A indústria farmacêutica tem como principal objetivo planejar, sintetizar e caracterizar compostos químicos que possuam atividade biológica e que sejam úteis no controle e combate de doenças e sintomas que acometem as populações. Estes compostos são referidos como ingredientes farmacêuticos ativos e podem, no estado sólido, apresentar diferentes arranjos de suas moléculas dentro de um cristal (polimorfismo). A cada um desses arranjos estão associadas propriedades físico-químicas que são de fundamental importância para o efeito terapêutico dos fármacos. Nesse contexto, este estudo teve como objetivo caracterizar as propriedades de estado sólido de novas formas cristalinas de três compostos farmacêuticos, Clorpropamida (CPA), Nevirapina (NVP) e Dietilcarbamazina (DEC). A CPA trata-se de um hipoglicemiante oral, utilizado no tratamento da Diabetes mellitus tipo II, apresentando na literatura cinco polimorfos conformacionais, dos quais dois foram caracterizados nesse estudo: as Formas IV e VI. A Forma IV cristaliza-se grupo espacial não centrossimétrico monoclínico P21, com Z = 2 e a Forma VI no grupo espacial centrossimétrico ortorrômbico Pbca, com Z = 8. Ambas apresentaram o mesmo padrão de interações intermoleculares clássicas, sendo que a principal diferença entre elas reside nas interações intermoleculares não clássicas, que levam a diferentes empacotamentos cristalinos. Por fim, as conformações moleculares dos cinco polimorfos da CPA foram comparadas entre si e as informações foram racionalizadas tomando como base os resultados provenientes de cálculos teóricos, que também indicaram a possibilidade de existência de novos polimorfos. A NVP, fármaco antiretroviral não nucleosídeo utilizado no tratamento da AIDS, também apresenta casos de polimorfismo na literatura. Neste trabalho, obteve-se um solvato não estequiométrico de butanol desse composto, que cristaliza no grupo espacial centrossimétrico triclínico P-1, com Z = 2, no qual as moléculas de butanol acomodaram-se em canais infinitos rodeados por moléculas de NVP. Esse tipo de empacotamento cristalino, diferente do reportado na literatura até o presente momento, possibilitou-nos propor que novos solvatos poderiam ser obtidos variando-se o solvente, proposta tal confirmada posteriormente. A DEC é amplamente utilizada na forma de um sal de citrato no tratamento da filariose linfática, não apresentado na literatura nenhuma caracterização de estado sólido. Assim, caracterizou-se não somente o sal utilizado nas formulações (DEC citrato), como também o composto puro (DEC). A forma pura, instável à temperatura ambiente, cristaliza no grupo espacial centrossimétrico P21/n à 250K. O sal, preferido como API por sua estabilidade, cristaliza à temperatura ambiente no grupo P21/c, porém com presença de desordem nas cadeias etílicas das moléculas de DEC. Para reduzir essa desordem, efetuou-se um estudo em função da temperatura, que acabou revelando a presença de três transições de fase sólido-sólido, gerando quatro fases cristalinas diferentes. Duas das transições exibiram efeito de histerese de acordo com a direção da rampa de temperatura. A terceira transição só foi obtida por resfriamento rápido do sistema. Estes dados foram comparados com os obtidos por DSC e espectroscopia Raman / One of the main goals of the pharmaceutical industry is to plan, synthesize and characterize chemical compounds with biological activity that can be useful in controlling diseases and symptoms that affect populations. These compounds are referred to as active pharmaceutical ingredients and may, in the solid state, present different crystal arrangements of its molecules (polymorphism). To each one of these crystalline arrays are associated physicochemical properties that are of fundamental importance for the therapeutic effect of the pharmaceutical drugs. In this context, the focus of this study was to characterize the solid state properties of new crystalline forms of three pharmaceutical compounds, Chlorpropamide (CPA), Nevirapine (NVP) and Diethylcarbamazine (DEC). CPA is an oral hypoglicemiant used in the treatment of type II Diabetes mellitus, and presents five conformational polymorphs reported in the literature, two of which were characterized in this study: Forms IV and VI. Form IV crystallizes in the monoclinic non-centrosymmetric space group P21, with Z = 2 and the Form VI in the orthorrombic centrosymmetric space group Pbca, with Z = 8. Both exhibited the same classical intermolecular interaction pattern; the main difference between them lay in the non-classical intermolecular interactions, which leads to different crystal packing. Finally, the molecular conformations of the five polymorphs of CPA were compared to each other and the information was rationalized taking as basis the results from theoretical calculations, which also indicated the possible existence of new polymorphs. NVP, a non-nucleoside antiretroviral pharmaceutical compound used in the treatment of AIDS, also presents polymorphism cases reported in the literature. In this study, we obtained a non-stoichiometric buthanol solvate of this compound, which crystallizes in the triclinic centrosymmetric space group P-1, with Z = 2, in which the buthanol molecules are positioned in infinite channels surrounded by NVP molecules. This kind of crystal packing, which is different from the one reported in the literature until now, has allowed us to propose that new solvates could be obtained by varying the solvent, being this proposal subsequently confirmed. DEC is largely used as a citrate salt form in the treatment of the lymphatic filariasis, not having any solid state characterization in the literature. Thus, we characterized not just the salt (DEC citrate) used in the formulation, but also the pure compound (DEC). The pure form, unstable at room temperature, crystallizes in the monoclinic centrosymmetric space group P21/n at 250K. The salt, preferred as API because of its stability, crystallizes at room temperature in the monoclinic centrosymmetric space group P21/c, but with the presence of disorder in the ethyl chains of the DEC molecules. To reduce this disorder, we performed a study in function of temperature, which revealed the presence of three solid-solid structural phase transitions, generating four different crystalline phases. Two of these transitions showed a hysteresis effect according to the direction of the temperature ramp. The third transition was only obtained by fast cooling of the system. These data where compared with the ones obtained by DSC and Raman spectroscopy.

A hipertensão é uma doença crônica não transmissível e mais freqüente na população sendo o principal fator de risco para complicações cardiovasculares, tais como acidente vascular cerebral e infarto agudo do miocárdio. Na presente pesquisa estão sendo estudados os fármacos utilizados no tratamento da hipertensão mais especificamente, os bloqueadores do canal de cálcio do grupo diidropiridínicos: besilato de anlodipino, nifedipino e nimodipino. O objetivo desse trabalho foi verificar a estabilidade intrínseca dos fármacos besilato de anlodipino, nifedipino e nimodipino, para isto foram utilizadas as seguintes técnicas: testes indicativos de estabilidade utilizando as técnicas de espectrofotometria na região do Ultravioleta/Visível (UV/VIS) e Cromatografia em fase Líquida de Alta Eficiência (CLAE). Termogravimetria/ Termogravimetria Derivada (TG/DTG), Calorimetria Exploratória Diferencial (DSC), Difração de Raios X (DRX), Espectroscopia de absorção na região do Infravermelho com Transformada de Fourier (FTIR) e Microscopia Eletrônica de Varredura (MEV). Para o fármaco besilato de anlodipino (AB) pelo método de degradação forçada, analisado por espectrofotometria no UV/VIS, as condições para a análise espectrofotométrica foram metanol e água a uma proporção de (5:45 v/v) e a segunda diluição com água. A leitura foi efetuada a 364,4nm. A linearidade foi estabelecida na faixa de 40,0-65,0 µg/mL e o coeficiente de correlação foi (r) 0,9992. O método cromatográfico, mostrou o diferente comportamento das substâncias nifedipino e nimodipino diante dos meios básicos, ácido, neutro e oxidativo. As condições para a substância nifedipino foram coluna LiChrospher®100 RP-18 (5µm) Merck® fase móvel constituída por metanol e água (45:55v/v), fluxo 1.0 mL/min, tempo de retenção 5,1min, detecção UV a 234nm e vazão de 1.0 mL/min. Foi obtida uma linearidade no intervalo de 5.0-55.0 µg/mL coeficiente de correlação (r) =0,9964. E para a substância nimodipino foram coluna LiChrospher®100 RP-18 (5µm) Merck® fase móvel constituída por acetonitrila e água (55:45v/v), fluxo 1.0mL/min, tempo de retenção 5,8 min, detecção UV a 235 nm e vazão de 1.0mL/min. Foi obtida uma linearidade no intervalo de 5.0-55.0 µg/mL coeficiente de correlação (r) =0,9964. Os resultados obtidos das curvas TG/DTG e DSC mostraram o perfil da decomposição térmica das substâncias estudadas pela Calorimetria Exploratória Diferencial. A análise dos resultados de DRX e DSC mostraram que não há evidências de polimorfismo nessas substâncias. No entanto nas análises de Espectroscopia de absorção na região do infravermelho com Transformada de Fourier (FTIR) não foram encontradas diferenças significativas na matéria-prima e no padrão de referência. As análises de MEV permitiram observar a cristalinidade das substâncias estudadas. / Hypertension is the most frequent non-communicable chronic disease in the population being the main factor of risk for cardiovascular complications, such as stroke and acute myocardial infarction. In this work, active pharmaceutical ingredients used to treat hypertension were studied, more specifically the blockers calcium channel dihydropyridine group: amlodipine besylate, nifedipine and nimodipine. The aim of this study was to determine the intrinsic stability of amlodipine besylate, nifedipine and nimodipine. For this purpose the following stability test techniques were used: UV/VIS spectrophotometry and chromatography Net phase High Performance. Thermogravimetry/Derivative Thermogravimetry (TG/ DTG), Differential Scanning Calorimetry (DSC), X-Ray Diffraction (XRD), Fourier Transformed Infrared absorption (FTIR) and Scanning Electron Microscopy (MEV). For drug amlodipine besylate (AB) by forced degradation method analyzed by spectrophotometry UV/VIS spectrophotometric conditions for the analysis were methanol and water at a ratio (5:45v/v) and the second dilution with water. The reading was made at 364,4nm. The linearity was established in the range of 40.0 to 65.0 mg/mL and the correlation coefficient was (r) 0.9992. The chromatographic method showed different behavior of nifedipine and nimodipine substances on the basic means, acid, neutral and oxidative. The conditions for nifedipine were LiChrospher®100 RP-18 column (5µm) Merck® mobile phase consisting of methanol and water (45:55v/v), flow 1.0 mL/min, retention time 5,1min, UV detection at 234 nm and flow of 1.0 mL/min. Linearity was obtained within the range of 5.0-55.0 mg/mL correlation coefficient (r) = 0.9964. And for nimodipine the parameters were: LiChrospher®100 RP-18 column (5µm) Merck® mobile phase consisted of acetonitrile: water (55:45v/v), flow 1.0 mL/min, retention time 5,8min, UV detection at 235nm and flow of 1.0 mL/min. The linearity was obtained within the range of 5.0- 55.0 mg/mL correlation coefficient (r) = 0.9964. The results of TG/DTG and DSC curves presented the profile of the thermal decomposition of the substances studied by DSC. The results of XRD and DSC presented no evidence of polymorphism in these analyzes, however, according to analyzes of absorption spectroscopy in the infrared (FTIR) there were no significant differences in the raw materials and standard reference. SEM analyzes allowed to observe the crystallinity of the studied substances.

No presente trabalho, AFLPs e marcadores baseados em retrotransposons foram utilizados para a construção de um mapa de ligação integrado em cana-de-açúcar. Dois retrotransposons encontrados no genoma da cana-de-açúcar, denominados SURE e Garapa, foram estudados. Os princípios da técnica NBS-profiling foram usados para gerar marcadores direcionados às sequências desses retrotransposons. Os marcadores foram analisados numa população F1 de cana-de-açúcar, composta por 188 indivíduos, oriunda do cruzamento entre os genitores IAC66-6 e TUC71-7. O mapa integrado foi construído usando-se o software OneMap, especialmente desenvolvido para mapear espécies não endogâmicas. Excelentes padrões de AFLP e de marcas direcionadas ao retrotransposon SURE foram obtidos; entretanto, para o Garapa, ainda são necessários ajustes na técnica. Um total de 600 marcadores de dose única foi obtido a partir de 22 combinações de enzimas de restrição/primers de AFLP e seis combinações otimizadas para amplificar marcas direcionadas ao retrotransposon SURE. Construiu-se um mapa com 107 grupos de ligação, com tamanho de 4.316,5 cM e densidade de 8,74 cM/marcador. O mapeamento dos marcadores derivados do retrotransposon SURE revelou que esse elemento não está uniformemente distribuído nos grupos de ligação e confirmou o seu baixo número de cópias no genoma da cana, conforme foi sugerido na literatura. / In the presenty study, AFLPs and retrotransposon-based markers were used for the construction of an integrated linkage map of sugarcane. Two retrotransposons described in the sugarcane genome, named SURE and Garapa were studied. The principles of NBS-profiling technique were used to generate markers based on these retrotransposon sequences. The markers were analyzed in a F1-population, composed of 188 individuals, derived from a single cross between the IAC66-6 and TUC71-7 parents. The integrated genetic map was constructed using the software OneMap, specially designed for mapping outcrossing species. Excellent gel profiles of AFLP and retrotransposon-derived markers were obtained; however, for the Garapa element, technical adjustments are still needed. A total of 600 single-dose markers were obtained from 22 AFLP restriction enzyme/primer combinations and six combinations optimized to amplify the SURE-based markers. A map with 107 linkage groups was constructed, spanning 4,316.5 cM, with a marker density of 8.74 cM. Mapping of SURE-based markers revealed that this element is not uniformly distributed across the linkage groups, and confirmed its low copy number in the sugarcane genome, as suggested in the literature.

O Brasil se mantém na posição de maior exportador mundial de carne de frango, e o terceiro em produção, abaixo apenas dos Estados Unidos e da China. O grande avanço na produtividade avícola é devido principalmente ao melhoramento genético. As novas tecnologias na área da genômica animal podem oferecer ferramentas que auxiliam na seleção assistida por marcadores. O presente trabalho teve como objetivo identificar polimorfismos em seis genes (FGFBP1, FGFBP2, CAPN1, CAPN3, MyoG e MSTN) de uma população experimental, validá-los em uma população comercial e realizar a associação dos polimorfismos com características de interesse econômico na avicultura. Para a identificação de polimorfismos foram utilizadas 11 aves (F1) pertencentes à uma população experimental da Embrapa Suínos e Aves, e um polimorfismo de cada gene foi selecionado para a genotipagem em média de 180 animais da geração F2 da população experimental da Embrapa e 311 frangos de corte de uma população comercial através de sondas TaqMan®. SNP 2014G>A (FGFBP1) foi associado com peso eviscerado (PE), e os genótipos do SNP 651G>A (FGFBP2) foram associados com perdas de água por exsudação (EXSU) e teor de vermelho da carne (a*). O polimorfismo g.2554T>C (CAPN1) foi associado com peso vivo aos 35 a 42 dias, peso da coxa, peso do peito, peso da carcaça e luminosidade da carne. O SNP g.15486C>T (CAPN3) foi associado com rendimento da coxa, perdas de água por cozimento da carne e força de cisalhamento da carne. O polimorfismo g.2947A>G (MyoG) foi associado com porcentagem de proteína e cinzas na matéria mineral, porcentagem de cinzas na matéria seca, peso eviscerado, peso de pernas e peso à seleção aos 38 dias. Para o SNP g.324C>T (MSTN) foram observados associações com peso do fígado, conversão alimentar, peso ao abate e peso eviscerado. Os resultados obtidos neste trabalho permitem a indicação dos polimorfismos do tipo SNP, aqui apresentados, como marcadores moleculares, os quais são ferramentas importantes para programas de melhoramento avícola que objetivam seleção de animais geneticamente superiores para características de desempenho, carcaça, desenvolvimento muscular e qualidade de carne. / Brazil is the worlds largest chicken meat exporter, and the third in production, behind the United States and China. The great advance in productivity is due mainly to poultry breeding. New technologies in the field of animal genomics that have provided tools such as marker-assisted selection. This study aimed to identify polymorphisms in six genes (FGFBP1, FGFBP2, CAPN1, CAPN3, MyoG and MSTN) of an experimental population, validate them in a business population and perform the association of polymorphisms with traits of economic interest in poultry. To identify polymorphisms, we used 11 birds (F1) belonging to an experimental population of Embrapa Swine and Poultry, and a polymorphism of each gene was selected for genotyping on average of 180 chickens from an F2 experimental population from Embrapa and 311 broilers from a commercial population using TaqMan® probes. SNP 2014G>A (FGFBP1) was associated with gutted weight (GW), and the genotypes of SNP 651G>A (FGFBP2) were associated with water loss by exudation (EXSU) and meat red content (a*). Polymorphism g.2554T>C (CAPN1) was associated with body weight at 35 to 42 days, thigh weight, breast weight, carcass weight and meat lightness content. The SNP g.15486C>T (CAPN3) was associated with thigh performance, water losses by cooking the meat and meat shear force. Polymorphism g.2947A>G (MyoG) was associated with protein percentage and ash in the mineral matter, ash percentage in dry matter, gutted weight, leg weight and weight to selection for 38 days. The SNP g.324C>T (MSTN) showed associations with liver weight, feed conversion, slaughter weight and gutted weight. The results indicate that the type polymorphisms SNP shown here as molecular markers, which are important tools for breeding programs aimed at poultry selection of genetically superior animals for of performance, carcass, muscle development and meat quality traits.

Sunitinib, an oral vascular endothelial growth factor receptor, is a first-line option for metastatic renal cell carcinoma and widely used in clinical practice. Despite the proven benefit of sunitnib in metastatic renal cell carcinoma, patients may suffer from a variety of adverse events including hypertension, fatigue, hypothyroidism, hand?foot skin reactions, rash, depigmentation, and myelosuppression. Myelosuppression is usually mild, transient and resolves during the two weeks at the end of each cycle where no drug is taken. We present a case of severe and early grade 3 neutropenia and thrombocytopenia occurring two weeks into a six-week cycle. Because of the extreme nature of the toxicity, CYP 3A4 polymorphisms were explored. The patient was found to be heterozygous for CYP 3A4*22, at least partially explaining the early-onset and severity of myelosuppression. This pharmacogenetics information resulted in a rechallenge of dose-reduced sunitinib, which was well tolerated by the patient. The current state of pharmacogenomics concerning sunitinb is also presented, and the need for greater research in this area is highlighted.

Papilio dardanus displays female-limited polymorphic mimicry of multiple model species. Butterfly wing shape is species-specific and can influence mimetic signaling, but has not been characterized in this species. We used elliptical fourier analysis to investigate whether mimetic P. dardanus female forms have converged on the wing shape of their respective models. Although both models and mimics varied in forewing and hind wing shape, we found no evidence of forewing shape convergence between them. Overall, forewings did not differ in shape between sexes in P. dardanus, nor in four non-mimetic Papilio used for comparison. Similarly, there were no hind wing differences between the sexes in the four non-mimetic Papilio. However, P. dardanus hind wings varied significantly between mimetic females and non-mimetic individuals suggesting that, in addition to wing color pattern, the evolution of mimicry has led to changes in hind wing shape in P. dardanus.

La vigne est soumise à de nombreuses maladies impliquant l'utilisation de produits phytosanitaires en grande quantité dont l'utilisation est néfaste pour l'environnement et la santé des utilisateurs. Un enjeu est donc de développer des méthodes alternatives à la lutte chimique. La protéine codée par le gène NPR1 (Nonexpressor of pathogenesis-related gene 1) joue un rôle clef dans la résistance à large spectre chez les plantes. Des éliciteurs tels que l'acide salicylique ou des agents pathogènes influencent l'activation de NPR1 dans le cytoplasme. La translocation de NPRl dans le noyau et son interaction avec des facteurs de transcription TGA induit l'expression des gênes PR (Pathogenesis-related). Nous avons identifié sept homologues potentiels des gènes NPR1 et TGA chez Vitis vinifera (VvNPR1.1, VvNPR1.2, VvTGA1 à 5). L'étude de la diversité de séquences dans les exons de 15 accessions de Vitaceae indique qu'ils sont soumis à une forte pression de sélection purificatrice. De plus, l'analyse in silico des régions promotrices des VvNPR1 montre la présence, d'éléments cis-régulateurs potentiels, en réponse aux stress biotiques et abiotiques ainsi que des motifs de liaison à des facteurs de transcription. Une étude plus poussée des introns montre quelques éléments transposables et un faible polymorphisme dans six accessions de Vitis vinifera. Ces résultats argumentent en faveur d'une pression de sélection forte agissant sur ces gènes. Ceci nous a mené à formuler des hypothèses fonctionnelles et à réaliser une étude d'interaction avec les facteurs de transcription VvTGA1 et VvTGA4 par la technique du double hybride. Ces derniers n'interagissent pas avec VvNPR 1.1. / Numerous diseases affect grapevine, resulting in the use of phytochemicals in large quantities that are harmful for environment and user's health. In the long term, the aim is to develop alternative methods to chemicals. The protein encoded by NPR1 (Nonexpressor of pathogenesis-related gene 1) plays a pivotal role in conferring broad spectrum pathogen resistance in plants. Activation of NPR 1 in the cytoplasm is influenced by elicitors such as salicylic acid or pathogens associated with the accumulation of reactive oxygen species. Translocation of NPR1 into the nucleus and interaction with TGA transcription factors induce the expression of PR (Pathogenesis-related) genes. Using a candidate gene approach, we have identified seven putative homologs to NPR1 and TGA in the grapevine genome (VvNPR1.1, VvNPR1.2, VvTGA1 to 5). The study of sequence diversity in exons of 15 accessions of the Vitaceae family indicates that these exons are subjected to a strong purifying selection pressure. Moreover, in silico analysis in the promoters of VvNPR1 shows putative cis-regulator elements, in answer to biotic and abiotic stresses as well as link patterns to transcription factors. An intron study shows transposable elements and a low polymorphism in six accessions of Vitis vinifera. These results suggest a strong selection pressure on these genes. Functional hypotheses were formulated, and an interaction study with transcription factors VvTGA1 and VvTGA4 was conducted using a method based on yeast two hybrid, showing that they do not interact with VvNPR1.1.

Une large hystérèse centrée autour de la température ambiante constitue l'un des objectifs principaux de la recherche sur les matériaux commutables fonctionnels. Dans le domaine très étudié de la conversion de spin, un tel comportement apparaît très rarement. Un nouveau composé, le complexe [Fe(PM-PeA)2(NCSe)2] présentant une large hystérèse autour de la température ambiante a été synthétisé, sous forme de monocristal et de poudre. Ce composé a été la base de deux axes de recherche. Le premier concerne l’étude multi-échelles des relations structure-propriétés en combinant la diffraction des rayons X sur poudre et sur monocristal, à température variable. Un focus tout particulier a été fait sur l’échelle microstructurale, très peu explorée à ce jour. Pour la première fois,la taille des domaines cohérents et le taux de microdéformations, ont été quantifiés pour un composé moléculaire discret à conversion de spin. Le deuxième axe concerne l’investigation de la transition de spin induite par la pression. L’étude in situ par diffraction des rayons X sur monocristal a permis une caractérisation complète de la structure cristalline des deux états de spin, sous pression. Par la suite, un suivi fin de la transition de spin, révélant une piezo-hystérèse, a été fait grâce à la diffraction des rayons X sur poudre, sous pression in situ à l’aide du rayonnement synchrotron. Les expériences couplant pression et température ont donné accès à des informations cruciales, telles que la variation des modules d’élasticité avec la température, les effets de la température sur la pression de transition, le caractère coopératif de la transition et la largeur de la piezo-hystérèse. / Spin crossover (SCO) compounds with a large hysteresis centered around room temperature (RT) are being constantly pursued although such behavior is very rare and most often noticed in coordination networks. In this context, a new molecular discrete compound, the complex [Fe(PM-PeA)2(NCSe)2], showing a large SCO hysteresis spanning RT has been synthesized in both singlecrystal and powder forms. Then, two research lines emerged. The first one concerns the multi-scales study of the structure-properties relationships, combining single-crystal and powder X-ray diffraction at variable temperature. A particular focus has been made on the microstructural scale, almostunexplored until now. For the first time, the coherent domain sizes and the micro-deformation rate has been quantified for a molecular discrete SCO compound. The second part of this work investigates in detail the pressure-induced SCO. The in situ single-crystal X-ray diffraction studies has been carried out to perform a complete characterization of the crystal structure under pressure. Thereafter, an accurate high-pressure X-ray diffraction measurement on the powder, with a synchrotron radiation,provides a fine track of the pressure-induced SCO and showed a piezo-hysteresis. Experiments coupling pressure and temperature brought crucial information pertaining to variation of bulk moduli withtemperature, the piezo-hysteresis width, the temperature dependence of the pressure transition and pressure-induced SCO abruptness, are provided in detail for the first time.

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.

Frequency-dependent selection has long been a popular heuristic explanation for the maintenance of genetic diversity in natural populations. Indeed, a large body of theoretical and empirical work has already gone into elucidating the causes and consequences of frequency-dependent selection. Most theoretical work, to date, has focused either on the diallelic case, or dealt with only very specific forms of frequency-dependence. A general model of the maintenance of multiallelic genetic diversity has been lacking. Here we extend a flexible general model of frequency-dependent selection, the pairwise interaction model, to the case of multiple alleles. First, we investigate the potential for genetic variation under the pairwise interaction model using a parameter-space approach. This approach involves taking a large random sample of all possible fitness sets and initial allele-frequency vectors of the model, iterating each to equilibrium from each set of random initial conditions, and measuring how often variation is maintained, and by which parameter combinations. We find that frequency- dependent selection maintains full polymorphism more often than classic constant-selection models and produces more skewed equilibrium allele frequencies. Fitness sets with some degree of rare advantage maintained full polymorphism most often, but a variety of non-obvious fitness patterns were also found to have positive potential for polymorphism. Second, we further investigate some unusual dynamics uncovered by the parameter-space approach above. Long-period allele-frequency cycles and a small number of aperiodic trajectories were detected. We measured the number, length and domains of attraction of the various attractors produced by the model. The genetic cycles produced by the model did not have periods short enough to be observable on an ecological time scale. In a real world system, allele-frequency cycling is likely to be indistinguishable from stable equilibrium when observed over short time scales. Third, we use a construction approach to model frequency-dependent selection with mutation under the pairwise interaction model. This approach involves the construction of an allelic polymorphism by bombarding an initial monomorphism with mutant alleles over many generations. We find that frequency-dependent selection is able to generate large numbers of alleles at a single locus. The construction process generates a wide range of allele- frequency distributions and genotypic fitness relationships. We find that constructed polymorphisms remain permanently invasible to new mutants. Analysis of constructed fitness sets may even reveal a signature of positive frequency dependence. Finally, we examine the numbers and distributions of fitnesses and alleles produced by construction under the pairwise interaction model with mutation from existing alleles, using several different methods of generating mutant fitnesses. We find that, relative to more general construction models, generating mutants from existing alleles lowers the average number of alleles maintained by frequency-dependent selection. Nevertheless, while the overall numbers of alleles are lower, the polymorphisms produced are more stable, with more natural allele-frequency distributions. Overall, frequency-dependent selection remains a powerful mechanism for the maintenance of genetic variation, although it does not always work in intuitively obvious ways.