The isoprenoid biosynthetic pathway (IBP) has been highly implicated in a number of cellular malignancies, including proliferation, invasion, and migration. Epidemiological studies have found clinically relevant inhibitors of the IBP, such as the statin family and nitrogenous bisphosphonates, reduce the risk of prostate cancer advancement. In vitro work has implicated statin's and nitrogenous bisphosphonate's inhibition of GGPP and protein geranylgeranylation as the components responsible for their reduction of prostate cancer progression. However, their depletion of nearly all isoprenoid intermediates as well as their organ specificities make understanding the specific role of protein geranylgeranylation in prostate cancer metastasis impossible. Consequently, we have developed a novel library of seven alkyl bisphosphonate ethers found to potently reduce GGDPS with little to no activity against the related FDPS enzyme. Inhibition of GGDPS in three human prostate cancer cell lines reduced GGPP and protein geranylgeranylation without affecting protein farnesylation, translating into a reduction in cell migration and invasion. Interestingly, the GGDPS inhibitors reduced protein geranylgeranylation at lower concentrations in the highly metastatic PC3 cell line as compared to the less metastatic LNCaP and 22Rv1 cell lines. Additionally, the PC3 cell line was found to have higher levels of endogenous IBP intermediates as compared to the less metastatic cells. Translation in vivo using two murine models of human prostate cancer metastasis found a reduction in soft tissue tumor burden that corresponded to a biochemical reduction in protein geranylgeranylation. In conclusion, selective reduction of GGPP and protein geranylgeranylation was sufficient to reduce the metastatic potential of prostate cancer in vitro and in vivo.

Peripheral nerve injuries are often associated with significant loss of nervous tissue leading to poor restoration of function following repair of injured nerves. Although the injury gap could be bridged by autologous nerve graft, the limited access to donor material and additional morbidity such as loss of sensation and scarring have prompted a search for biosynthetic nerve transplants. The present thesis investigates the effects of a synthetic matrix BD™ PuraMatrix™ peptide (BD)hydrogel, alginate/fibronectin gel and fibrin glue combined with cultured rat Schwann cells or human bone marrow derived mesenchymal stem cells (MSC) on neuronal regeneration and muscle recovery after peripheral nerve injury in adult rats. In a sciatic nerve injury model, after 3 weeks postoperatively, the regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel if compared with the alginate/fibronectin gel. The addition of rat Schwann cells to the BD hydrogel drastically increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. However, at 16 weeks the number of regenerating spinal motoneurons was decreased to 49% and 31% in the BD hydrogel and alginate/fibronectin groups respectively. The recovery of the gastrocnemius muscle was also inferior in both experimental groups if compared with the nerve graft. The addition of the cultured Schwann cells did not further improve the regeneration of motoneurons and muscle recovery. The growth-promoting effects of the tubular conduits prepared from fibrin glue were also studied following repair of short and long peripheral nerve gaps. Retrograde neuronal labeling demonstrated that fibrin glue conduit promoted regeneration of 60% of injured sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20 mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed a more atrophic appearance and the mean area and diameter of fast type fibres were significantly worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. The combination of fibrin conduit with human MSC and daily injections of cyclosporine A enhanced the distance of regeneration by four fold and the area occupied by regenerating axons by three fold at 3 weeks after nerve injury and repair. In addition, the treatment also significantly reduced the ED1 macrophage reaction. At 12 weeks after nerve injury the treatment with cyclosporine A alone or cyclosporine A combined with hMSC induced recovery of the muscle weight and the size of fast type fibres to the control levels of the nerve graft group. In summary, these results show that a BD hydrogel supplemented with rat Schwann cells can support the initial phase of neuronal regeneration across the conduit. The data also demonstrate an advantage of tubular fibrin conduits combined with human MSC to promote axonal regeneration and muscle recovery after peripheral nerve injury.

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.

During seed development, myo-inositol (Ins) hexakisphosphate or phytic acid (PA) is stored in the form of phytin with mineral cations, and is mobilized following germination, releasing these nutrients that are required for seedling growth. Outside its role in seeds, PA and other phosphoylated Ins derivatives play critical roles in biological processes in many eukaryotes. PA also has negative influences on nutrition in both non-ruminant animals and humans due to its lack of digestibility. There have been two parallel PA biosynthetic pathways proposed, yet, the pathway is still poorly understood in terms of its regulation and enzymes involved. Here, genes encoding enzymes putatively implicated in castor bean PA biosynthetic pathways were identified in the genome and expression followed. Isolated castor bean embryos have the ability to resynthesize PA following germination if exogenous phosphate is available. It was found that the genes purported to act in PA synthesis were constitutively expressed in the embryos regardless of the availability of phosphate. Castor bean Ins 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1), catalyzing the final reaction in PA biosynthesis, regardless of pathway, was chosen for further study. Even though only one copy of the RcIPK1 gene was present in the genome, numerous RNA variants were found, most likely due to alternative splicing events. These variants encoded six closely related protein isoforms based on in silico analysis. Functional analyses using yeast mutant strains lacking the IPK1 gene revealed that only three of the mRNA variants could rescue the temperature-sensitive growth phenotype, and it was demonstrated by HPLC analysis of Ins phosphates that their ability to complement the missing yeast IPK1 enzyme was associated with enzyme activity. Only these three isoforms possessed conserved motif III and motif IV important for IPK1 catalytic activity.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

Orientador: Cláudia Valéria Seullner Brandão / Banca: Maria Jaqueline Mamprim / Banca: Nilza Maria Freres Mascarenhaz / Resumo: O objetivo deste trabalho foi avaliar a aplicação de membrana biossintética de celulose (MBC) nacional, após a realização de trocleoplastia experimental em cães, com intuito de verificar se o uso desta poderia favorecer a migração de células com potencial condrogênico, assim como ocorre no tecido ósseo. A evolução pós-operatória dos cães foi analisada com especial interesse nos processos de reparação frente ao defeito osteocondral, estabelecendo as vantagens e desvantagens do uso do biomaterial. Foram utilizados 12 cães (Canis familiaris) adultos, sadios e sem alterações no aparelho locomotor. Todos os animais foram submetidos ao procedimento de trocleoplastia bilateral, sendo que a MBC foi aplicada no membro esquerdo. Os animais foram avaliados clínica e radiograficamente aos 15, 30, 60 e 90 dias de pós-operatório. A função locomotora do membro foi avaliada em escores pré-definidos, nos referidos momentos. A avaliação macroscópica da articulação foi realizada, nos momentos pré-estabelecidos, seguida da artrotomia exploratória, bem como da coleta de biópsia para o exame microscópico. Todos os animais apresentaram função normal dos membros nos momentos avaliados. Não houve diferença clínica e radiográfica entre os grupos controle (GC) e tratado (GT). Na avaliação histopatológica, aos 30 dias, notou-se intensa celularidade em ambos os grupos, sendo que no GC esta era constituída por fibroblastos ativos e no GT por condrócitos imaturos, formando um tecido conjuntivo mais organizado. O GC apresentava fibrose e muitos fibroblastos aos 60 dias, enquanto o GT apresentava maior número de condrócitos. Aos 90 dias, constatou-se formação de tecido do tipo fibrocartilaginoso maduro em ambos os grupos. Histomorfometricamente, o GT apresentou melhor resposta ao processo de reparação nos momentos iniciais quanto ao número de células e espessura do tecido...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the application of national biosynthetic cellulose membrane after experimental trochleoplasty, to verify if its utilization could support chondrogenic cells migration like occurs in osseous tissue. The advantages and disadvantages of the use of this biomaterial were evaluated through the observation of the reparative process of the osteochondral injury. Twelve adult healthy dogs (Canis familiaris) were used. All dogs were submitted to trochleoplasty in both pelvic limbs and the biosynthetic cellulose membrane was applied in the left limb. Clinical and radiographic evaluation was performed at 15, 30, 60 and 90 postoperatively. The locomotor function of the limb was evaluated in scores on the differents moments. The joint macroscopic evaluation was performed before exploratory arthrotomy and biopsy for microscopic exam. All dogs showed normal function of the limbs in all differents moments. Radiographic results showed no difference in control (CG) and treated groups (TG). Microscopic results showed at 30 days an increase of the celularity in both groups was observed. In the CG it was constituted by active fibroblasts and in TG by immatures condrocytes forming a more organized connective tissue. In the CG a fibrous tissue and many fibroblasts appear at 60 days and in the TG more condrocytes appear. A mature fibrocartilaginous tissue formed in both groups at 90 days. Histomorphometrically, TG presents better response of repair process in the fist moments as the number of cells and the tissue thickness. The biosynthetic cellulose membrane didn't cause deleterious effects showing good adaptation in intrarticular environment...(Complete abstract, access undermentioned eletronic address) / Mestre

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.