Durante o preparo químico-cirúrgico são utilizados instrumentos e substâncias químicas, que constituem um binômio indivisível e necessário para alcançar a modelagem e a sanificação dos canais radiculares. Assim, propõe-se com este trabalho avaliar in vivo, por meio de método molecular de PCR quantitativo, baseado em DNA (qPCR), a eficiência do preparo químico-cirúrgico empregando como agente de irrigação o Hipoclorito de Sódio (NaOCL) a 2,5% ou o Gel de Endo PTC associado ao Hipoclorito de Sódio a 1,0% na redução bacteriana de canais radiculares de dentes portadores de periodontite apical primária. Foram selecionados 30 pacientes portadores de infecção endodôntica primária, totalizando 30 dentes, com rarefação óssea periapical visível na radiografia, sem tratamento endodôntico prévio. Os pacientes foram divididos de forma randomizada em dois grupos distintos, de acordo com a substância química auxiliar utilizada durante a instrumentação, NaOCL 1% + Endo-PTC leve ou NaOCL 2,5%. Em todos os casos empregou-se instrumentos Reciproc R40 ou R50 e as coletas foram realizadas antes (S1) e após o prepare químico-cirúrgico (S2). A análise de aderência foi realizada por meio do teste de Kolmogorov-Smirnov, as análises intragrupo foram realizadas com teste de Wilcoxon para amostras relacionadas e as comparações entre os dois grupos foram realizadas com o teste de Mann-Whitney, para a análise quantitativa de bactérias. Em ambos os grupos, houve diminuição significativa no número de bactérias entre S1 e S2 (p<0,05). No grupo NaOCL 1% + Endo-PTC leve, houve redução de 3,7x105(S1) para 5,7x104 (S2). No grupo NaOCl 2,5%, redução de 1,3x105 (S1) para 1,1x104(S2). Na comparação entre grupos, o NaOCL a 2,5% (91,62%) promoveu maior redução bacteriana do que o grupo NaOCL 1% + Endo-PTC (84,60%) (p<0,05). / During the chemomechanical preparation, instruments and chemical substances are used, which constitute an indivisible and necessary binomial to achieve modeling and sanification. Knowing the auxiliary chemical substances, understanding their mechanisms of action, being able to use them efficiently, is fundamental, so that the chemical-surgical preparation is well performed by the clinician. Thus, the purpose of this study is to evaluate in vivo, the efficiency of the chemomechanical preparation using as the irrigant agent 2,5% sodium hypochlorite and Endo-PTC gel, associated to 1% sodium hypochlorite, to assess the bacterial reduction of root canals of teeth with primary apical periodontitis, using a molecular quantitative method DNA-based - polymerase chain reaction (qPCR). Were selected 30 patients with primary infection totaling 30 teeth, with visible periapical bone rarefaction on the radiography, without previous endodontic treatment. Patients were randomly divided into two distinct groups according to the auxiliary chemical substances used during the instrumentation, 1% sodium hypochlorite associated with Endo-PTC gel or 2,5% sodium hypochlorite. In all cases, reciproc instruments R40 or R50 were used and the samples were taken before (S1) and after chemical surgical preparation (S2). The adherence analysis was performed using the Kolmogorov-Smirnov test, intragroup analysis were performed with Wilcoxon test for related samples and comparisions between the two groups were performed with the Mann-Whitney test for the quantitative analysis of bacteria. In the both groups, there was a significant decrease in the number of bacteria between S1 and S2 (p<0,05), the inicial sample (S1) of the group Endo-PTC, the median 3,7x105, reduced to 5,7x104. In the other group of NaOCl, the median in S1 was 1,3x105 that reduced to 1,1x104 . In the comparision between groups, the 2,5% NaOCl promoted a greater microbial reduction of 91,62%, than the Endo-PTC associated with 1% NaOCl (p<0,05) 84,60%.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Objectives: The aim of this study was to determine if exposure to volatile organic compounds can lead to DNA damage and impaired DNA repair capacity. Nail cosmetics is a fast growing industry around the world where employees and clients are subjected to various chemical substances which may be harmful to their health: such as formaldehyde, toluene, acetone, xylene, ethylmethacrylate, methylmethacrylate and n–buthyl acetate. These chemicals have the potential to be harmful to their health and exposure to these chemicals should be actively controlled. Formaldehyde is classified as a human carcinogen by the IARC, whereas, toluene and xylene are group three carcinogens, classified in 1999 (not classified as carcinogenic to humans), and various studies have linked DNA damage and impaired DNA repair to the above mentioned substances. Methods: Fifteen nail technicians were monitored by means of personal air sampling, measuring formaldehyde, toluene, xylene, acetone and ethylmethacrylate exposure. Fifteen unexposed subjects were chosen and matched for age and smoking habits with the exposed group. Heparinised blood samples were obtained from each test subject with which the Comet Assay was performed on lymphocytes to determine DNA damage and repair ability. Results: Exposure to ethylmethacrylates and methylmethacrylates leads to DNA damage. Methylmethacrylate causes DNA damage by specifically targeting pyrimidine (fpg) bases. N–buthyl acetate, xylene and acetone exposure impaired DNA repair capacity. The exposed group showed signs of Class III and Class IV DNA damage, whereas the control group had little Class III damage and no indication of Class IV damage. The overall DNA repair ability of the nail technicians was slightly impaired when compared to that of the control group, which is in concurrence with previous studies. Smoking habits and age did not show significant influences on the level of DNA damage and repair when compared with the control group. Conclusion: Exposure to volatile organic compounds such as ethylmethacryale and methylmethacrylate may lead to DNA damage and altered DNA repair in some individuals, although further studies are recommended. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2011.

Objectives: The aim of this study was to determine if exposure to volatile organic compounds can lead to DNA damage and impaired DNA repair capacity. Nail cosmetics is a fast growing industry around the world where employees and clients are subjected to various chemical substances which may be harmful to their health: such as formaldehyde, toluene, acetone, xylene, ethylmethacrylate, methylmethacrylate and n–buthyl acetate. These chemicals have the potential to be harmful to their health and exposure to these chemicals should be actively controlled. Formaldehyde is classified as a human carcinogen by the IARC, whereas, toluene and xylene are group three carcinogens, classified in 1999 (not classified as carcinogenic to humans), and various studies have linked DNA damage and impaired DNA repair to the above mentioned substances. Methods: Fifteen nail technicians were monitored by means of personal air sampling, measuring formaldehyde, toluene, xylene, acetone and ethylmethacrylate exposure. Fifteen unexposed subjects were chosen and matched for age and smoking habits with the exposed group. Heparinised blood samples were obtained from each test subject with which the Comet Assay was performed on lymphocytes to determine DNA damage and repair ability. Results: Exposure to ethylmethacrylates and methylmethacrylates leads to DNA damage. Methylmethacrylate causes DNA damage by specifically targeting pyrimidine (fpg) bases. N–buthyl acetate, xylene and acetone exposure impaired DNA repair capacity. The exposed group showed signs of Class III and Class IV DNA damage, whereas the control group had little Class III damage and no indication of Class IV damage. The overall DNA repair ability of the nail technicians was slightly impaired when compared to that of the control group, which is in concurrence with previous studies. Smoking habits and age did not show significant influences on the level of DNA damage and repair when compared with the control group. Conclusion: Exposure to volatile organic compounds such as ethylmethacryale and methylmethacrylate may lead to DNA damage and altered DNA repair in some individuals, although further studies are recommended. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2011.

Dentre as etapas do tratamento endodôntico, a limpeza e modelagem tem especial atenção, já que a conformação geométrica satisfatória e a sanificação dos canais radiculares propiciam condições favoráveis ao controle da infecção e à adequada obturação do espaço endodôntico. O desenvolvimento de instrumentos fabricados a partir da liga de níquel-titânio (NiTi) contribuiu de maneira importante para a melhora na qualidade e previsibilidade do preparo endodôntico. Os diferentes sistemas mecanizados que são propostos constantemente no mercado devem ser avaliados com detalhes, pois sua performance pode variar de acordo com as características de fabricação e design de cada instrumento. O objetivo deste estudo foi avaliar, por meio da microtomografia computadorizada (micro-CT), o preparo de canais mesiais de molares inferiores com o sistema XP-endo Shaper (XPS), comparando-o ao preparo realizado com instrumentos do sistema Reciproc (REC), quanto ao volume de debris dentinários remanescentes, área de paredes não tocadas, dentina excisada, aumento do volume e superfície do canal radicular e desvio do canal original no terço apical. Vinte e quatro raízes mesiais de molares inferiores, com dois canais e um forame, foram anatomicamente pareadas e divididas em 2 grupos experimentais (n=12) de acordo com o sistema utilizado para o preparo químico cirúrgico: Grupo XPS e Grupo REC. Os espécimes foram escaneados por um microtomógrafo de raios-X (SkyScan 1176) com resolução de 17.42 ?m pré e pós preparo. Com o auxílio dos softwares Dataviewer, CTan e CTVol foi possível a avaliação tridimensional do canal para observação das mudanças nos parâmetros estudados. Os dados obtidos foram comparados e, onde se identificou distribuição normal e homogeneidade das variâncias, foi utilizado o Teste t de student para analisar as hipóteses experimentais. Para os dados onde se observou distribuição não-normal, optou-se pelo teste não-paramétrico numérico (Mann- Whitney). Todos as análises foram realizadas com nível de significância de 5%. Nos resultados, em toda extensão do canal radicular, assim como para as análises por terços, não houve diferença estatisticamente significativa entre os grupos quanto ao transporte do canal, volume de dentina excisada e aumento do volume e superfície do canal. Identificou-se diferença significativa (p < 0,05) entre os grupos quanto a área de superfície não tocada, onde observou-se que, no canal total e nos terços cervical e médio, o grupo XPS preparou maior área do canal do que o grupo REC. Quanto ao volume e percentual de debris dentinários acumulados, o grupo REC apresentou valores estatisticamente maiores na totalidade do canal e nos terços médio e apical, evidenciando maior acúmulo de debris intra-radiculares do que o grupo XPS. Concluiuse que as técnicas de preparo avaliadas se comportaram de maneira semelhante quanto ao aumento de volume e superfície do canal, volume de dentina removida e desvio apical. Em contrapartida, o sistema XPS, por possuir cinemática rotatória excêntrica, conseguiu tocar maior quantidade de paredes e eliminar maior volume percentual de debris acumulados em relação ao sistema que se vale de cinemática reciprocante cêntrica (REC). / Among the stages of endodontic treatment, cleaning and shaping should have special attention, since a satisfactory geometry and sanification of the root canals provide favorable conditions for infection control and adequate endodontic obturation. The development of the Niquel-Titanium (NiTi) instruments contributed to the improvement in quality and predictability of the endodontic preparation. The new mechanized systems that are constantly proposed in the market must be evaluated, as their performance can vary according to the manufacturing and design features of each instrument. The purpose of this study was to evaluate, using microcomputed tomography (micro-CT), the preparation of mesial root canals of mandibular molars with the new XP-endo Shaper (XPS) system comparing it to the preparation performed with Reciproc instruments (REC), evaluating the volume of remaining dentin debris, area of untouched walls, dentin removed, increase in volume and surface of the root canal and apical tranportation. Twenty-four mandibular molar mesial roots with two canals and one foramen were anatomically matched and divided into 2 experimental groups (n = 12) according to the system used for shaping: XPS Group and REC Group. The specimens were scanned pre and post preparation by a X-ray microtomograph (SkyScan 1176) with a resolution of 17.42 ?m. With Dataviewer, CTan and CTVol softwares, it was possible a tridimensional evaluation of the root canal in order to observe changes in the studied parameters. The data were compared and, where normal distribution and homogeneity of variances were identified, Student\'s t test was used to analyze the experimental hypotheses. For the data where non-normal distributions were observed, the non-parametric numerical test (Mann-Whitney) was chosen. All analyzes were performed with a significance level of 5%. In the results, in all extension of the root canal, as well as for analyzes by thirds, there was no statistically significant difference between the groups regarding apical transportation, volume of removed dentin and increase of volume and canal surface. A significant difference (p <0.05) was found between the groups regarding the untouched surface area, where in the total canal and in the cervical and middle thirds, the XPS group prepared a larger area of the canal than the group REC. As to the volume and percentage of accumulated dentinal debris, the REC group presented statistically higher values in the whole canal and in the middle and apical thirds, evidencing a higher accumulation of intra-radicular debris than the XPS group. It was concluded that the preparation techniques evaluated behaved similarly to the changes in volume and surface of the canal, the volume of dentin removed and the apical tranportation. The XPS system by its eccentric rotational kinematics was able to touch a larger number of walls and eliminate a greater percentage volume of accumulated debris compared to the system that uses a centralized reciprocating motion.

Durante o preparo químico-cirúrgico são utilizados instrumentos e substâncias químicas, que constituem um binômio indivisível e necessário para alcançar a modelagem e a sanificação dos canais radiculares. Assim, propõe-se com este trabalho avaliar in vivo, por meio de método molecular de PCR quantitativo, baseado em DNA (qPCR), a eficiência do preparo químico-cirúrgico empregando como agente de irrigação o Hipoclorito de Sódio (NaOCL) a 2,5% ou o Gel de Endo PTC associado ao Hipoclorito de Sódio a 1,0% na redução bacteriana de canais radiculares de dentes portadores de periodontite apical primária. Foram selecionados 30 pacientes portadores de infecção endodôntica primária, totalizando 30 dentes, com rarefação óssea periapical visível na radiografia, sem tratamento endodôntico prévio. Os pacientes foram divididos de forma randomizada em dois grupos distintos, de acordo com a substância química auxiliar utilizada durante a instrumentação, NaOCL 1% + Endo-PTC leve ou NaOCL 2,5%. Em todos os casos empregou-se instrumentos Reciproc R40 ou R50 e as coletas foram realizadas antes (S1) e após o prepare químico-cirúrgico (S2). A análise de aderência foi realizada por meio do teste de Kolmogorov-Smirnov, as análises intragrupo foram realizadas com teste de Wilcoxon para amostras relacionadas e as comparações entre os dois grupos foram realizadas com o teste de Mann-Whitney, para a análise quantitativa de bactérias. Em ambos os grupos, houve diminuição significativa no número de bactérias entre S1 e S2 (p<0,05). No grupo NaOCL 1% + Endo-PTC leve, houve redução de 3,7x105(S1) para 5,7x104 (S2). No grupo NaOCl 2,5%, redução de 1,3x105 (S1) para 1,1x104(S2). Na comparação entre grupos, o NaOCL a 2,5% (91,62%) promoveu maior redução bacteriana do que o grupo NaOCL 1% + Endo-PTC (84,60%) (p<0,05). / During the chemomechanical preparation, instruments and chemical substances are used, which constitute an indivisible and necessary binomial to achieve modeling and sanification. Knowing the auxiliary chemical substances, understanding their mechanisms of action, being able to use them efficiently, is fundamental, so that the chemical-surgical preparation is well performed by the clinician. Thus, the purpose of this study is to evaluate in vivo, the efficiency of the chemomechanical preparation using as the irrigant agent 2,5% sodium hypochlorite and Endo-PTC gel, associated to 1% sodium hypochlorite, to assess the bacterial reduction of root canals of teeth with primary apical periodontitis, using a molecular quantitative method DNA-based - polymerase chain reaction (qPCR). Were selected 30 patients with primary infection totaling 30 teeth, with visible periapical bone rarefaction on the radiography, without previous endodontic treatment. Patients were randomly divided into two distinct groups according to the auxiliary chemical substances used during the instrumentation, 1% sodium hypochlorite associated with Endo-PTC gel or 2,5% sodium hypochlorite. In all cases, reciproc instruments R40 or R50 were used and the samples were taken before (S1) and after chemical surgical preparation (S2). The adherence analysis was performed using the Kolmogorov-Smirnov test, intragroup analysis were performed with Wilcoxon test for related samples and comparisions between the two groups were performed with the Mann-Whitney test for the quantitative analysis of bacteria. In the both groups, there was a significant decrease in the number of bacteria between S1 and S2 (p<0,05), the inicial sample (S1) of the group Endo-PTC, the median 3,7x105, reduced to 5,7x104. In the other group of NaOCl, the median in S1 was 1,3x105 that reduced to 1,1x104 . In the comparision between groups, the 2,5% NaOCl promoted a greater microbial reduction of 91,62%, than the Endo-PTC associated with 1% NaOCl (p<0,05) 84,60%.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-10T11:30:57Z No. of bitstreams: 2 Dissertação - Vinicius Caixeta de Sousa - 2016.pdf: 6443318 bytes, checksum: 09bb12a456ef519b05b09ed648a75811 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-10T11:31:28Z (GMT) No. of bitstreams: 2 Dissertação - Vinicius Caixeta de Sousa - 2016.pdf: 6443318 bytes, checksum: 09bb12a456ef519b05b09ed648a75811 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-10T11:31:28Z (GMT). No. of bitstreams: 2 Dissertação - Vinicius Caixeta de Sousa - 2016.pdf: 6443318 bytes, checksum: 09bb12a456ef519b05b09ed648a75811 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Abstract: Objective: To evaluate the effectiveness of complementary protocols of sanitization in the decontamination of infected root canals. Methods: Fifty uniradicular maxilary anterior human teeth were selected. The crowns were removed and the lengths standardized at 16 mm. The specimens were inoculated with Enterococcus faecalis and incubated at 37 °C for sixty days. Thirty teeth were performed with BioRace instruments until diameter corresponding to #60.02, and then complemented with Self-Adjusting File (SAF); XP-endo Finisher (XPF) and passive ultrasonic irrigation (PUI). Ten samples were used as positive control and 10 were not contaminated. Initial and final samples were collected and incubated at 37 °C for a period of 48 hours. The bacterial growth was analyzed in culture, determining the presence or absence of bacteria. The optical density of the culture medium was interpreted by UV spectrophotometry. The specimens were sectioned and prepared for evaluation in SEM. The images of the root surfaces were analyzed and classified by scores according to the presence of debris. The Kruskal-Wallis test was used for statistical analysis. The level of significance was 5%. Results: The mean optical density (μm) of the sanification protocols showed bacterial reduction in all groups. The experimental groups did not present statistically significant differences (p = 0.196). The analysis of SEM images revealed no significant difference (p = 0.414) between the scores of the groups. Conclusion: Complementary sanitization protocols reduced bacterial contamination. / Resumo: Objetivo: Avaliar a efetividade de protocolos complementares de sanificação na descontaminação de canais radiculares infectados. Materiais e métodos: Cinquenta dentes humanos anteriores superiores unirradiculares foram selecionados. As coroas foram removidas e os comprimentos padronizados em 16 mm. Os espécimes foram inoculados com Enterococcus faecalis e incubados a 37°C por sessenta dias. Trinta dentes foram preparados com instrumentos BioRace até alcançar o diâmetro correspondente ao #60.02, e a seguir complementados com Self-Adjusting File (SAF); XP-endo Finisher (XPF) e irrigação ultrassônica passiva (PUI). Dez amostras foram usadas como controle positivo e 10 não foram contaminadas. Amostras inicial e final foram coletadas e incubadas a 37°C por um período de 48 horas. O crescimento bacteriano foi analisado em cultura, determinando a presença ou ausência de bactérias. A densidade óptica do meio de cultura foi interpretada por espectrofotometria UV. Os espécimes foram seccionados e preparados para avaliação em MEV. A análise das imagens das superfícies radiculares foram analisadas e classificadas em scores de acordo com a presença de debris. O teste de Kruskal-Wallis foi utilizado para as análises estatísticas. O nível de significância foi 5%. Resultados: A média da densidade óptica (μm) dos protocolos de sanificação revelou redução bacteriana em todos os grupos. Os grupos experimentais não apresentaram diferenças estatisticamente significante (p=0,196). A análise das imagens de MEV revelou ausência de diferença significante (p=0,414) entre os scores dos grupos. Conclusão: Os protocolos complementares de sanificação favoreceu a redução da contaminação bacteriana.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities. / AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W) geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1 geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie van die geen sodoende in sommige rasse voorkom. Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens, asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk. Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van wyngiste te verbeter. Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme van regulering van toepassing op PGU1 te identifiseer. As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes. The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol. The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types. This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene.