Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-04-19T17:09:39Z No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-23T11:47:10Z (GMT) No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-23T11:47:10Z (GMT). No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-13 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Paracoccidioidomycosis (PCM) is an important mycosis of Latin America, caused by thermodymorphic fungi of the genus Paracoccidioides. Homeostasis of metals such as copper, zinc and iron is important for the survival of fungi in the host environment. In this context, copper is an important cofactor for several enzymes, such as as superoxide dismutases and cytochrome c oxidase. The excess free copper in the cell promotes accumulation of reactive oxygen species, causing damage to nucleic acids, lipids and proteins. Thus, organisms shall to maintain cytoplasmic levels of that micronutrient at non-toxic levels, just sufficient for cell growth and vital metabolic processes. The metabolism of that metal is strictly controlled by high and low affinity uptake systems. The objective of this work is to perform heterologous expression of the Atx1 protein, described in the literature as a copper chaperone, and to analyze its cellular location, as well as the protein behavior upon copper deprivation in Paracoccidioides spp. Heterologous expression was performed on electrocompetent cells of Escherichia coli strain BL21. Identity of the recombinant protein was confirmed by LC-MS / MS. BALB/c mice were immunized to obtain anti-Atx1 polyclonal antibodies. After performing immunoblotting the produced antibodies were used in immunofluorescence assays. Analysis by qRT-PCR allowed us to evaluate the levels of the transcript encoding the Atx1 protein during copper deprivation in Paracoccidioides spp. / A paracoccidioidomicose (PCM) é uma importante micose da América Latina, causada por fungos termodimórficos do gênero Paracoccidioides. A homeostase de metais como cobre, zinco e ferro é importante para a sobrevivência dos fungos no ambiente do hospedeiro. Nesse contexto, o cobre é um importante cofator para várias enzimas, como as superóxido dismutases e a citocromo c oxidase. O excesso de cobre livre na célula promove o acúmulo de espécies reativas de oxigênio, causando danos a ácidos nucléicos, lipídeos e proteínas. Assim, os organismos devem manter concentrações citoplasmáticas desses micronutrientes em níveis não tóxicos, apenas suficiente para o crescimento celular e processos metabólicos vitais. O metabolismo desse metal é rigorosamente controlado por sistemas de captação de alta e baixa afinidade. O objetivo do trabalho é realizar a expressão heteróloga da proteína Atx1, descrita na literatura como uma chaperona citosólica de cobre e analisar a sua localização celular, bem como o comportamento diante da privação de cobre em Paracoccidioides spp. A expressão heteróloga foi realizada em células eletrocompetentes de Escherichia coli, cepa BL21. A identidade da proteína recombinante foi confirmada por LC-MS/MS. Camundongos BALB/c foram imunizados para obtenção de anticorpos policlonais anti-Atx1. Após realização de imunoblotting, os anticorpos produzidos foram utilizados para ensaios de imunofluorescência em células leveduriformes. Análise por qRT-PCR nos permitiram avaliar os níveis do transcrito codificante da proteína Atx1 durante a privação de cobre em Paracoccidioides spp.

No description available.

<p>Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. </p><p>The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.</p><p>In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.</p><p>In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. </p><p>In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.</p>

Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development. In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

No description available.

Aquesta tesi es centra en la caracterització funcional d'una proteïna de xoc de calor de baix pes molecular (Small Heat Shock Protein - sHSP) de classe I de surera pel que fa a la seva capacitat per protegir les cèl·lules de l'estrès i per estabilitzar les membranes. Les sHsps són proteïnes que s'expressen en condicions d'estrès cel·lular. Encara que certs aspectes funcionals de les sHsps són ben coneguts, el nostre treball aporta informacions noves sobre el paper de les diferents regions de la proteïna, especialment de la regió N-terminal.L'objectiu concret d'aquest treball és determinar la funció termoprotectora de QsHsp17.4-CI, una sHsp de classe I oobtinguda a partir de les cèl·lules de fel·lema d'alzina surera, en un model bacterià i analitzar la importància de les diferents regions de la proteïna en aquesta funció. Amb aquesta finalitat s'han dissenyat dues proteïnes parcials derivades de QsHsp17.4-CI: una a la que li falta la regió N-terminal (C105) i una altra amb pràcticament tot el domini &#61537;-cristal·lí deleccionat (N61), i una tercera, derivada de QsHs10-CI, a la que li falta la meitat del domini &#61537;-cristal·lí (Hsp10). També s'estudia la possible capacitat estabilitzadora de membranes i la capacitat de modificar l'expressió d'altres Hsps quan s'expressa de forma heteròloga.Els nostres resultats demostren que l'expressió de QsHsp17.4-CI protegeix a les cèl·lules d'E.coli de l'estrès tèrmic alhora que la regió N-terminal i la regió consens II del domini &#61537;-cristal·lí són imprescindibles per aquesta funció de protecció. En relació a un possible paper en les membranes, els estudis de localització subcel·lular mostren que QsHsp17.4-CI colocalitza amb la fracció membranes i que la regió N-terminal de la proteïna és responsable d'aquesta colocalització. No s'ha pogut demostrar, però, que la localització amb la membrana estigui associada a un efecte protector d'aquesta: en cap cas la sobrexpressió de les proteïnes modifica la composició d'àcids grassos i només N61, que no té acció termoprotectora, altera l'estat fisico-químic de la membrana. En estudis d'expressió de novo en E.coli s'ha observat que, a diferència de les altres proteïnes heteròlogues, N61 activa l'expressió de la majoria de Hsps d'E.coli fent pensar en una possible relació entre l'estat físic de la membrana i l'activació de la resposta a l'estrès.En resum, en aquest treball hem provat la capacitat protectora de QsHsp17.4 i aportem noves dades sobre la importància de la regió N-terminal i la regió consens II del domini &#61537;-cristal·lí en aquesta funció. Per altra banda, es suggereix que QsHsp17.4 podria interaccionar amb la membrana d'E.coli i que la regió N-terminal seria imprescindible per aquesta interacció. Finalment hem determinat que les proteïnes que provoquen variacions en l'estat de fluïdesa de la membrana poden activar la resposta al xoc de calor per part de la cèl·lula bacteriana. / This thesis is focused in the functional studies of a Small Heat Shock Protein (sHsp). sHsps are expressed under stress conditions. Although some functional aspects of these proteins are known, our work aport new data about the role of the different protein regions, especially the N-terminal region. The aim of this work is to demonstrate a thermotolerance effect of QsHsp17.4-CI in bacterial cells and to analyze the importance of the protein regions in this function. To achieve this objective two deletion mutants derived from QsHsp17.4-CI were designed: a protein lacking the N-terminal region (C105) and a protein where the entire &#61537;-cristallin domain is missing (N61) and a third mutant, derived from QsHsp10-CI, that bears half of the &#61537;-cristallin domain (Hsp10). To better understand the functional mechanism of sHsps we study the membrane stabilizing capacity of QsHsp17.4-CI as well as its capacity to modify other Hsps expression.Our results demonstrate that the expression of QsHsp17.4-CI protects E.coli cells from a heat shock and that the N-terminal region and the consensus region II of the &#61537;-cristallin domain are necessary for this protective function. Related to a possible role in membranes, location studies suggest that QsHsp17.4-CI colocalizes with cell membrane fraction and that N-terminal region is important for this location. However, no relation between membrane localization and a protective effect has been demonstrated: Protein overexpression does not modify membrane fatty acid composition and only N61, which has no thermoprotection, changes membrane physical state. Studies of E.coli de novo synthesis show that, unlike the other recombinant proteins, the overexpression of N61 activates the expression of almost all E.coli Hsps suggesting a possible relation between membrane physical state and the activation of the heat shock response.As summary, in this work we have demonstrated the thermoprotective capacity of QsHsp17.4-CI and we contribute with new data about the importance of N-terminal region and consensus region II of &#61537;-cristallin domain for this function. On the other hand, we suggest the possibility that QsHsp17.4-CI interacts with membrane and that N-terminal region is important for this interaction. Lastly, we have observed how changes in membranes fluidity state can activate heat shock response in bacterial cells.

No description available.

Les glycosidases sont des enzymes impliquées dans de nombreux processus biologiques. Entre autres, elles sont responsables de la dégradation des déchets polysaccharidiques de nos cellules. Lorsqu’une modification génétique touche un gène qui code pour une de ces enzymes, des pathologies graves regroupées sous l’appellation de « maladies lysosomales » peuvent être déclenchées. L'objectif de ce projet a été de proposer une méthode de synthèse efficace de molécules potentiellement actives spécifiquement sur l'une ou l'autre de ces maladies. Les molécules ciblées sont des inhibiteurs de glycosidases de la famille des aminocyclitols, utilisés dans une stratégie thérapeutique émergente « par molécules chaperonnes ». La méthode de synthèse développée s’appuie sur une étape enzymatique clé utilisant les aldolases comme catalyseurs et répondant aux contraintes environnementales actuelles de la chimie verte. Nous avons atteint nos objectifs grâce à l’utilisation de trois aldolases différentes, produites et purifiées pour la première fois au sein de notre laboratoire. Il s’agit de la fuculose-1-phosphate aldolase F1PA, de la rhamnulose-1-phosphate aldolase R1PA et de la nouvellement découverte fructose-6-phosphate aldolase FSA. La formation d’une quarantaine de nitrocyclitols, de stéréochimies définies, précurseurs des aminocyclitols correspondant, a ainsi été réalisée avec de très bons rendements de synthèse. / Glycosidases are enzymes involved in many biological processes. For example, they are responsible for breaking up polysaccharide waste materials of our cells. When a genetic mutation concerns a gene encoding for one of theses enzymes, acute pathologies named lysosomal storage disorders can appear. Aim of this work was to find an effective synthesis method of molecules potentially active specifically on one or others diseases. Target molecules are glycosidases inhibitors from the aminocyclitols family, used in an emergent strategy “by molecular chaperones”. The method of synthesis developed in the course of this work is based on an enzymatic key step using aldolases as catalyst, and follows current environment constraints of the green chemistry concept. Goals were reached thanks to the use of three different aldolases, produced and purified for the first time in our lab. It consists in fuculose-1-phosphate aldolase F1PA, rhamnulose-1-phosphate aldolase R1PA and the newly discovered fructose-6-phosphate aldolase FSA. Formation of around forty nitrocyclitols (aminocyclitols precursors) with a defined stereochemistry was realised with very good yields of synthesis.