de dissociação de 164 nM. Um dos clones isolados, o aptâmero B7, se ligou preferencialmente ao receptor P2Y2 (Kd 184 nM), em relação aos receptores P2Y1 e P2Y4 recombinantes expressos em células 1321N1. A interação deste aptâmero não foi dependente da espécie, uma vez que ele foi capaz de se ligar tanto ao receptor P2Y2 de origem humana como murina. A atividade biológica do aptâmero foi avaliada em células P19 (sabidamente expressando receptores P2Y2 endógenos), na qual a proteção do ATP contra a apoptose, provavelmente interagindo com o receptor P2Y2, foi anulada na presença deste aptâmero em uma concentração mil vezes menor do que a do ATP. Além de confirmar a viabilidade da técnica SELEX para identificar ligantes subtipos-específicos dos receptores purinérgicos, o aptâmero anti-P2Y2 serve como ferramenta fundamental para definir demais funções fisiológicas deste receptor. Passos de otimização das suas propriedades como ligante e biodisponibilidade tornarão este aptâmero um composto de alta relevância farmacêutica. / Many published studies have focused on purinergic signaling and receptors, represented by ionotropic (P2X1 P2X7) and metabotropic (P2Y1,2,4,6,11,12,14) subtypes as a universal system which is not only involved in neuronal signaling, but also in various other vital processes including immune response, inflammation, platelet aggregation as well as differentiation, proliferation and cell death occurring during development and tissue regeneration. In agreement with other published reports, results of our laboratory based on overlapping purinergic receptor pharmacology suggest the participation of the P2Y2 subtype in proliferation and in vitro neurogenesis of P19 embryonal carcinoma cells. In view of the lack of availability of specific agonists and antagonists for most purinergic receptors making the elucidation of exact functions of these receptors in their cellular context almost impossible, we used a combinatorial oligonucleotide library approach, denominated SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the development of high-affinity and specificity ligands for the P2Y2 receptor. This approach is based on re-iterative steps of in vitro selection of 2´-fluoro-pyrimidine-modified RNA molecules for receptor-binding affinity from an RNA pool containing 1013 different sequences and secondary and tertiary structures until this pool is purified to a homogeneous population of ligands with high affinity to the P2Y2 receptor. The combinatorial DNA library is in vitro transcribed into RNA followed by target presentation of the RNA library and elution of the target-binders, denominated aptamers, and RT-PCR amplification in order to restore the DNA library used for in vitro transcription for next SELEX cycle. Following nine SELEX cycles using 1321N1 human astroglial cells expressing recombinant human P2Y2 receptors as target, we isolated 46 aptamer sequences which, based on consensus sequence motifs, were grouped in three structural groups. The mixture of the isolated sequences bound themselves to human P2Y2 receptors with a dissociation constant (Kd) of 164 nM. One of the isolated clones, the aptamer denominated B7, bound itself to P2Y2 receptors in preference to P2Y1 and P2Y4 recombinant receptors expressed in 1321N1 cells. The binding activity of the aptamer was not limited to P2Y2 receptors of human origin, as the aptamer also interacted with mouse P2Y2 receptors. The capability of the aptamer of affecting the biological activity of P2Y2 receptors was verified in P19 cells in which ATP-induced protection against apoptosis, mediated by P2Y2 receptors, was abolished in the presence of the aptamer. In addition to providing a proof of principle for the feasibility of developing purinergic subtype-specific ligands by using the SELEX technique, the anti-P2Y aptamer provides a fundamental tool for gaining insights of physiological functions of this receptor in various cellular contexts. Moreover, steps of optimization of receptor binding properties and aptamer half-life in vivo will turn this aptamer into a compound of high pharmaceutical relevance.

Os receptores purinérgicos são divididos em receptores P1 e P2 de acordo com o seu agonista endógeno, os receptores metabotrópicos P1 são ativados por adenosina, enquanto os metabotrópicos P2Y e ionotrópicos P2X são estimulados através do ATP e outros nucleotídeos. Além de sua função bem estabelecida na neurotransmissão, a sinalização purinérgica tem despertado crescente interesse científico devido à sua importância nas funções no metabolismo celular, incluindo os processos de desenvolvimento embrionário e reparação de tecidos. Isso ocorre especialmente no sistema nervoso central, onde eles controlam a proliferação, diferenciação, apoptose e a liberação de fatores neurotróficos. Nesse trabalho nós focamos nas ações sinérgicas entre a sinalização purinérgica e o fator de crescimento de nervos (NGF), tendo em vista três formas conhecidas de interação entre o NGF e os receptores purinérgicos, a potencialização dos efeitos do NGF através de uma ligação cruzada entre as vias, a regulação da expressão dos receptores purinérgicos pelo NGF e regulação da liberação de NGF pela adenosina. Com o objetivo de investigar estes processos sinérgicos na regulação da proliferação, migração e determinação fenotípica, células precursoras neurais foram obtidas do telencéfalo de embriões de rato e cultivadas na forma de neuroesferas, sendo então submetidas a tratamentos com agonistas e antagonistas dos receptores purinérgicos, em associação com o NGF, ao longo da diferenciação. Os efeitos desses tratamentos foram analisados por citometria de fluxo. Nós mostramos que o NGF age sobre as células aumentando a proliferação, migração e população de células indiferenciadas e diminuindo a apoptose. A ativação dos receptores P1 levou à diminuição da gliogênese e ao aumento da proliferação e da migração. A estimulação de receptores P2 resultou em aumento da proliferação e redução da taxa de apoptose. Os receptores purinérgicos potenciaram a proliferação mediada por NGF, resultando assim num aumento da população de células indiferenciadas. Neste último efeito percebemos a participação do receptor P2Y2 na sinergia. Os resultados aqui apresentados revelam novos mecanismos para a interação entre o NGF e a sinalização purinérgica na biologia de células tronco neurais / Purinergic receptors are divided into P1 and P2 receptors according to agonist selectivity, G-protein- coupled P1 receptors are activated by adenosine, while metabotropic P2Y and ionotropic P2X subtypes are stimulated by ATP and other nucleotides. In addition to its well-established function in neurotransmission, purinergic signaling has raised increasing scientific interest due to its importance in essential cellular functions and metabolism including embryonic developmental processes and tissue repair, specially in the central nervous system, where they control proliferation, differentiation, apoptosis and the release of neurotrophic factors. Here we focus on synergistic actions between purinergic P1 and P2 receptor-mediated signaling and the nerve growth factor (NGF), in view of three recognized forms of interaction between NGF and purinergic signaling, the potencialization of NGF-mediated effects through a crosstalk, the regulation of purinergic receptor expression by NGF and the regulation of NGF release by adenosine. With the objective to investigate such synergistic processes in regulating proliferation, neural migration and phenotype determination, neurospheres obtained as proliferating neural stem and progenitor from rat embryonic telencephalon were subjected to treatments with purinergic receptor agonists and antagonists in association with NGF along differentiation. Effects of these treatments were analyzed by flow cytometry. We show that NGF acted on cells by increasing the population of undifferentiated cells, proliferation, migration and reducing apoptosis. P1 receptor activation led to decreased gliogenesis, increased proliferation and migration. Stimulation of P2 receptors resulted in increased proliferation and decreased apoptosis rates. Purinergic receptors potentiated NGF-mediated proliferation, thereby resulting in increased population of undifferentiated cells, in this latter effect, the P2Y2 receptor subtype participated in synergistic processes. The herein presented results reveal novel mechanisms for the interaction of NGF and purinergic signaling in neural stem cell biology

Lau Chui Pik. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 104-122). / Abstracts in English and Chinese. / Chapter Chapter 1 --- --- Introduction --- p.1 / Chapter 1.1 --- Importance of sinoatrial node in heart functions --- p.3 / Chapter 1.2 --- The importance of Adenosine 5'-triphosphate (ATP) --- p.5 / "ATP as a neurotransmitter, cotransmitter and neuromodulator" --- p.5 / Role of ATP in the heart --- p.7 / Chapter 1.3 --- Importance of delayed rectifier potassium channels (Ik) in the heart --- p.9 / Delayed rectifier potassium channel --- p.10 / Properties of Ik channels in the sinoatrial nodal (SAN)cells --- p.11 / Importance of Ik on heart function --- p.14 / Chapter 1.4 --- Drug/hormone/neurotransmitter modulation of Ik --- p.15 / Drugs modulations of Ik --- p.15 / Hormones/neurotransmitters modulations of Ik --- p.18 / Chapter 1.5 --- Problems encountered in using extracellular ATP on experiments --- p.23 / Chapter 1.6 --- Classification of P2-purinergic receptors --- p.24 / Major nucleotide receptors --- p.24 / p2X receptors --- p.26 / p2Y receptors --- p.28 / 1.7Objectives of the experiment --- p.30 / Chapter Chapter 2 --- --- Materials & Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Solutions --- p.32 / Chapter 2.1.2 --- Enzymes --- p.34 / Chapter 2.1.3 --- Drugs --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Isolation of guinea pig SAN cells --- p.35 / Chapter 2.2.2 --- Identification of SAN region --- p.36 / Chapter 2.2.3 --- Obtaining of single SAN cells --- p.38 / Chapter 2.2.4 --- Preparation of micro-pipettes --- p.40 / Chapter 2.2.5 --- The Patch Clamp Technique --- p.40 / Recording configurations --- p.41 / Electrical recordings --- p.44 / Formation of gigaseal on cell membrane and the development of whole-cell configuration --- p.45 / The changing of bathing solution and addition of drugs --- p.46 / The voltage clamp protocol --- p.47 / Data acquisition and analysis --- p.48 / Statistics --- p.48 / Chapter Chapter 3 --- --- Results --- p.49 / Chapter 3.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.50 / Chapter 3.1.1 --- Characterization of IKs currents --- p.50 / Chapter 3.1.2 --- Stimulatory effect of extracellular A TP on IKs current --- p.51 / Chapter 3.1.3 --- Current-Voltage relationship of ATP on IKs current --- p.57 / Chapter 3.1.4 --- Percentage increase of IKs current in the presence of different [ATP] o --- p.63 / Chapter 3.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.71 / Chapter 3.2.1 --- Effect of 100 μMATP-γS and adenosine on IKs --- p.71 / Chapter 3.2.2 --- Percentage increase of IKs in the presence of adenosine and ATP-γS --- p.76 / Chapter 3.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.80 / Chapter 3.3.1 --- Effects of GTP-γS alone on IKs --- p.80 / Chapter 3.3.2 --- Effect of 100 μM ATP in the presence of GTP-yS on IKs --- p.83 / Chapter Chapter 4 --- --- Discussion --- p.86 / Chapter 4.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.87 / Chapter 4.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.92 / Chapter 4.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.97 / Chapter 4.4 --- Limitations of this study --- p.102 / Chapter 4.5 --- Future studies --- p.102 / Chapter Chapter 5 --- --- References --- p.104

Wong Hau Yan Connie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 86-95). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.ix / Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Role of extracellular nucleotides in equine sweat gland epithelial cells --- p.1 / Chapter 1.2 --- Subdivision of P1 and P2 purinoceptor --- p.4 / Chapter 1.3 --- General properties of P2 purinoceptor --- p.5 / Chapter 1.3.1 --- P2X purinoceptor family --- p.5 / Chapter 1.3.2 --- P2Y purinoceptor family --- p.8 / Chapter 1.4 --- The diversity of P2Y purinoceptor --- p.10 / Chapter 1.4.1 --- P2Y1 receptor --- p.10 / Chapter 1.4.2 --- P2Y2 receptor --- p.10 / Chapter 1.4.3 --- P2Y4 receptor --- p.10 / Chapter 1.4.4 --- P2Y6 receptor --- p.10 / Chapter 1.4.5 --- P2Y11 receptor --- p.11 / Chapter 1.5 --- The importance of calcium --- p.13 / Chapter 1.6 --- General aspects of calcium signaling --- p.14 / Chapter 1.7 --- Calcium release from the intracellular calcium stores --- p.15 / Chapter 1.7.1 --- Metabolism of inositol phosphates --- p.15 / Chapter 1.7.2 --- Ca2+ release from the internal calcium store --- p.15 / Chapter 1.8 --- Store-operated calcium channels (SOCC) or Capacitative calcium entry (CCE) --- p.18 / Chapter 1.8.1 --- The nature of the signal for CCE --- p.18 / Chapter 1.8.1.1 --- Conformational coupling --- p.18 / Chapter 1.8.1.2 --- Diffusible messenger --- p.21 / Chapter 1.9 --- Mechanism of intracellular calcium measurement --- p.25 / Chapter 1.10 --- Background of E92/3 cell line --- p.28 / Chapter Chapter 2: --- Materials and methods --- p.29 / Chapter 2.1 --- Cell culture --- p.29 / Chapter 2.2 --- Preparation of the simultaneous measurement --- p.31 / Chapter 2.2.1 --- Cell seeding --- p.31 / Chapter 2.2.2 --- Dye loading --- p.33 / Chapter 2.3 --- The setup of simultaneous measurement --- p.36 / Chapter 2.4 --- Statistical analysis --- p.40 / Chapter Chapter 3: --- Results --- p.41 / Chapter 3.1 --- Major domain of Ca2+ influx is from the basolateral side --- p.41 / Chapter 3.1.1 --- Effect of store depletion by apical ATP --- p.41 / Chapter 3.1.2 --- Effect of store depletion by basolateral ATP --- p.43 / Chapter 3.1.3 --- Effect of store depletion by thapsigargin --- p.47 / Chapter 3.2 --- Differential effect of apical and basolateral nucleotides on [Ca2+]i and Isc --- p.51 / Chapter 3.2.1 --- Basolateral ATP activates an increase in [Ca2+]i but not Isc --- p.51 / Chapter 3.2.2 --- Apical and basolateral ATP activated distinct but partially overlapped internal Ca2+ pool --- p.51 / Chapter 3.2.3 --- "Dose-dependent effect of apical or basolateral ATP, UDP and UTP on [Ca2+]i i and Isc" --- p.54 / Chapter 3.3 --- P2Y receptors subtypes on the basolateral membrane --- p.60 / Chapter 3.3.1 --- "Possible involvement of P2X, P2Y1 and P2Y11 purinoceptors on the basolateral membrane" --- p.60 / Chapter 3.3.2 --- "Cross-desensitization of experiments of UTP, ATP and UDP" --- p.60 / Chapter 3.4 --- The ATP-activated Ca2+ pool and thapsigargin-activated Ca2+ pool are partially overlapped --- p.68 / Chapter 3.5 --- Anion secretion activated by Ca2+ -independent pathway --- p.74 / Chapter Chapter 4: --- Discussion --- p.76 / Chapter 4.1 --- The major membrane for the CCE is from the basolateral side --- p.76 / Chapter 4.2 --- Basolateral P2Y receptors --- p.80 / Chapter 4.3 --- Differential effects of apical and basolateral ATP --- p.82 / Chapter 4.3.1 --- Apical and basolateral ATP release Ca2+ from different pools --- p.83 / Chapter 4.3.2 --- Ca2+ -independent mechanism --- p.83 / Chapter 4.3.3 --- Other potential signaling molecules --- p.84 / Chapter Chapter 5: --- Reference --- p.86

Wong Lai Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 162-173). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Heterogeneity of mast cells --- p.3 / Chapter 1.3. --- Mast cell mediators --- p.5 / Chapter 1.3.1. --- Performed and granule associated mediators --- p.5 / Chapter 1.3.2. --- Newly synthesized mediators --- p.8 / Chapter 1.3.3. --- Cytokines --- p.10 / Chapter 1.4. --- Mast cell activation --- p.10 / Chapter 1.4.1. --- Aggregation of IgE Receptors (FcεRI) --- p.10 / Chapter 1.4.2. --- Activation of Phospholipase C --- p.11 / Chapter 1.4.3. --- Activation of Adenylate cyclase --- p.13 / Chapter 1.5. --- Adenosine --- p.14 / Chapter 1.5.1. --- Adenosine receptors --- p.14 / Chapter 1.5.2. --- Selective agonists and antagonists --- p.17 / Chapter 1.5.3. --- Physiological and pathological roles of adenosine --- p.20 / Chapter 1.6. --- Role of adenosine receptors in mast cell activation --- p.21 / Chapter 1.7. --- Aims of the study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Mast cells secretagogues --- p.31 / Chapter 2.1.2. --- Anti-allergic compounds --- p.31 / Chapter 2.1.3. --- Adenosine receptor agonists and antagonists --- p.31 / Chapter 2.1.4. --- Materials for buffers --- p.32 / Chapter 2.1.5. --- Materials for rat sensitization --- p.32 / Chapter 2.1.6. --- Materials for histamine assay --- p.33 / Chapter 2.1.7. --- Miscellaneous --- p.33 / Chapter 2.2. --- Buffers and stock solutions --- p.34 / Chapter 2.2.1 --- Buffer ingredients --- p.34 / Chapter 2.2.2 --- Stock solutions --- p.34 / Chapter 2.3. --- Source of mast cells --- p.35 / Chapter 2.3.1. --- Animals --- p.35 / Chapter 2.3.2. --- Sensitization of animals --- p.35 / Chapter 2.3.3. --- Isolation of rat peritoneal mast cells --- p.35 / Chapter 2.3.4. --- Mast cells purification --- p.36 / Chapter 2.3.5. --- Cell counting --- p.36 / Chapter 2.4. --- General protocol for histamine release --- p.37 / Chapter 2.4.1. --- Histamine assay --- p.37 / Chapter 2.4.2. --- Antagonist studies --- p.38 / Chapter 2.4.3. --- Determination of histamine contents --- p.38 / Chapter 2.4.4. --- Calculation of histamine levels --- p.39 / Chapter 2.5. --- Statistical analysis --- p.40 / Chapter Chapter 3 --- "Effects of adenosine, adenosine deaminase and adenosine receptor agonists on mast cell activation" --- p.42 / Chapter 3.1. --- Introduction --- p.43 / Chapter 3.2. --- Materials and methods --- p.44 / Chapter 3.3. --- Results --- p.45 / Chapter 3.3.1. --- Effects of adenosine on anti-IgE induced histamine release in HEPES buffer --- p.45 / Chapter 3.3.2. --- Effects of NECA on anti-IgE induced histamine release in HEPES buffer --- p.46 / Chapter 3.3.3. --- Effects of CCPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.4. --- Effects of CPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.5. --- Effects of CGS21680 on anti-IgE induced histamine release in HEPES buffer --- p.48 / Chapter 3.3.6. --- Effects of Cl-MECA on anti-IgE induced histamine release in HEPES buffer --- p.49 / Chapter 3.3.7. --- Effects of adenosine deaminase on anti-IgE induced histamine release from rat peritoneal mast cells --- p.50 / Chapter 3.3.8. --- Effects of NECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.50 / Chapter 3.3.9. --- Effects of Cl-MECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.53 / Chapter 3.3.10. --- Effects of CV1808 on anti-IgE induced histamine release in HEPES buffer --- p.55 / Chapter 3.4. --- Discussion --- p.76 / Chapter 3.5. --- Conclusion --- p.83 / Chapter Chapter 4 --- Effects of adenosine receptor antagonists on mast cell activation --- p.88 / Chapter 4.1. --- Introduction --- p.89 / Chapter 4.2. --- Materials and methods --- p.90 / Chapter 4.3. --- Results --- p.91 / Chapter 4.3.1. --- Effects of A1 receptor antagonist DPCPX on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.2. --- Effects of A2A receptor antagonist ZM241385 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.3. --- Effects of A2B receptor antagonist MRS 1706 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.92 / Chapter 4.3.4. --- Effects of A3 receptor antagonist VUF5574 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.93 / Chapter 4.3.5. --- Further characterization of adenosine mediated potentiation of anti-IgE histamine release using VUF5574 and ZM241385 --- p.93 / Chapter 4.3.6. --- Effects of theophylline on anti-IgE induced percentage potentiation in HEPES buffer --- p.95 / Chapter 4.4. --- Discussion --- p.130 / Chapter 4.5. --- Conclusion --- p.135 / Chapter Chapter 5 --- Further characterization of the effects of adenosine on mast cells --- p.138 / Chapter 5.1. --- Introduction --- p.139 / Chapter 5.2. --- Materials and methods --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- Effects of anti-IgE induced histamine release in calcium free and HEPES buffers --- p.142 / Chapter 5.3.2. --- Effects of adenosine on anti-IgE induced histamine release in calcium free buffer --- p.143 / Chapter 5.3.3. --- Effects of adenosine deaminase on compound48/80 induced histamine release from rat peritoneal mast cells --- p.143 / Chapter 5.3.4. --- Effects of adenosine on compound 48/80 induced histamine release in HEPES buffer --- p.144 / Chapter 5.3.5. --- Effects of adenosine deaminase on A23187 induced histamine release from rat peritoneal mast cells --- p.144 / Chapter 5.3.6. --- Effects of adenosine on calcium ionophore A23187 induced histamine release in HEPES buffer --- p.145 / Chapter 5.3.7. --- Effects of adenosine receptor antagonists on inosine mediated enhancement of anti-IgE induced histamine release --- p.145 / Chapter 5.4. --- Discussion --- p.157 / Chapter 5.5. --- Conclusion --- p.160 / References --- p.162

Trichomonas vaginalis é o protozoário flagelado que parasita o sistema urogenital humano causando a tricomoníase, a doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos a cada ano. O sucesso da colonização das células hospedeiras e desenvolvimento da infecção envolve um complexo processo que culmina em citoaderência e citotoxicidade. Nucleotídeos e nucleosídeos são liberados para o espaço extracelular por células em situações de estresse ou lesão tecidual e desencadeiam seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, a hidrólise sequencial de nucleotídeos pelas ectonucleotidases, nucleosídeo trifosfato difosfoidrolase (NTPDase) e ecto-5’-nucleotidase leva à formação de nucleosídeos que são essenciais para o metabolismo de purinas do parasito. Efeitos antagônicos são desencadeados por nucleotídeos e nucleosídeos, respectivamente próinflamatórios e anti-inflamatórios, na mediação de respostas imunes. A atividade dessas enzimas sobre os nucleotídeos da guanina e o efeito de restrição metabólica sobre a hidrólise de nucleotídeos foi avaliada. Além disso, a participação da sinalização mediada pelos nucleotídeos e nucleosídeos também foi avaliada em um modelo de citotoxicidade. Os resultados demonstram que os nucleotídeos GTP, GDP e GMP são substratos para as ectonucleotidases de T. vaginalis com parâmetros cinéticos compatíveis para enzimas dessa família. A condição de restrição de soro aumentou a atividade da NTPDase e da ecto-5’-nucleotidase e o aumento da expressão gênica das TvNTPDase 2 e 4 pode justificar o aumento da atividade. A recaptação de guanosina extracelular foi menor do que a recaptação de adenosina, demonstrada pela razão isotópica C12/C13 no nucleosídeo detectada no DNA dos parasitos. A fim de investigar um possível papel biológico para o acúmulo de guanosina extracelular, bem como avaliar o envolvimento da sinalização purinérgica na citotoxicidade mediada pelo parasito, diferentes isolados de T. vaginalis foram testados frente à capacidade de promover citólise. Todos os isolados foram capazes de promover efeito citolítico em alguma proporção, com destaque para o isolado TV-LACM6, que apresenta um perfil de hidrólise ATP, GTP > AMP > GMP. Quando nucleotídeos e nucleosídeos foram testados, o efeito citotóxico produzido pelo isolado foi potencializado na presença de ATP e GTP. Por outro lado, o efeito foi revertido na presença de eritro-9-(2-hidroxi-3-nonil) adenina (EHNA), um inibidor da adenosina deaminase. Importante, guanosina não foi capaz de reverter o efeito citotóxico produzido pelos trofozoítos, resultado que corrobora com o perfil de hidrólise de nucleotídeos e acúmulo de guanosina exracelular, sendo uma vantagem para o parasito. A possível participação dos receptores de adenosina foi avaliada, entretando os receptores ADORA1 e ADORA2A não parecem estar envolvidos no efeito protetor mediado pela adenosina. Considerando o potencial farmacológico desempenhado por essas enzimas no metabolismo de purinas em protozoários bem como no controle de respostas imunes, a modulação da hidrólise de nucleotídeos pode ser um alvo terapêutico importante e representar um mecanismo sinérgico na atividade antiparasiária de compostos ativos. Nesse sentido, o estudo demonstrou a atividade anti-T. vaginalis de três compostos, e em especial o isoaustrobrasilol B, com IC50 de 38 μM. O composto não apresentou efeitos hemolíticos frente a eritrócitos humanos e apesar de ter demonstrado efeito citotóxico in vitro frente à linhagem de células epiteliais vaginais humanas (HMVII), nenhuma citotoxicidade foi demonstrada no modelo in vivo. Isoaustrobrasilol B foi o único composto que inibiu significativamente as atividades da NTPDase e ecto-5’-nucleotidase e o efeito imune atribuído ao acúmulo extracelular de nucleotídeos foi avaliado. A produção de espécies reativas de oxigênio e interelucina-6 (IL-6) por neutrófilos estimulados por T.vaginalis não foi afetada pelo tratamento com o composto. Por outro lado, a liberação de interleucina-8 (IL-8), a principal interleucina produzida por neutrófilos na tricomoníase, foi aumentada. O efeito sinérgico de redução de viabilidade de trofozoítos e modulação da NTPDase e ecto-5’-nucleotidase pode aumentar a suscetibilidade do T. vaginalis frente à resposta imune do hospedeiro e consequentemente, sua eliminação do sítio de infecção. / Trichomonas vaginalis is a flagellate protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease, infecting approximately 276 million people worldwide annually. To achieve success in parasitism trichomonads develop a complex process against the host cells that culminate in cytoadherence and cytotoxicity. Nucleotides and nucleosides are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing nucleosides that are essential to the parasite purine metabolism. The enzymatic cascade mediated by ectonucleotidases is relevant in controlling nucleotides and nucleosides levels as these molecules play antagonistic roles in inflammation, as proinflamatory and anti-inflammatory mediators, respectively. This study investigated the hydrolysis profile of guanine nucleotides in T. vaginalis as the effect of serum limitation condition in the enzymatic cascade. Furthermore, we investigated the influence of extracellular nucleotides and nucleosides on the modulation of the host cell cytotoxicity mediated by T. vaginalis. Results show that guanine nucleotides GTP, GDP, GMP are substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. The metabolic restriction condition enhanced NTPDase and ecto-5’-nucleotidase activities and the highest gene expressions found for TvNTPDase 2 and 4 which may explain the enzymatic activity enhance. The extracellular guanosine uptake was lower than that observed for adenosine into parasite DNA measured by isotopic ratio C12/C13 of the nucleosides. In order to investigate the possible biological role for extracellular guanosine accumulation as well as to evaluate the involvment of purinergic signaling in the citotoxicity promoted by the parasite, a collection of T. vaginalis isolates were tested against a human epithelial vaginal cell line (HMVII). Fresh clinical T. vaginalis isolates produced cytolytic effect against human vaginal epithelial cells in a heterogeneous profile. The most cytotoxic isolate, TV-LACM6, presents the hydrolysis profile ATP, GTP > AMP > GMP. When the nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased in presence of nucleotides ATP and GTP. In contrast, the cytotoxicity was reversed by adenosine in presence of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), but not by guanosine, which is in agreement with the accumulation of extracellular guanosine and the hydrolysis profile, acting as an advantage for the parasite. ADORA1 and ADORA2A are not involved in the protective mechanism of adenosine. Considering the pharmacological potential that ectonucleotidases play in the context of purine metabolism and in the imune response modulation, nucleotide hydrolysis may represent a therapeutic target as an additional mechanism in association with anti T.vaginalis compounds. The study demonstrated promissing activities for three derivatvies with isoaustrobrasilol B the most activity compound with IC50 38 μM. The compound did not demonstrate any hemolytic activity and although induced cytotoxicity against human epithelial vaginal cells (HMVII), absence of toxicity was obtained when in vivo model was studied. Isoaustrobrasilol B was the only compound that significantly inhibited NTPDase and ecto-5’-nucleotidase activities and the immune modulation attributed to extracellular nucleotide accumulation was evaluated. Reactive oxygen species production and interleukin-6 (IL-6) release by T.vaginalis stimulated neutrophils were not affected by phloroglucinol treatment. On the other hand, interleukin-8 (IL-8), the primarily cytokine produced by neutrophils during trichomoniasis, was significantly enhanced. The associative mechanism of trophozoites death and NTPDase and ecto-5’-nucleotidase modulation may increase the susceptibility of T. vaginalis to host immune responses and, consequently, the elimination from the infection site.

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.

A criptococose é uma infecção fúngica sistêmica, predominantemente oportunista, causada por leveduras encapsuladas do gênero Cryptococcus. As infecções por Cryptococcus neoformans são comuns em nível mundial, e as formas graves são observadas nos pacientes imunocomprometidos. A principal fonte de infecção da criptococose são fezes de pássaros (principalmente pombos) contaminadas com o fungo, sendo a inalação de basidiósporos a principal via de infecção. O estabelecimento e a propagação da infecção são altamente dependentes da imunidade do hospedeiro, sendo o sistema imune celular o mecanismo primário de defesa do organismo contra C. neoformans. Nos últimos anos têm-se descrito outros elementos como ativadores e moduladores da resposta imune, destacando o sistema purinérgico e o sistema colinérgico. Desta maneira, este estudo buscou avaliar a influência da criptococose experimental sobre a atividade do sistema purinérgico e colinérgico, através de três objetivos: (1) avaliar a atividade da ecto-difosfoidrolases (E-NTPDase) e ecto-adenosina deaminase (E-ADA) em linfócitos e soro; (2) atividade da acetilcolinesterase (AChE) no cérebro e linfócitos, e butirilcolinesterase (BChE) no soro; e (3) avaliar os níveis de purinas no soro. Os resultados da avaliação do sistema purinérgico demonstraram que a hidrólise do trifosfato de adenosina (ATP) e difosfato de adenosina (ADP) foram diminuídas, bem como a atividade da E-ADA também esteva diminuída. Em relação a dosagem das colinesterases, observou-se um aumento na atividade da AChE nos linfócitos e no cérebro, e diminuição da BChE. Na dosagem do nível de purinas no soro, verificou-se um aumento nos níveis de ATP e adenosina (ADO) no dia 20 pós-infecção (PI), aumento de ATP e diminuição da ADO, inosina e ácido úrico no dia 50 PI. A avaliação da atividade da E-NTPDase e E-ADA levou a conclusão de que seus comportamentos hidrolíticos seriam compensatórios enquanto a E-NTPDase teria uma ação pró-inflamatória a E-ADA teria uma ação anti-inflamatória, gerando mecanismo de proteção contra danos teciduais secundários, possivelmente gerados respostas exacerbadas à infecção por C. neoformans. Adicionalmente, os dados da atividade da AChE, em amostras correspondentes, comprovaram o estabelecimento de uma resposta pró-inflamatória, corroborando com a hipótese da necessidade de um mecanismo de modulação. Por fim, observou-se um aumento nos níveis extracelulares de ATP caracterizando uma resposta pró-inflamatória. Desta forma, foi possível observar que existe uma participação direta dos sistemas purinérgico e colinérgico na imunomodulação da criptococose experimental, contribuindo para a instalação de uma resposta imune celular adequada para combater a proliferação da levedura, e um mecanismo de redução de danos teciduais associados à resposta imune exacerbada. / Cryptococcosis is a systemic fungal infection predominantly opportunistic, caused by encapsulated yeast from Cryptococcus genus. Cryptococcus neoformans infections are common worldwide, and the severe forms are observed in immunocompromised patients. The main source of cryptococcosis infection are bird droppings (especially pigeons) contaminated with the fungus, and the inhalation of basidiospore is the main route of infection. The establishment and spread of infection are highly dependent of the host immunity, and the cellular immune system is the primary mechanism for defense against C. neoformans. In recent years it has been described other elements as activators and modulators of the immune response, highlighting the purinergic and the cholinergic system. Thus, this study aimed to evaluate the influence of experimental cryptococcosis on the activity of purinergic and cholinergic systems through three objectives: (1) to evaluate the activity of the ecto-diphosphohydrolases (E-NTPDase) and ecto-adenosine deaminase (E-ADA ) in lymphocytes and serum; (2) the activity of acetylcholinesterase (AchE) in the brain and lymphocytes, and butyrylcholinesterase (BChE) in serum; and (3) evaluate the serum levels of purines. The results of the evaluation in the purinergic system demonstrated that the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was decreased as well as the E-ADA activity. For the dosage of the cholinesterase, there was an increase in AChE activity in lymphocytes and in the brain, and a decreased in BChE. The measurement of serum purine level demonstrate an increase in the levels of ATP and adenosine) on day 20 post-infection (PI), an increased in ATP and decreased in ADO, inosine and uric acid on day 50 PI. The assessment of the E-NTPDase and E-ADA activity led the conclusion that their hydrolytic behavior would be compensatory while the E-NTPDase would have a pro-inflammatory action, E-ADA would have an anti-inflammatory action, generating protective mechanism against secondary damage tissue, producing possibly exacerbated responses to C. neoformans infection. In addition, data of AChE activity in corresponding samples confirmed the establishment of a pro-inflammatory response, corroborating the hypothesis of the need for a modulation mechanism. Finally, there was an increase in extracellular levels of ATP featuring a pro-inflammatory response. In this way, it was observed a direct involvement of the purinergic and cholinergic systems in immunomodulation of experimental cryptococcosis, contributing to the installation of an immune cell response suitable to combat the proliferation of yeast, and a reduction mechanism of tissue damage associated with response immune exacerbated.

Receptores purinérgicos são expressos em neurônios e glia e, por certo, participam dos processos de sinalização entre estes células. Nos gânglios da raiz dorsal o soma dos neurônios sensoriais é envolto por uma camada de células satélites (CSt), que são células de glia, cuja função é desconhecida. Até o momento a natureza dos receptores purinérgicos presentes nas células satélites foi pouco investigada. No presente trabalho demonstra-se a presença dos receptores P2Y1, P2Y2 e/ou P2Y4 e P2Y6 nas CSt de gânglios da raiz dorsal de ratos recém-nascidos. A prova da existência dos receptores foi a elevação na concentração intracelular do Ca2+, induzida pela aplicação de um agonista dos receptores. Nas culturas de mais de 24 horas, estimuladas por soro bovino fetal, proliferam de forma notável células de glia de aspecto predominantemente fusiforme. Determinou-se que as células fusiformes expressam receptores P2Y1, P2Y2 e/ou P2Y4 e P2Y6. As informações obtidas contribuem para o esclarecimento da sinalização entre neurônios e glia nos gânglios sensoriais da raiz dorsal. / Purinergic receptors are expressed and play role in the sinalization between neurons and glia. In dorsal root ganglia the soma of the sensory neurons is surrounded by a layer of satellite glial cells, whose function is unclear. There are evidences that ATP is released by neurons to act on receptors in satellite cells. So far, the nature of the purinergic receptors of satellite cells was not fully investigated. This study shows the presence of metabotropics purinergic receptors P2Y1, P2Y2 and/or P2Y4, e P2Y6 in satellite cells from dorsal root ganglia of newborn rats. The demonstration was carried on following the transient increases in intracellular calcium concentration induced by a purinergic agonist. As time goes by, in the presence of fetal bovine serum, there is a remarkable proliferation of glial cells, with predominant fusiform shapes. The fusiform cells also express P2Y1, P2Y2 and/or P2Y4, e P2Y6 receptors. These informations add on the understanding of the complex phenomena of neuron-glia interaction.