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Com clima diversificado e terras férteis, o Brasil tem vocação natural para o desenvolvimento agropecuário e todas as suas vertentes. Assim, o agronegócio é hoje a principal locomotiva da economia brasileira, representando cerca de um terço do nosso Produto Interno Bruto (PIB). Nesse contexto, o Brasil é o terceiro maior produtor de frutas do planeta, com destaque para a produção de laranjas. Particularmente, o país lidera a produção mundial de suco de laranja e conta com uma participação de 85% nas exportações deste produto. Porém, um dos principais fatores atuais que restringem os lucros e a expansão da citricultura é o controle fitossanitário. Atualmente, dentre as principais doenças podemos destacar o HLB e o cancro cítrico. Ambas, doenças bacterianas que não têm cura comprometem a produção e desenvolvimento da fruta e levam à morte da árvore. Dessa maneira, o monitoramento destas é fundamental para evitar danos aos frutos e a necessidade da erradicação de plantações inteiras. O presente trabalho avaliou o emprego das técnicas de Espectroscopia de Fluorescência Induzida por Laser (LIFS) e Espectroscopia de Emissão Óptica com Plasma Induzido por Laser (LIBS) como forma de diagnóstico destas doenças, se apresentando como uma alternativa às inspeções visuais e ao PCR utilizados atualmente. Para isso, folhas de citros in natura provenientes de plantas sadias, com HLB ou com cancro foram amostradas e medidas com ambos os sistemas. Através do sistema LIFS foi realizado um estudo de precocidade no diagnóstico do HLB. Este sistema fotônico pôde detectar a doença até 21 meses antes do aparecimento dos primeiros sintomas visuais. Foram utilizados classificadores sazonais, criados a partir de Regressão por Mínimos Quadrados Parciais (PLSR) e conjuntos de calibração previamente avaliados. Quanto ao cancro cítrico e o sistema LIFS, taxas de acerto superiores a 90% foram alcançadas nos melhores casos de validação cruzada dos dados. A diferenciação do cancro e do HLB também foi possível pela mesma técnica ao avaliar um conjunto pequeno de dados, que atingiu uma taxa de acerto de 82%. Através dos espectros obtidos pelo sistema LIBS, avaliou-se as variações nutricionais causadas na planta devido à doença. Por meio de tais variações e métodos de PLSR foi construído um modelo para o diagnóstico de HLB alcançando taxas de acerto da ordem de 75%. Em relação ao cancro cítrico e o sistema LIBS, um estudo preliminar foi realizado, e uma taxa de acerto superior a 90% foi atingida. Por fim, os resultados corroboraram com a ideia inicial de se realizar o diagnóstico do HLB e do cancro cítrico através de técnicas fotônicas. Dentre outras vantagens estas permitem uma análise rápida, in loco, sem a necessidade de preparo de amostra. Além disso, para o HLB, as técnicas fotônicas demonstraram menor sensibilidade à distribuição não homogênea da doença, quando comparada com a técnica de referência (qPCR). / The diverse climate and fertile soils make Brazil a country with a natural vocation for agricultural development. Thus, agribusiness is now the main locomotive of the Brazilian economy, accounting for about one-third of our Gross Domestic Product (GDP). In this context, Brazil is the third largest fruit producer in the world, with emphasis on the orange production. Particularly, the country leads the world production of orange juice, and with a stake of 85% in exports of this product. However, one of the main factors that restrict current profits and the expansion of citrus production is phytosanitary control. Currently, among the major diseases we highlight the HLB (Greening) and Citrus Canker, two bacterial diseases that have no cure and affect production and fruit development. Therefore, monitoring is essential to prevent damage to the fruits and the complete eradication of infected orchards. The present study evaluated the use of Laser-Induced Fluorescence Spectroscopy (LIFS) and Laser-Induced Breakdown Spectroscopy (LIBS) techniques as alternative diagnostic methods to visual inspection and PCR technique, currently used. For that, citrus leaves from healthy, HLB or citrus canker infected plants, were sampled and measured with both systems. Through LIFS system, a study of the HLB early diagnosis was done. This photonic system could detect the disease up to 21 months before the tree show the visual symptoms. Seasonal classifiers were used, which were designed from Partial Least Square Regression (PLSR) methods and calibration database previously acquired. Regarding citrus canker and LIFS system, success rates higher than 90% were achieved in the best cases. The differentiation between citrus canker and HLB was also possible using the same technique reaching a success rate around 82%. Through the spectrum obtained by LIBS system, it was evaluated the nutritional variations caused in the plant due to HLB, and based on these data, the diagnosis was done. The average success rate was 75%, which was achieved by PLSR model. Regarding on citrus canker and LIBS system, a preliminary study was carried out and a success rate greater than 90% was achieved. Finally, the results corroborated with the use of photonic techniques for the HLB and citrus canker diagnosis. Among other advantages, they allow rapid analysis, in loco and without the need of sample preparation. In addition, for the HLB, photonics techniques showed lower sensitivity to the non-homogeneous distribution of the disease when compared with the reference technique (qPCR).

Les gliomes sont des tumeurs cérébrales infiltrantes difficilement curables, notamment à cause de la difficulté à visualiser toutes les infiltrations au bloc opératoire. Dans cette thèse, nous réalisons une étude clinique de spectroscopie de fluorescence de la protoporphyrine IX (PpIX) dans les gliomes de 10 patients selon l’hypothèse que les spectres collectés proviennent de la contribution de 2 états de la PpIX dont les proportions varient suivant la densité en cellules tumorales. Après avoir présenté le développement du système interventionnel proposant une excitation multi-longueurs d’onde, nous présentons son utilisation sur fantômes de PpIX mimant les propriétés des gliomes. Ceci permet tout d’abord d’obtenir les spectres émis par les 2 états séparément puis de proposer un modèle d’ajustement des spectres comme une combinaison linéaire des 2 spectres de référence sur la bande spectrale 608-637 nm. Ensuite, nous présentons la mise en place de l’étude clinique, notamment l’analyse de risques, avant d’appliquer ce système in vivo. Les mesures in vivo détectent de la fluorescence dans des tissus où le microscope chirurgical n’en détecte pas, ce qui pourrait s’expliquer par un changement d’état de la PpIX entre le cœur des gliomes et leurs infiltrations. L’intérêt de l’excitation multi-longueurs d’onde est démontré par la décroissance de la corrélation des spectres acquis aux trois excitations suivant la densité en cellules tumorale. Enfin, nous soulevons des pistes d’étude de l’identification peropératoire des zones de fonctionnalité cérébrale à l’aide d’une caméra optique ainsi que l’étude du temps de vie de fluorescence et de la fluorescence deux photons de la PpIX sur fantômes / Gliomas are infiltrative tumors of the brain which are yet hardly curable, notably because of the difficulty to precisely delimitate their margins during surgery. Intraoperative 5-ALA induced protoporphyrin IX (PpIX) fluorescence microscopy has shown its relevance to assist neurosurgeons but lacks sensitivity. In this thesis, we perform a spectroscopic clinical trial on 10 patients with the assumption that collected fluorescence is a linear combination of the contribution of two states of PpIX which proportions vary with the density of tumor cells. This work starts with the development of the intraoperative, portable and real time fluorescence spectroscopic device that provides multi-wavelength excitation. Then, we show its use on PpIX phantoms with tissues mimicking properties. This first enables to obtain a reference emitted spectrum for each state apart and then permits the development of a fitting model to adjust any emitted spectrum as a linear combination of the references in the spectral band 608-637 nm. Next, we present the steps led to get approvals for the clinical trial, especially the risk analysis. In vivo data analysis is then presented, showing that we detect fluorescence where current microscopes cannot, which could exhibit a change in PpIX state from glioma center to its margins. Besides, the relevance of multi-wavelength excitation is highlighted as the correlation between the three measured spectra of a same sample decreases with the density of tumor cells. Finally, the complementary need to intraoperatively identify cerebral functional areas is tackled with optical measurements as a perspective and other properties of PpIX on phantoms are also raised

Ce travail présente une étude, par microscopie optique et de fluorescence, de matériaux hybrides, basés sur des monolithes de silice contenant des dérivés du cyano-anthracène : 9,10-dicyano-anthracène (DCA) ou 9,14-dicyano-benzo(b)triphénylène (DBTP), qui sont des photo-sensibilisateurs de l'oxygène singulet. Alors que ces matériaux sont bien caractérisés du point de vue de la photo-oxydation des sulfures sous des conditions hétérogènes par des études macroscopiques, certaines propriétés concernant l'association du photosensibilisateur avec l'absorbant peuvent être masquées, dominées ou encore mal interprétées par uniquement des mesures d'ensemble. Ici, nous combinons la spectroscopie d'ensemble et la microscopie optique et de fluorescence, et développons des protocôles expérimentaux concernant des échantillons solides épais, dans le but d'étudier la distribution spatiale et la mobilité des photosensiblisateurs dans la matrice hôte ainsi que d'analyser les interactions entre ces deux entités. La microscopie optique montre dans tous les cas des inhomogénéités localisées à l'interface du monolithe et attribuées à la formation de bulles pendant la synthèse et une accumulation locale du DBTP. A partir de simulations Monte-Carlo de lancer de rayons, nous développons un protocôle pour corriger les artéfacts de réfraction dans les profils d'intensité de fluorescence en fonction de la profondeur, obtenus par des mesures confocales, pour déterminer la distribution axiale du photosensibilisateur ce qui permet de mettre en évidence une nette augmentation de la concentration en photosensibilisateurs dans les premiers 50—100 m dessous la surface. L'analyse FRAP montre la très lente mobilité de tous les photosensibilisateurs et un retour partiel de l'intensité ce qui signifie que les photosensibilisateurs se trouvent dans des régions compartimentées, probablement dues à des contraintes aléatoires du réseau de pores. De plus, l'analyse FLIM montrent des propriétés photo physiques semblables pour le DBTP inclus et greffé ce qui permet d'envisager l'inefficacité de la fonctionnalisation. Ces observations soulignent que les monolithes à base de silice sont des systèmes hors d'équilibre et correspondent à un instantané des inhomogénéités gelées pendant les derniers instants du processus de condensation-hydrolyse des monomères de silice. Enfin, corréler la spectroscopie classique avec nos observations confocales sur différentes formes de DBTP, nous permet d'établir que la bande d'émission de fluorescence non-structurée et fortement déplacée vers le rouge est probablement due à la formation d'excimères. / This work presents an optical and fluorescence microscopy study of hybrid materials based on porous silica monoliths containing derivatives of cyano-anthracene: 9,10-dicyano-anthracene (DCA) or 9,14-dicyano-benzo(b)triphenylene (DBTP), photo-sensitizers of singlet oxygen. While these materials are well known from bulk studies for the efficient photo-oxidation of sulphides under heterogeneous conditions, some characteristics of the association of the photo-sensitizer and the absorbent may be masked, overlooked or otherwise misinterpreted by bulk investigations alone. Here, we combine classical bulk spectroscopy with optical and fluorescence microscopy, and develop experimental protocols for thick solid state samples, to study the spatial distribution and the mobility of the guest in the host matrix, and analyse guest-host interactions. Optical microscopy shows in all cases localised inhomogeneities at monolith interface, ascribed to bubble formation during synthesis; wide-field fluorescence microscopy shows that these features are associated with local accumulation of the larger, more hydrophobic of the two photo-sensitizers, DBTP. Photo-sensitizer lateral distribution at the monolith interface is otherwise homogeneous. Based on Monte Carlo ray-tracing simulations, we develop a protocol for correcting refraction artefacts in measured confocal fluorescence depth profiles, to obtain the photo-sensitizer axial distribution. While it in general exhibits a sharp increase in concentration in the first 50—100 m below the surface compared to the bulk, this layer contributes negligibly to the total content of the monoliths. FRAP analysis shows mobility of the photo-sensitizers in all cases, but with diffusion constants implying months or years to equilibrate the centimetre-sized monoliths. Classical bulk and confocal spectroscopy with FLIM analysis show similar photo-physical properties of DBTP included and grafted. The main effects of funcionalization in this photo-sensitizer are to slow down diffusion and to counter its aggregation. Incomplete FRAP recovery implies photo-sensitizer mobility is compartmented, probably due to random constrictions in the pore network. These observations underline that silica-based monoliths are non-equilibrium systems encapsulating a snapshot of any homogeneities frozen in during the later stages of hydrolysis-condensation of silicate units. Correlating classical bulk spectroscopy with our confocal observations on the different DBTP forms, conclude that its unusual structureless, red-shifted emission is probably due to excimer emission.

Les triterpènes sont un groupe important et structurellement diversifié de produits naturels issus du squalène. Les progestatifs et les glucocorticoïdes sont des triterpènes oxygénés ayant un squelette tétracyclique et reconnus pour leurs diverses propriétés thérapeutiques. Par ailleurs, l'érythrodiol et l'uvaol sont des triterpènes pentacycliques reconnus pour leurs effets bénéfiques dans l'alimentation humaine. Dans ce travail, l'interaction avec les membranes des vésicules lipidiques et la liaison à la sérum albumine humaine de ces molécules sont étudiées dans le but de mieux comprendre leurs propriétés pharmacologiques. Les vésicules lipidiques ont été caractérisées par DSC, spectroscopie Raman, FTIR et polarisation de fluorescence du DPH pour comprendre l'effet des molécules sélectionnées sur la fluidité membranaire.Également, nous avons étudié la liaison du cholestérol, d'une série de progestatifs et de glucocorticoïdes à l'albumine humaine par la spectroscopie de fluorescence.Les résultats ont démontré que les progestatifs, les glucocorticoïdes, l'érythrodiol et l'uvaol altèrent les propriétés physiques de la bicouche lipidique.Les progestatifs et les glucocorticoïdes démontrent un attachement modéré à l'albumine. Par ailleurs, la dydrogestérone présente la constante de liaison la plus importante. Enfin, notre étude a démontré que la constante de liaison du cholestérol à l'albumine est faible en comparaison avec les autres molécules étudiées. Notre étude a conduit à une connaissance approfondie des mécanismes moléculaires et des caractéristiques structurales impliqués dans l'interaction des triterpènes avec les protéines et les membranes synthétiques / The triterpenoids are a large and structurally diverse group of natural products derived from squalene. Progesterone derivatives and glucocorticoids are a group of oxygenated triterpenes having a tetracyclic skeleton and identified for their therapeutic properties. Whereas, erythrodiol and uvaol are pentacyclic triterpenes, known for their beneficial effects on human diet. In this thesis, we studied their interaction with the membranes of lipid vesicles and with human serum albumin to better understand their pharmacological properties. DSC, Raman spectroscopy, FTIR and fluorescence polarization of DPH were used to investigate the effect of triterpenes on the membrane fluidity. Besides, we used fluorescence spectroscopy to study the binding of cholesterol, a series of progesterone derivatives and another series of glucocorticoids to albumin. The results revealed that progesterone derivatives, glucocorticoids, erythrodiol and uvaol changed the physical properties of the bilayers. Progesterone derivatives and glucocorticoids have been proven to bind moderately to albumin. Dydrogesterone showed the highest binding constant. Finally, our study demonstrated that cholesterol exhibited a much weaker interaction with albumin compared to progesterone derivatives and glucocorticoids.Our work has led to a better understanding of triterpenes molecular mechanisms of their interaction with proteins and biological membranes and structural features controlling these interactions

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-13T21:33:03Z No. of bitstreams: 2 Dissertação - Adriana Pereira Mundim Guedes - 2013.pdf: 2999561 bytes, checksum: 755cb864a8446e6ff5c334be00ea5367 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-16T18:47:44Z (GMT) No. of bitstreams: 2 Dissertação - Adriana Pereira Mundim Guedes - 2013.pdf: 2999561 bytes, checksum: 755cb864a8446e6ff5c334be00ea5367 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-16T18:47:44Z (GMT). No. of bitstreams: 2 Dissertação - Adriana Pereira Mundim Guedes - 2013.pdf: 2999561 bytes, checksum: 755cb864a8446e6ff5c334be00ea5367 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-09-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Motivated by the perspective of ruthenium complexes to be used in cancer treatment, our research group has tested the hipotesis that some complexes of Ru (III) are able to interact with serum proteins, particularly albumin and transferrin. The Complex cis- [RuCl2(NH3)4]Cl (CTRu(III)) have been tested against different kind of tumor cells, obtaining good results. Starting from promising results obtained with this compound, subsequent studies are required to understanding the mechanism by which it exerts specificity for tumor cells. In this article, we report the first application of absorption UV-Vis, Fluorescence and Electron Paramagnetic Resonance (EPR) spectroscopy, to study the complex CTRu(III) interaction with human serum albumin (hsA) and bovine serum albumin (bsA). Fluorescence measurements revealed strong proteinsbound complex with Ksv of 1.32 x 105 and 3.71 x 105 for hsA and bsA, respectively. EPR spectra from mono-nuclear Ru(III) complexes in buffer, showed a significant decrease in the overall signal intensity following the first aquation step, is consistent with the formation of oxo-bridged Ru(III) dimers. EPR spectra revealed that the BSA very rapid binding to the protein via covalent binding through ligand-exchange with protein side chains, likely with histidine imidazoles. On the other hand, the complex binds non-covalently in hsA, probably as a product of the oligomerization of the complex in hemin-biding pocket. Furthermore, two species are slowly formed by covalent binding of the complex with the histidine residues, producing a species of axial symmetry and the other rhombic symmetry. These bonds seem to arise from the interaction of the complex with the histidine residue located in the binding Sudlow’s site II. / Motivado pela perspectiva de complexos de rutênio podem ser utilizados no tratamento do câncer, o nosso grupo de pesquisa testou a Hipótese que alguns complexos de Ru (III) são capazes de interagir com as proteínas do soro, particularmente albumina e transferrina. O complexo de cis-[RuCl2(NH3)4]Cl (CTRu(III)) foi testado contra diferentes tipos de células tumorais, obtendo bons resultados. A partir de resultados promissores obtidos com este composto, estudos subsequentes são necessários para a compreensão do mecanismo pelo qual ele exerce sua especificidade para células de tumor. Neste artigo, apresentamos a aplicação de espectroscopia de absorção UV-vis, fluorescência e ressonância paramagnética eletrônica (RPE), para estudar a interação do complexo CTRu(III) com albumina sérica humano (hsA) e a albumina sérica bovina (bsA). Medidas de fluorescência revelaram uma forte ligação do complexo com as proteínas com Ksv de 1,32 x 105 e 3,71 x 105 para hsA e bsA, respectivamente. Espectros de RPE de complexos de Ru (III) mono-nucleares em tampão mostraram um decréscimo significativo na intensidade do sinal global após a primeira passo de aquação, que é consistente com a formação de dímeros de oxo complexos de Ru (III). Os espectros de RPE revelaram que a ligação à bsA é muito rápida, a ligação covalente à proteína ocorre através de troca dos ligantes com cadeias laterais de proteínas, provavelmente com o imidazol da histidina. Por outro lado, o complexo se liga não covalentemente na hsA, provalente como produto da oligomerização do complexo no bolso de ligação hemin. Além disso, duas espécies são formadas lentamente por ligação covalente do complexo com os resíduos histidina, produzindo uma espécie de simetria axial e a outra de simetria rômbica. Essas ligações parecem surgir pela interação do complexo com o resíduo histidina localizado no sítio de ligação Sudlow II.

The peripheral skin microcirculation reflects the overall health status of the cardiovascular system and can be examined non-invasively by laser methods to assess early cardiovascular disease (CVD) risk factors, i.e. oxidative stress and endothelial dysfunction. Examples of methods used for this task are the laser Doppler flowmetry (LDF) and laser fluorescence spectroscopy (LFS), which respectively allow tracing blood flow and the amounts of the coenzyme NAD(P)H (nicotamide adenine dinucleotide) that is involved in the cellular production of ATP (adenosine triphosphate) energy. In this work, these methods were combined with iontophoresis and PORH (post-occlusive reactive hyperaemia) reactive tests to assess skin microvascular function and oxidative stress in mice and human subjects. The main focus of the research was exploring the nonlinear dynamics of skin LDF and NAD(P)H time series by processing the signals with the wavelet transform analysis. The study of nonlinear fluctuations of the microcirculation and cell energy metabolism allows detecting dynamic oscillators reflecting the activity of microvascular factors (i.e. endothelial cells, smooth muscle cells, sympathetic nerves) and specific patterns of mitochondrial or glycolytic ATP production. Monitoring these dynamic factors is powerful for the prediction of general vascular/metabolic health conditions, and can help the study of the mechanisms at the basis of the rhythmic fluctuations of micro-vessels diameter (vasomotion). In this thesis, the microvascular and metabolic dynamic biomarkers were characterised <i>in-vivo</i> in a mouse model affected by oxidative stress and a human cohort of smokers. Data comparison, respectively, with results from control mice and non-smokers, revealed significant differences suggesting the eligibility of these markers as predictors of risk associated with oxidative stress and smoke. Moreover, a relevant link between microvascular and metabolic oscillators was observed during vasomotion induced by α-adrenergic (in mice) or PORH (in humans) stimulations, suggesting a possible role of cellular Ca²⁺oscillations of metabolic origin as drivers of vasomotion which is a theory poorly explored in literature. As future perspective, further exploration of these promising nonlinear biomarkers is required in the presence of risk factors different from smoke or oxidative stress and during vasomotion induced by stimuli different from PORH or α-adrenergic reactive challenges, to obtain a full picture on the use of these factors as predictors of risk and their role in the regulation of vasomotion.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand". Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

Les isoenzymes de nucléoside diphosphate kinase (NDPK) sont connues depuis maintenant presque 60 ans et n'ont été considérées que pour leur activité catalytique de transfert de groupement phosphoryle. La découverte du gène nme, un gène antimétastatique codant une NDPK, a renouvelé l'intérêt scientifique pour cette famille d'enzymes. Il est désormais connu que la multiplication des gènes durant l'évolution a été accompagnée de diversifications structurales et fonctionnelles. J'ai étudié la fixation des NDPK-A, -B et –D (retrouvées associées aux membranes biologiques, bien que le rôle de cette association soit encore méconnu) à des membranes modèles, et j'ai trouvé des différences dans les mécanismes de fixation. J'ai montré la capacité de la NDPK-D, isoforme mitochondriale, à interagir avec des membranes anioniques ou zwitterioniques, à augmenter leur fluidité et à former des domaines protéolipidiques en présence de CL, lipide anionique spécifique de la membrane mitochondriale interne. J'ai observé cette capacité à former des domaines protéolipidiques avec d'autres protéines interagissant avec la CL, comme la créatine kinase mais pas le cytochrome C. La NDPK-A ne se fixe pas aux phospholipides du feuillet interne de la membrane plastique, ce qui suggère un autre partenaire in vivo. La NDPK-B n'interagit qu'avec des membranes anioniques via un processus en deux étapes, provoque une diminution de fluidité et est capable de former des domaines protéolipidiques. La ségrégation des lipides anioniques induite par la fixation de protéines pourrait contribuer à la formation de plateformes au sein de la membrane susceptibles de servir de point d'ancrage à de nombreuses molécules, modulant ainsi les fonctions cellulaires / Nucleoside diphosphate kinase isoenzymes (NDPK) have been known for nearly 60 years and, until recently, have been considered as housekeeping enzymes. The discovery of a nme gene, an antimetastatic gene that codes for a NDPK, revived the interest for this family. It is now known that the multiplication of nme genes throughout evolution has been accompanied with structural and functional diversification. I studied the binding of NDPK-A, -B and –D (which ae retrieved associated to cellular membranes where they are thought to play several roles) to model membranes and found differences in their behavior towards different compositions of phospholipids. I showed the ability of the NDPKD mitochondrial isoform to interact with both anionic and zwitterionic membranes, to modify their fluidity and to form proteolipidic domains in presence of CL, a mitochondrial inner membrane specific anionic lipid. I observed this ability to form proteo-cardiolipin domains with other CL interacting protein like creatine kinase but not with cytochrome c. NDPK-A was not able to bind to inner leaflet plasma membrane mimicking systems suggesting another partner in vivo. Concerning NDPK-B, it interacted only with anionic membranes via a two step-process, induced a decrease of the membrane fluidity and was able to form proteolipidic domains. Such anionic lipid segregation triggered by protein binding may contribute to platforms formation within membranes. Those platforms are then susceptible to provide a functional docking platform for numerous molecules and thus to modulate cellular functions