A soja (Glycine max) contém uma variedade de compostos com comprovada atividade biológica, tais como as isoflavonas, que estão presentes em diferentes formas, glicosiladas e agliconas. Além disso, a soja contém uma grande quantidade de proteínas, que são consideradas fontes de peptídeos bioativos. As isoflavonas agliconas, daidzeína e genisteína, possuem maior atividade antioxidante que as glicosiladas, daidzina e genistina. No entanto, os grãos de soja são ricos nas formas glicosiladas das isoflavonas. Estudos mostram que a biotransformação da soja, por micro-organismos e enzimas, leva ao aumento dos teores das isoflavonas agliconas, as quais são liberadas pela ação de enzimas -glicosidases, que clivam as ligações -glicosídicas das isoflavonas glicosiladas, e também pode possibilitar a hidrólise das proteínas da soja. Além disso, pesquisadores têm demonstrado aumento na atividade antioxidante e na prevenção e/ou supressão de certos cânceres após biotransformação da soja. Neste contexto, foi realizada a biotransformação da soja pelo fungo A. awamori, e por uma mistura enzimática, proveniente do processo fermentativo deste fungo na soja. Os extratos da soja biotransformada, não biotransformada, e o extrato comercial isoflavin beta®, rico em isoflavonas, foram avaliados quanto aos perfis cromatográficos, teores de daidzeína, genisteína, proteínas, aminoácidos e/ou peptídeos, potencial antioxidante e atividade citotóxica frente a células de fibroblasto e melanoma. O modo de morte celular das células de melanoma, necrose ou apoptose, também foi avaliado. A biotransformação da soja, pelos dois processos, resultou em extratos enriquecidos com isoflavonas agliconas e aminoácidos e/ou peptídeos, e com maior atividade antioxidante que o extrato da soja não biotransformada. Os dois processos de biotransformação da soja resultaram em extratos com características químicas e biológicas diferentes. O conteúdo de daidzeína, proteínas, aminoácidos e/ou peptídeos encontrados no extrato da soja biotransformada pelo fungo foram 6%, 56% e 357%, respectivamente, superiores ao extrato da soja biotransformada pela mistura enzimática. Ao contrário do observado para o teor de genisteína que foi 48% maior no extrato da soja biotransformada pela mistura enzimática. O extrato da soja biotransformada pelo fungo apresentou maior atividade antioxidante que o extrato da soja biotransformada pela mistura enzimática, além disso, foi o único dos extratos aqui estudados que apresentou citotoxicidade seletiva para as células de melanoma, induzindo morte celular por apoptose destas células cancerosas. Sendo assim, os resultados obtidos pelo extrato da soja biotransformada pelo fungo A. awamori fornecem boas perspectivas para futura utilização deste extrato como antitumoral. / Soybean (Glycine max) contains a variety of compounds with proven biological activity, such as isoflavones, which are present in different forms, glycosides and aglycones. In addition, soybean contains a lot of proteins, which are considered sources of bioactive peptides. The aglycone isoflavones, daidzein and genistein, have higher antioxidant activity than the glucoside ones, daidzin and genistin. However, soybean grains are rich in the glycosylated forms of isoflavones. Studies have shown that the soybean biotransformation, by microorganisms and enzymes, lead to increased levels of aglycone isoflavones, which are released by the action of -glycosidase enzymes, which cleave the -glycosidic bonds of isoflavone glucosides, and can also allow the hydrolysis of soybean proteins. Additionally, researchers have shown an increase in the antioxidant activity and in the prevention and/or suppression of certain cancers after soybean biotransformation. In this context, it was performed the biotransformation of soybeans with the fungus A. awamori, and with an enzyme mixture, from the fermentation process of the fungus in soybean. The biotransformed, the non biotransformed soybean extracts and the marketed isoflavin beta® extract rich in isoflavones, were evaluated regarding their chromatographic profiles, levels of daidzein, genistein, proteins, amino acids and/or peptides, the antioxidant potential and the cytotoxic activity against melanoma cells and fibroblasts. The mode of cell death of melanoma cells, necrosis or apoptosis, was also evaluated. The biotransformation of soybean by the two processes resulted in extracts enriched with aglycone isoflavones and aminoacids and/or peptides, and with antioxidant activity higher than the non biotransformed soybean extract. The two processes of soybean biotransformation resulted in extracts with different chemical and biological characteristics. The contents of daidzein, proteins, aminoacids and/or peptides found in soybean extract biotransformed by the fungus were 6%, 56% and 357%, respectively, higher than the soybeam extract biotransformed by the enzyme mixture. Contrary to what was observed with the genistein content that was 48% higher in the soybean extract biotransformed by the enzyme mixture. The soybean extract biotransformed by the fungus had a higher antioxidant activity than the soybean extract biotransformed by the enzyme mixture, moreover, it was the unique extract among the ones studied in this work that showed selective cytotoxicity to melanoma cells, inducing cell death by apoptosis of these cancer cells. Thus, the obtained results of the soybean extract biotransformed by the fungus A. awamori provide good prospects for future use of this extract as antitumoral.

The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost of the fungal enzymes usually employed in this process remains a significant bottleneck for manufacturing low value-added products from biomass. A potential route to increase hydrolysis yield, and thereby to reduce hydrolysis cost, would be to supplement the fungal enzymes with their lacking enzymatic activities, such as Beta-glucosidase. To produce such enzymes at a low cost, the bacterium Escherichia coli is a strong contender, owing to its ability to grow rapidly on simple and inexpensive media, and to achieve high levels of productivity. Nevertheless, there is hardly any techno-economic analysis of low-value protein production using E. coli in the literature, and, more generally, there are very few techno-economic analyses of low-value protein production ever reported, with the exception of cellulase production by Trichoderma reesei. In particular, the biotechnological application of recombinant E. coli platforms equipped with toxin-antitoxin systems to ensure plasmid stability remains largely unexplored, and its economic impact, unknown. As such, this work presents a comprehensive techno-economic analysis of the industrial production of a low-cost enzyme (Beta-glucosidase) using both E. coli BL21(DE3) and E. coli SE1, a modified BL21(DE3) strain equipped with a toxin-antitoxin system for plasmid maintenance. Moreover, this study describes the actual cloning and expression of a Beta-glucosidase enzyme into E. coli BL21(DE3) and E. coli SE1, and the development of a novel inoculum production scheme that exploits the features of the SE1 strain, based on repeatedly recycling a fraction of the inoculum cells. The results of the techno-economic analysis project an enzyme production cost of 316 US$/kg in the baseline scenario, which is considerably higher than the values reported in the literature for the fungal cocktails. The facility-dependent cost, which is strongly associated with the cost of equipment, accounts for roughly half of the estimated cost, while the cost of raw materials, especially IPTG and glucose, and the cost of consumables are all quite significant. However, the simulation of multiple scenarios and optimization measures suggest that the enzyme cost can be substantially reduced on many fronts, such as: substituting the carbon source for cheaper alternatives; reducing the amount of IPTG used for induction; using an E. coli strain capable of extracellular production; or eliminating the steps of concentration and stabilization of the enzyme, in the case of on-site enzyme utilization. Developing E. coli strains capable of high rEnzyme volumetric productivities can also significantly reduce the cost of the enzyme, up to approximately 135 US$/kg in the scenario of highest productivity. In addition, based on the experimental results with the E. coli SE1 system, an inoculum recycle strategy that avoids the need of an extensive seed train was simulated, resulting in a significant reduction of the enzyme cost. Finally, the combination of multiple process improvements could lead to an enzyme cost near 20 US$/kg of protein, which comes close to the cost of fungal cellulases and demonstrates the great biotechnological potential of recombinant E. coli platforms. / A conversão enzimática de biomassa lignocelulósica em açúcares fermentescíveis é uma via promissora para a produção de combustíveis e produtos químicos renováveis. No entanto, o custo das enzimas fúngicas usualmente empregadas nesse processo permanece um gargalo significativo para a fabricação de produtos de baixo valor agregado a partir de biomassa. Uma possível estratégia para aumentar o rendimento da hidrólise e, assim, reduzir seu custo, seria suplementar as enzimas fúngicas com suas atividades enzimáticas deficientes, tais como a enzima Beta-glicosidase. Para produzir tais enzimas a um baixo custo, a bactéria Escherichia coli é uma forte candidata, dada a sua capacidade de crescer rapidamente em meios simples e baratos e de alcançar altos níveis de produtividade. No entanto, na literatura quase não há análises técnico-econômicas de produção de proteínas de baixo valor agregado utilizando E. coli e, de forma mais geral, há muito poucas análises técnico-econômicas de produção de proteínas de baixo valor agregado publicadas, com exceção da produção de celulases por Trichoderma reesei. Em particular, a aplicação biotecnológica de plataformas recombinantes baseadas em E. coli dotadas de sistemas toxina-antitoxina para garantir a estabilidade plasmidial segue em larga medida inexplorada, e seu impacto econômico, desconhecido. Assim, este trabalho apresenta uma análise técnico-econômica abrangente da produção industrial de uma enzima de baixo custo (Beta-glicosidase) usando E. coli BL21 (DE3) e E. coli SE1, uma cepa de BL21 (DE3) modificada que possui um sistema toxina-antitoxina para manutenção plasmidial. Além disso, este estudo descreve a clonagem e expressão de uma Beta-glicosidase em E. coli BL21 (DE3) e E. coli SE1, assim como o desenvolvimento de um novo método de produção de inóculo que tira proveito das peculiaridades da linhagem SE1, baseado em reciclar repetidamente uma fração das células do inóculo. Os resultados da análise técnico-econômica apontam para um custo de produção da enzima de 316 US$/kg no cenário-base, valor consideravelmente superior àqueles relatados na literatura para os coquetéis fúngicos. Os custos de overhead da planta, que estão fortemente associados ao custo de aquisição dos equipamentos, são responsáveis por aproximadamente metade do custo total, enquanto o custo de matérias-primas, especialmente IPTG e glicose, e o custo de consumíveis são bastante significativos. Porém, a simulação de múltiplos cenários e medidas de otimização sugerem que o custo da enzima pode ser substancialmente reduzido em muitas frentes, tais como: a substituição da fonte de carbono por alternativas mais baratas; a redução da quantidade de IPTG usado para indução; a utilização de cepas capazes de produzir a enzima extracelularmente; ou a eliminação das etapas de concentração e estabilização da enzima, em caso de utilização da enzima in situ. O desenvolvimento de cepas de E. coli capazes de atingir altas produtividades volumétricas de rEnzima também pode reduzir significativamente o seu custo, chegando a US$ 135/kg no cenário de maior produtividade. Com base nos resultados experimentais com a linhagem E. coli SE1, uma estratégia de reciclagem de inóculo que evita a necessidade de um extenso trem de inoculação também foi simulada, gerando significativa diminuição do custo da enzima. Por fim, a combinação de múltiplas melhorias no processo poderia levar a um custo de enzima em torno de 20 US$/kg de proteína, valor que se aproxima do custo das celulases fúngicas e que demonstra o grande potencial biotecnológico de plataformas de expressão baseadas em E. coli recombinante.

An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch. / Doctor of Philosophy (PhD)

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

The overwhelming occurrence and structural diversity of carbohydrates in Nature indicate their importance in a range of fundamental life processes. Indeed, it is this diversity that has lead to the two equally diverse groups of carbohydrate-processing enzymes, namely the glycoside hydrolases and glycosyl transferases. Thus, understanding the role of both carbohydrates and their processing enzymes in biological systems has attracted significant attention. This thesis, firstly, describes endeavours towards the synthesis of an inositol ?- amino acid, along with a series of sugar α-substituted carboxylic acid esters, utilising an extension of the modified Corey-Link reaction. The emphasis of the thesis is then shifted towards the synthesis of a putative inhibitor of a family GH26 lichenase from Clostridium thermocellum (CtLic26A). The preparation of 2-deoxy-2-fluoro-β-laminarbiosyl fluoride 1 is described, along with elaboration into oligosaccharides utilising AbgE358G glycosynthase technology. Crystallographic investigations indicated that the transition state adopted by CtLic26A is in stark contrast to that utilised by the related family GH26 mannanase from Pseudomonas cellulose (Man26A). ... Following on from this work, expanding the role of the AbgE358G glycosynthase acceptor repertoire to accommodate inositol substrates was explored, furthering the synthetic utility of this enzyme. Thus, a number of inositol acceptors bearing an aryl anchor, for example 2, were prepared and shown to be surrogates for carbohydrate acceptors. ... The thesis then describes the synthesis of an acetamide derivative of 1-epivalienamine, namely 3, a putative inhibitor of β-N-acetylglucosaminidases. Both the synthesis of 3, along with kinetic data for four β-N-acetylglucosaminidases, is reported; as well, Western blot analysis indicated no inhibition of a recombinant OGTase. ... Related to the preparation of a putative inhibitor of β-N-acetylglucosaminidases was the synthesis of a conformationally rigid carbocycle derivative of PUGNAc 4, along with two other derivatives 5 and 6. These compounds were also tested against four β-N-acetylglucosaminidases and a recombinant OGTase. ... Finally, the synthesis of a mechanism-based inhibitor of family GH3 β-Nacetylglucosaminidases, namely 2-acetamido-2-deoxy-5-fluoro-β-D-glucopyranosyl fluoride 7, is described. The incorporation of an azido moiety allows for the utilisation of 8 as an effective probe of β-N-acetylglucosaminidases. ...

Η ποσοτική ή ποιοτική ανεπάρκεια του λυσοσωματικού ενζύμου γλυκοκερεβροσιδάση οδηγεί στην παθολογία της νόσου Gaucher με κεντρικό το ρόλο της συσσώρευσης του υποστρώματός της, του γλυκοκερεβροσιδίου, στα μακροφάγα. Για περισσότερες από δύο δεκαετίες χρησιμοποιούνται γ-ρετροϊικά οχήματα στην ερευνητική προσέγγιση της διόρθωσης του ελλείμματος μέσω γονιδιακής μεταφοράς του γονιδίου της γλυκοκερεβροσιδάσης. Όπως έχει αναδειχθεί και σε κλινικό επίπεδο, είναι επιτακτική η ανάγκη για το σχεδιασμό γ ρετροϊικών οχημάτων, τα οποία θα είναι όχι μόνο αποτελεσματικά αλλά και ασφαλή, με κύριο στόχο τον περιορισμό της πιθανότητας εξαλλαγής του κυττάρου από τις συνέπειες της ενσωμάτωσης («μεταλλαξιγένεση κατά την ενσωμάτωση»). Η παρούσα μελέτη έχει ως στόχο τον έλεγχο της αποτελεσματικότητας νέων και θεωρητικά ασφαλέστερων σε σχέση με προηγουμένως χρησιμοποιηθέντα σε προκλινικό και κλινικό επίπεδο γ-ρετροϊικών οχημάτων για τη γονιδιακή μεταφορά του γονιδίου της γλυκοκερεβροσιδάσης σε προγονικά αιμοποιητικά κύτταρα του ανθρώπου. Στο πρώτο μέρος παρουσιάζεται η μελέτη έξι νέων και θεωρητικά ασφαλέστερων γ ρετροϊικών οχημάτων με το γονίδιο της γλυκοκερεβροσιδάσης, τα οποία είναι Self Inactivating (SIN) οχήματα και φέρουν αλληλουχίες για τη βελτίωση της ολοκλήρωσης της μεταγραφής, είτε από τον ιό WHV (WPRE, Woodchuck hepatitis virus Post-transcriptional Regulatory Element) είτε από τον ιό SV40 (2xSV40 USE, Simian Virus 40 Upstream Sequence Element σε δύο διαδοχικά αντίγραφα). Καταδεικνύεται ότι αυτά τα νέα SIN γ ρετροϊικά οχήματα είναι ικανά να μεταφέρουν ένα ενεργό φυσιολογικό γονίδιο γλυκοκερεβροσιδάσης σε CD34+ ανθρώπινο πρωτογενή προγονικό αιμοποιητικό κυτταρικό πληθυσμό. Τα οχήματα αυτά εμφάνισαν διαφορές στην αύξηση της ενζυμικής δραστικότητας, που προέκυψε από τη διαγονιδιακή έκφραση, οι οποίες οφείλονταν εν μέρει σε διαφορές στην αποδοτικότητα της διαμόλυνσης. Το δεύτερο μέρος της μελέτης περιλαμβάνει τέσσερα νέα SIN γ-ρετροϊικά οχήματα με το γονίδιο της γλυκοκερεβροσιδάσης με αντίστοιχο αριθμό οχημάτων-μαρτύρων, τα οποία είναι σχεδιασμένα με στόχο την κυτταροειδική έκφραση στα μονοκύτταρα / μακροφάγα, ώστε να περιοριστούν οι κυτταρικοί πληθυσμοί που εκτίθενται στην πιθανότητα ενεργοποίησης πρωτογκογονιδίων από τη δραστικότητα ενός εσωτερικού υποκινητή. Τα οχήματα φέρουν τους υποκινητές των ανθρώπινων γονιδίων CD11b και CD68 σε αλληλουχίες που είχαν επιδείξει στο παρελθόν μυελοειδοειδική έκφραση σε πειράματα γονιδιακής μεταφοράς. Τα νέα αυτά οχήματα ελέγχθηκαν ως προς την ικανότητά τους να παρουσιάζουν ισχυρότερη έκφραση του διαγονιδίου μετά από μονοκυτταρική / μακροφαγική διαφοροποίηση διαμολυσμένου CD34+ ανθρώπινου πρωτογενή προγονικού αιμοποιητικού κυτταρικού πληθυσμού σε σχέση με την έκφραση σε ένα λιγότερο διαφοροποιημένο διαμολυσμένο κυτταρικό πληθυσμό. Τα αποτελέσματα του δεύτερου μέρος της μελέτης σε συνδυασμό με τα ήδη υπάρχοντα βιβλιογραφικά δεδομένα ενθαρρύνουν την περαιτέρω διερεύνηση του υποκινητή του CD68 στο πλαίσιο των προσπαθειών για μυελοειδοειδική γονιδιακή μεταφορά του γονιδίου της γλυκοκερεβροσιδάσης, χωρίς να ενθαρρύνουν την περαιτέρω διερεύνηση του υποκινητή του CD11b. / Quantitative or qualitative deficiency of the lysosomal enzyme glucocerebrosidase results in the accumulation of its substrate, glucocerebroside, in macrophages, leading to the pathology of Gaucher disease. For more than two decades of research, gammaretroviral vectors have been used for gene transfer of the glucocerebrosidase gene for the correction of the enzyme’s deficit. It has been shown, even on clinical level, that the design of efficient as well as safe gammaretroviral vectors, aiming mainly at eliminating “insertional mutagenesis”, is an imperative need. The present study aims at the determination of the efficiency of new and theoretically safer, gammaretroviral vectors, compared to previously used ones on preclinical and clinical level, for the gene transfer of the glucocerebrosidase gene into human hematopoietic progenitor cells. In the first part of the study, six new and theoretically safer gammaretroviral vectors of the glucocerebrosidase gene have been evaluated. All six vectors are Self-Inactivating (SIN) and bear sequences for the improvement of transcriptional termination. These are either the WPRE (“Woodchuck hepatitis virus Post-transcriptional Regulatory Element”) from the virus WHV or the 2xSV40 USE (two copies of “Simian Virus 40 Upstream Sequence Element” in tandem repeat) from the virus SV40. It is shown that these new gammaretroviral vectors are efficient in transferring an active wild type glucocerebrosidase gene copy into a CD34+ human primary hematopoietic progenitor cell population. All six vectors showed increased enzyme activity, as compared to the untransduced cells, albeit with differences amongst them, partly due to differences in the transduction efficiency. The second part of the study focuses on the analysis of four new SIN gammaretroviral vectors of the glucocerebrosidase gene and the corresponding number of control vectors, designed for cell-specific expression in monocytes / macrophages. The aim is to thus limit the range of cell populations to be exposed to possible proto-oncogene activation from the internal promoter activity. In these vectors the expression of a glucocerebrosidase gene is driven by one of the promoters of the human genes for CD11b and CD68, which have exhibited myeloid specific expression in gene transfer experiments in the past. These new vectors were studied for their effectiveness in leading to stronger transgene expression after monocytic / macrophagic differentiation of a transduced CD34+ human primary hematopoietic progenitor cell population, compared to a less differentiated transduced cell population. The results of this study, taken together with the current bibliographical data, are encouraging for further study mainly of the CD68 promoter in the context of the research approach to myeloid specific gene transfer of the glucocerebrosidase gene.

A Doença de Gaucher (DG) é a Doença Lisossômica de Depósito mais frequente com prevalência de 1:50.000. Ela é causada pela deficiência da betaglicosidase ácida (GBA), gerando acúmulo de glicosilceramidas (glicocerebrosídio) nos lisossomos. Outra enzima relacionada é a quitotriosidase (QT), onde a atividade está aumentada nos pacientes em até 1000 vezes. Atualmente o diagnóstico é baseado na atividade específica de enzimas em leucócitos ou fibroblastos. O uso de sangue impregnado em papel filtro (SPF) vem sendo ampliado, porém somente como forma de triagem, pois não há forma validada de estabelecer a atividade específica da enzima. A utilização de sangue em papel filtro tem diversas vantagens, tais como: fácil transporte, fácil armazenagem das amostras, menor volume de reação e segurança de manipulação das amostras. Esse estudo tem como objetivo validar técnicas fluorimétricas para o estabelecimento da atividade específica da GBA e QT em sangue impregnado em papel filtro eluido com tampão universal (20 mmol/L de fosfato de sódio, pH 7,0) através da correção do volume amostral pela quantificação de proteínas totais. Além disso, foram miniaturizadas as técnicas padrão para a triagem em SPF e as técnicas confirmatórias padrão ouro em leucócitos da DG. Foram estabelecidos novos valores de referência para todas as técnicas estabelecidas. Foi possível diferenciar os controles saudáveis dos pacientes com DG utilizando as técnicas miniaturizadas em SPF e leucócitos. A atividade específica em SPF se mostrou valida e os coeficientes de variação foram considerados aceitáveis. A estabilidade enzimática foi analisada por 21 dias de armazenamento a 4°C e foi observado que a há um decaimento da atividade da GBA, mas não da QT. Deste modo a atividade específica em SPF pode ser utilizada de forma confiável como método de triagem e confirmação do diagnóstico de DG. / Gaucher disease (GD) is the most frequent Lysosomal Storage Disorder with a prevalence of 1:50,000. It is caused by the deficiency of acid betaglucosidase (GBA), generating glucosylceramide (glucocerebroside) accumulation in the lysosomes. Another enzyme related to GD is chitotriosidase (CT), which activity is increased in patients up to 1000 times. Currently the diagnosis is based on the specific activity of enzymes in leukocytes or fibroblasts. The use of dried blood spots (DBS) has been extended, but only as screening, because there is no validated specific activity of the enzymes. The use of DBS has several advantages, such as easy transport and easy storage of samples, smaller reaction volume and safety of handling samples. This study aims to validate fluorometric techniques for establishing specific activity of the GBA and CT in DBS eluted with universal buffer (20 mmol/L sodium phosphate, pH7.0) by correcting for sample volume quantification of total protein. Moreover, standard screening techniques in DBS and standard diagnosis techniques in leukocytes for GD have been miniaturized. New reference values and cut-off points were established for all techniques. It was possible to differentiate healthy controls from patients with GD using miniaturized techniques in DBS and leukocytes. The specific activity of DBS proved valid and coefficients of variation were acceptable. The enzymatic stability was analyzed by 21 days of storage at 4° C and it was observed that there is a decrease of the activity of GBA, but not of CT. Thus the specific activity on DBS can be used as a reliable screening method and as diagnosis of GD.