L'acide ascorbique (AsA, vitamine C) est l'un des composés parmi les plus importants chez les eucaryotes. En raison de son potentiel antioxydant élevé, l'AsA représente un trait important de la qualité nutritionnelle des végétaux. Au-delà de sa valeur bénéfique pour la santé, une augmentation de la teneur en AsA des fruits bénéficierait probablement à la qualité post-récolte et à la résistance aux pathogènes. Pour mieux comprendre ces régulations chez les plantes et leurs impacts sur la qualité des fruits, une collection de tomates EMS hautement mutée (cv. Micro-Tom) a été criblée pour identifier des mutants dont les fruits sont enrichis en AsA. Cette stratégie de génétique directe associant le criblage à une approche de cartographie par séquençage devait permettre d’identifier de nouveaux gènes liés au caractère AsA+. L'un des mutants, noté P21H6, présentait un enrichissement en AsA de 2 à 4 fois supérieur à celui du WT, et fut le premier à être génétiquement caractérisé. Cette étude a permis de mettre en évidence une nouvelle classe de photorécepteurs impliqués dans la détection de la lumière bleue, appelée SlPLP, en tant que régulateur négatif de l'accumulation d'AsA dans la tomate. Le rôle de PLP dans le phénotype AsA+ du fruit a été confirmé par une stratégie de mutagenèse dirigée, avant d’entreprendre sa caractérisation fonctionnelle. Nous avons démontré que SlPLP interagit avec SlGGP (GDP-L-galactose phosphorylase), une enzyme clé de la voie du L-Galactose, sous contrôle de la lumière bleue et que cette interaction a lieu dans le cytoplasme et le noyau. Nos résultats renforcent le rôle central du GGP dans la biosynthèse de l'AsA et suggèrent un nouveau mécanisme de régulation par la lumière bleue de la fonction du GGP, en plus de son activité métabolique. Parallèlement, nous avons entrepris la caractérisation d’un autre mutant, le P17C5-3, qui présentait le plus fort taux d'AsA (jusqu'à 10 fois le WT). Outre le phénotype AsA+, le mutant P17C5 présentait de fortes altérations morphologiques, notamment l’absence de graines, rendant la mise en place de la stratégie de cartographie difficile. Grâce à un croisement avec la variété commerciale M82, la mutation causale pu être identifiée dans un ORF cis-régulateur en amont de GGP. Ce résultat confirme le rôle clé de GGP dans la voie L-Galactose. Des études préliminaires liées au phénotype parthénocarpique suggèrent un problème de stérilité mâle associé aux processus de développement du pollen. Enfin, dans l’étude de la qualité des fruits après la récolte, des expériences de stress froid effectuées avec les fruits P21H6 semblent démontrer que l’augmentation de la teneur en AsA améliore la durée de conservation et la capacité de maturation des fruits. Dans l'ensemble, nos résultats confirment la position clé de la protéine GGP dans la voie de biosynthèse de l'AsA, et fournissent des outils et du matériel végétal précieux pour décortiquer la régulation de l'AsA et son rôle physiologique dans la qualité des fruits et les caractères post-récolte. / Ascorbic acid (AsA, vitamin C) is one of the most important biochemical in living organisms. Due to its high antioxidant potential, AsA represents an important trait of nutritional quality in fruits and vegetables. In addition to its beneficial health value in fruit consumption, increasing fruit AsA content would likely affect postharvest quality and resistance to pathogens. Thus, understanding the regulation of AsA accumulation in order to improve crop species of agronomical interest is an important issue in plant breeding for many fleshy fruit species. To get a better understanding of the regulation of AsA level in plants and its impact on fruit quality, a highly mutagenized EMS tomato collection (cv. Micro-Tom) was screened for AsA+ fruit mutants. This forward genetic strategy combined with a mapping-by-sequencing approach, had allowed identifying new genes related to the AsA+ trait. One of the mutant line named P21H6, displayed an AsA-enrichment 2 to 4 fold that of the WT, and was the first to be genetically characterized. It allowed highlighting a new class of photoreceptor involved in blue light sensing named SlPLP as a negative regulator of AsA accumulation in tomato. We confirmed the role of the PLP in the fruit AsA+ phenotype using a directed mutagenesis strategy, undertaking its functional characterization. We demonstrate that PLP interacts with GGP (GDP-L-galactose phosphorylase), a key enzyme of the L-Galactose pathway, under blue light control and that this interaction takes place in the cytoplasm and the nucleus. Our results strengthen the central role of GGP in the AsA biosynthesis and suggest a new regulation mechanism by blue light of the GGP function in addition to its metabolic activity. Besides we started the characterization another mutant, the P17C5-3, which displayed the highest level of AsA (up to 10 times the WT). Beyond its AsA+ content, the P17C5 mutant showed strong morphological alterations including a seedless phenotype making the mapping difficult at first. Thanks to the crossing with the commercial M82 tomato cultivar, the causal mutation was identified in a cis-acting ORF, upstream of the GGP gene. This result confirmed the key role of GGP in the L-Galactose pathway. Preliminary studies related to the parthenocarpic phenotype suggest a problem of male sterility associated with pollen development processes. Finally, in the study of the post-harvest fruit quality, chilling stress experiments carried out with the P21H6 fruits seem to demonstrate that increasing AsA content improve the fruit shelf life and its maturation capacity. Taken as a whole, our results confirmed the key position of the GGP protein in the AsA biosynthesis pathway and they provided precious tools and plant material for deciphering the regulation of AsA and its physiological role in fruit quality and post-harvest traits.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

O gene nuclear GAL1 de Saccharomyces cerevisiae codifica uma galactoquinase induzida por galactose e reprimida por glicose. Três evidências indicam que a transcrição de GAL1 é dependente da atividade mitocondrial. Linhagens petite, com deleção no DNA da organela (ρ-) ou rompimento em gene nuclear, que codifica a farnesil transferase mitocondrial, são incapazes de induzir GAL1. Os inibidores de respiração antimicina-A e azoteto de sódio (NaN3), que atuam, respectivamente, nos complexos III e IV da cadeia de transporte de elétrons, impedem a indução de GAL1. Em células crescidas em glicose ou glicerol, o oligômero formado pela proteína URF13, na presença de metomil, produz um poro na membrana mitocondrial interna que reduz o potencial de membrana ΔΨ e os níveis do mRNA de GAL1. A regulação dependente da atividade da mitocôndria ocorre a nível transcricional, pois o gene repórter GUS, sob controle de GAL1, não é induzido na presença de galactose, após tratamento prévio das células com antimicina-A ou NaN3. Mig1p é um repressor que atua diretamente no promotor de GAL1 e inibe a transcrição da galactoquinase. Os resultados obtidos indicam que Mig1p media a repressão da indução de GAL1, na presença do inibidor da cadeia respiratória antimicina-A. / The nuclear gene GAL1 of Saccharomyces cerevisiae encodes a galactokinase induced by galactose and repressed by glucose. Three lines of evidence indicate that expression of GAL1 transcript is dependent on mitochondrial activity. Petite strains in which mitochondrial DNA was partialy deleted (ρ-) or cells containing a disruption in the nuclear gene COX10, which encodes the mitochondrial farnesil transferase, are unable to induce GAL1. Respiratory inhibitors such as antimycin-A or sodium azide (NaN3), that inhibit complexes III and IV of the electron transport chain, respectively, affect GAL1 induction. Functional expression of the maize protein URF13, which is translocated to the mitochondrial inner membrane, forming a pore that leads to a reduction of the mitochondrial membrane potential ΔΨ and reduces the leveis of GAL1 transcripts. Experiments using a heterologous gene fusion showed that the inhibition of GAL1 expresion, by treatment of cells with antimycin-A or NaN3, controls the expression of GAL1 at the transcrptional level. Mig1p is a repressor that binds GAL1 promoter. Our results indicate that Mig1p mediates the represion of GAL1 induction, observed in the presence of the mitochondrial inhibitor antimycin-A.

Depuis peu, des travaux ont montré que chez l’Homme, la transcétolase (TK, EC 2.2.1.1.) dont le cofacteur est la thiamine diphosphate (forme active de la vitamine B1), est une enzyme impliquée dans de nombreuses maladies telles que, le diabète, certains cancers, ou encore des maladies neurologiques, comme le syndrome de Wernicke-Korsakoff et la maladie d’Alzheimer. Pour des applications thérapeutiques, des inhibiteurs spécifiques de cette enzyme sont actuellement conçus et synthétisés dans les milieux académiques et industriels. Afin de déterminer l’activité de la TK (dans un but de diagnostic) d’une part, et de détecter des inhibiteurs potentiels de cette enzyme (dans un but thérapeutique) d’autre part, il est nécessaire de disposer de tests alliant rapidité, sensibilité et faible coût. Nous avons envisagé d’utiliser des biocapteurs ampérométriques qui combinent l’ensemble de ces avantages, et qui, de plus, n’ont jamais été mis en oeuvre avec la TK. Pour la détermination de l’activité des TK d’E. coli et humaine libres en solution, nous avons tout d’abord élaboré un premier biocapteur à galactose oxydase (GAOx, EC 1.1.3.9), dans lequel cette enzyme est immobilisée sur la laponite. Puis, dans le but de detecter des inhibiteurs de la TK, avec un système réutilisable, nous avons developpé un biocapteur à GAOx-TK d’E. coli, les deux enzymes étant co-immobilisées à la surface de l’électrode. Pour cela la TK a été immobilisée dans des Hydroxydes Doubles Lamellaires (HDL). Ce biocapteur bicouche et bi-enzymatique GAOx-TK, nous a permis d’évaluer l’effet d’inhibiteurs, tels que différents analogues du cofacteur et de substrats pris comme modèles. / Some recent studies have shown that human transketolase (TK, EC 2.2.1.1.), which thiamine diphosphate (active form of vitamin B1) is the cofactor, is involved in numerous disease such as diabete, some cancers and neurodegenerative diseases as Alzheimer’s disease and Wernicke-Korsakoff syndrome. For therapeutic purposes, TK inhibitors have been designed and synthesized in both academic and industrial fields. To determine TK activity (diagnostic) on the one hand, and to detect potential inhibitors of this enzyme (therapeutic) on the other hand, it is necessary to develop fast, sensitive and low cost assays. In this context, we designed some original amperometric biosensors that combine these advantages and were never studied with TK from now. We performed a first galactose oxidase (GAOx, EC 1.1.3.9) biosensor for E. coli and human TK activities detection. For that purpose, GAOx was immobilized on laponite matrix. Then, we designed a GAOx-TK biosensor by co-immobilization of GAOX and TK on the electrode surface that enabled the detection TK inhibitors with a reusable system. Thence, TK was immobilized in Layered Double Hydroxides (HDL). This bilayer and bi-enzymic biosensors, allowed us to determine the inhibitor potencies of several cofactors and substrates analogues as model compounds.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

O gene nuclear GAL1 de Saccharomyces cerevisiae codifica uma galactoquinase induzida por galactose e reprimida por glicose. Três evidências indicam que a transcrição de GAL1 é dependente da atividade mitocondrial. Linhagens petite, com deleção no DNA da organela (ρ-) ou rompimento em gene nuclear, que codifica a farnesil transferase mitocondrial, são incapazes de induzir GAL1. Os inibidores de respiração antimicina-A e azoteto de sódio (NaN3), que atuam, respectivamente, nos complexos III e IV da cadeia de transporte de elétrons, impedem a indução de GAL1. Em células crescidas em glicose ou glicerol, o oligômero formado pela proteína URF13, na presença de metomil, produz um poro na membrana mitocondrial interna que reduz o potencial de membrana ΔΨ e os níveis do mRNA de GAL1. A regulação dependente da atividade da mitocôndria ocorre a nível transcricional, pois o gene repórter GUS, sob controle de GAL1, não é induzido na presença de galactose, após tratamento prévio das células com antimicina-A ou NaN3. Mig1p é um repressor que atua diretamente no promotor de GAL1 e inibe a transcrição da galactoquinase. Os resultados obtidos indicam que Mig1p media a repressão da indução de GAL1, na presença do inibidor da cadeia respiratória antimicina-A. / The nuclear gene GAL1 of Saccharomyces cerevisiae encodes a galactokinase induced by galactose and repressed by glucose. Three lines of evidence indicate that expression of GAL1 transcript is dependent on mitochondrial activity. Petite strains in which mitochondrial DNA was partialy deleted (ρ-) or cells containing a disruption in the nuclear gene COX10, which encodes the mitochondrial farnesil transferase, are unable to induce GAL1. Respiratory inhibitors such as antimycin-A or sodium azide (NaN3), that inhibit complexes III and IV of the electron transport chain, respectively, affect GAL1 induction. Functional expression of the maize protein URF13, which is translocated to the mitochondrial inner membrane, forming a pore that leads to a reduction of the mitochondrial membrane potential ΔΨ and reduces the leveis of GAL1 transcripts. Experiments using a heterologous gene fusion showed that the inhibition of GAL1 expresion, by treatment of cells with antimycin-A or NaN3, controls the expression of GAL1 at the transcrptional level. Mig1p is a repressor that binds GAL1 promoter. Our results indicate that Mig1p mediates the represion of GAL1 induction, observed in the presence of the mitochondrial inhibitor antimycin-A.

Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

by Chan Sau Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references. / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.ii / ACKNOWLEDGMENT --- p.iii / CONTENTS --- p.iv / ABBREVIATIONS --- p.vi / Chapter CHAPTER 1 --- General Introduction / Chapter 1-A. --- Role of Copper in Biology --- p.1 / Chapter 1-B. --- A Brief Review on Radical Copper Proteins --- p.4 / Chapter 1-C. --- Objectives of This Work --- p.12 / Chapter 1-D. --- References --- p.13 / Chapter CHAPTER 2 --- Copper(II) and Zinc(II) Complexes containing N3O Tetradentate Ligands / Chapter 2-A. --- Introduction Results and Discussion --- p.14 / Chapter 2-B. --- Preparation of Tetradentate Ligands and Complexes --- p.27 / Chapter 2-C. --- Characterization --- p.36 / Chapter 2-D. --- Generation of Metal Phenoxyl Radical Species --- p.51 / Chapter 2-E. --- Summary --- p.57 / Chapter 2-F. --- References --- p.59 / Chapter CHAPTER 3 --- Copper(I) Complexes with N30 Tetradentate Ligands / Chapter 3-A. --- Introduction Results and Discussion --- p.62 / Chapter 3-B. --- Preparation of Copper(I) Complexes with N30 Tetradentate Ligands --- p.75 / Chapter 3-C. --- Characterization --- p.79 / Chapter 3-D. --- Reactivities of 86，87 and 88 toward Dioxygen --- p.88 / Chapter 3-E. --- Summary --- p.93 / Chapter 3-F. --- References --- p.94 / Chapter CHAPTER 4 --- Experimental Sections / Chapter 4-A. --- General Preparations and Physical Measurements --- p.97 / Chapter 4-B. --- Compounds Described in Chapter2 --- p.99 / Chapter 4-C. --- Compounds Described in Chapter3 --- p.113 / Chapter 4-D. --- Oxo-Transfer to Triphenylphosphine as Described in Chapter3 --- p.117 / Chapter 4-E. --- References --- p.119 / Chapter APPENDIX A --- 1H and13 C̐ưث1H ̐ưحNMR Spectra / Chapter A-1. --- Compounds Described in Chapter2 --- p.120 / Chapter A-2. --- Compounds Described in Chapter3 --- p.127 / Chapter APPENDIX B --- Crystallographic Data / Chapter B-1. --- X-ray Crystal Structure Data for Complexes in Chapter2 --- p.131 / Chapter B-2. --- X-ray Crystal Structure Data for Complexes in Chapter3 --- p.133 / Chapter APPENDIX C --- GC-MS Spectra / Chapter C-1. --- GC-MS Spectra for Standard Samples --- p.134 / Chapter C-2. --- GC-MS Spectra for the Reactions with Triphenylphosphine Described in Chapter3 --- p.136

Altos níveis de galactose circulantes e cerebrais são encontrados em portadores da galactosemia clássica não tratada, cujos pacientes comumente desenvolvem problemas cognitivos e motores ao longo da vida. Entretanto pouco se conhece a respeito dos mecanismos da disfunção celular e molecular responsáveis por estes sintomas. Assim, o objetivo do presente trabalho foi de investigar o efeito da injeção intracerebroventricular de galactose sobre a memória (aversiva e de reconhecimento de objetos) e a coordenação motora em ratos Wistar. Além disso, a atividade, o imunoconteúdo e a expressão gênica da acetilcolinesterase no hipocampo e córtex cerebral foram também avaliados. No cerebelo, foram medidos parâmetros histológicos (contagem de células e imunohistoquímica) e bioquímicos (imunoconteúdo de caspase-3 ativa e BDNF, atividade e imunoconteúdo da acetilcolinesterase, níveis de glutationa e sulfidrilas, bem como o índice de dano ao DNA). Ratos Wistar receberam uma injeção intracerebroventricular de galactose (4 mM) ou salina (controles) sendo esses submetidos às tarefas comportamentais e/ou decapitados em diferentes tempos (1 h, 3 h ou 24 h), logo após, o hipocampo, córtex cerebral e cerebelo foram dissecados. Os resultados mostraram que a galactose prejudicou a memória aversiva e de reconhecimento de objetos, quando injetada antes do treinamento, bem como alterou a atividade e a expressão gênica da acetilcolinesterase em hipocampo. Em relação ao comportamento motor e aos parâmetros histológicos e bioquímicos realizados no cerebelo, a administração intracerebroventricular de galactose prejudicou a coordenação motora e reduziu o número de células e a imunomarcação de neurônios e astrócitos. A galactose, também aumentou o imunoconteúdo de caspase-3 ativa, a atividade da acetilcolinesterase e o índice de dano ao DNA, bem como diminuiu o imunoconteúdo de BDNF e acetilcolinesterase e os níveis de glutationa e sulfidrilas no cerebelo. Tomados em conjunto, nossos resultados mostram que a administração intracerebroventricular de galactose prejudicou a memória e a coordenação motora. Além disso, o modelo experimental utilizado mostrou diversas alterações a nível celular e molecular, os quais podem contribuir pelo menos em parte com o entendimento da fisiopatologia da galactosemia clássica. / Non-treated patients with classical galactosemia present high levels of galactose in the bloodstream and brain. Patients usually develop cognitive and motor impairments during life. However, little is known about the cellular and molecular mechanisms responsible for these symptoms. Thus, the aim of this study was to investigate the effect of intracerebroventricular galactose injection on memory (aversive and object recognition) and motor coordination in Wistar rats. Acetylcholinesterase activity, immunocontent and gene expression were investigated in hippocampus and cerebral cortex. In the cerebellum, we performed histological (cell counting and immunohistochemistry) and biochemical (active caspase-3 immunocontent, BDNF, acetylcholinesterase activity and immunocontent, glutathione and sulfhydryl levels, as well as DNA damage index) parameters. Wistar rats received a single intracerebroventricular injection of galactose (4 mM) or saline (control). The animals performed behavioral tasks and/or were decapitated at different times (1 h, 3 h or 24 h) after injection. The hippocampus, cerebral cortex and cerebellum were dissected. The results showed that injecting galactose before training provokes impairment on aversive and object recognition memories, as well as altered the activity and gene expression of acetylcholinesterase in hippocampus. Regarding to the histological and biochemical parameters measured in the cerebellum, intracerebroventricular galactose injection impaired motor coordination, reduced the number of cells and immunostaining of neurons and astrocytes. In the cerebellum, galactose also increased active capase-3 immunocontent, acetylcholinesterase activity and DNA damage index, as well as decreased BDNF and acetylcholinesterase immunocontent, and glutathione and sulfhydryl levels. Altogether, our results show that intracerebroventricular injection of galactose impaired memory and motor coordination. Moreover, the experimental model used showed several alterations at cellular and molecular levels. These findings may contribute at least in part with the understanding of the physiopathology in classical galactosemia.

This study investigated the role of exopolysaccharide (EPS) in cheese moisture retention. Analysis of low-fat Mozzarella cheese made with different combinations of EPS-producing (Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii ssp. bulgaricus MR-lR) and non-EPS-producing (S. thermophilus TA061 and L. helveticus LH100) starters showed significantly higher moisture levels in cheese made with S. thermophilus MR-1C. To determine if the S. thermophilus MR-1C EPS was responsible for increased moisture retention, gene replacement was used to inactivate the epsE gene in this bacterium. Low-fat Mozzarella cheese made with L. helveticus LH100 plus the EPS-negative mutant, S. thermophilus DM1O, had significantly lower moisture content than cheese made with LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-lC EPS indicated that it had a repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1. Interestingly, carbohydrate utilization tests showed that DMlO had acquired the ability to ferment galactose.