5 resultados para 8-Hydroxyguanin, DNA Reparatur, Oxidativer Stress, Antioxidantien
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Resumo: Os profissionais de sade podem estar expostos a vrios factores indutores de stress crnico nomeadamente de natureza profissional destacando-se, entre os seus possveis efeitos, a diminuio da resposta de anticorpos aps administrao de vacinas, entre as quais, a vacina contra a gripe. Uma vez que os trabalhadores da sade esto expostos a factores indutores de stress e, simultaneamente, a agentes biolgicos cujos efeitos podero ser prevenidos pela vacinao, pertinente estudar a influncia do stress na resposta imunitria vacina contra a gripe em enfermeiros. Constituram objectivos deste trabalho: (1) estudar a associao entre a presena de stress crnico em enfermeiros hospitalares e a insuficiente resposta imunitria vacina contra a gripe, avaliada um ms aps a vacinao (T1); (2) estudar a associao entre a presena de stress crnico em enfermeiros hospitalares e a reduo dos ttulos de anticorpos dirigidos s hemaglutininas seis meses aps a vacinao (T6) e (3) identificar algumas caractersticas das unidades de internamento e do trabalho dos participantes que possam estar associadas presena de stress crnico e estudar a sua possvel associao com a resposta imunitria vacina contra a gripe. Realizou-se um estudo caso-controlo incorporado num estudo de coortes e a amostra em estudo foi constituda por 136 enfermeiros saudveis (83,8% sexo feminino; mdia de idades de 33anos) de um hospital universitrio. Realizaram-se entrevistas individuais e aplicaram-se as verses portuguesas dos questionrios The General Health Questionnaire (GHQ12) e Maslach Burnout Inventory Human Services Survey (MBI-HSS) para determinao da presena de stress crnico pelo mtodo da triangulao, no incio do estudo (T0) e realizou-se a recolha de dados relativos caracterizao de elementos de trabalho nas unidades de internamento. Foi administrada a vacina contra a gripe e determinou-se os ttulos de anticorpos dirigidos s hemaglutininas de cada estirpe componentes da vacina contra a gripe utilizada em 2007, antes da vacinao, um ms e seis meses aps a vacinao. No se encontrou associao, ao nvel de significncia de 5%, entre a presena de stress e a insuficiente resposta vacina contra a gripe, avaliada pela taxa de indivduos que apresentaram um aumento, ao fim de um ms, inferior a quatro vezes os ttulos de anticorpos antes da vacinao. No entanto, encontrou-se uma maior proporo de indivduos com stress no grupo de participantes em que ocorreu uma diminuio do ttulo de anticorpos dirigidos hemaglutinina AH1 (ac AH1) em T6, quando comparado com o respectivo grupo controlo. A diferena entre grupos foi estatisticamente significativa, quando se avaliou a presena de stress pelo mtodo da triangulao usando a entrevista (p=0,006), pelo mtodo da triangulao usando o GHQ12 (p=0,045) e ainda usando a combinao dos trs critrios (p=0,001). Aps anlise multivariada, verificou-se que a associao entre a presena de XXVI stress e a reduo dos ac AH1 em T6 manteve significado estatstico (respectivamente, p= 0,010, p= 0,042 e p=0,002) e apresentou odds ratio ajustados, em funo de cada um dos mtodos de avaliao da presena de stress, de 3,643, de 2,733 e de 5,223. A quantidade de trabalho percepcionada como sobrecarga constituiu o factor indutor de stress mais vezes referido (58,8% da amostra e 61,8% dos enfermeiros de unidades de internamento), seguida dos conflitos entre profissionais. O contacto com o sofrimento e a morte de doentes foram identificados em quarto lugar pela amostra, mas em segundo pelos enfermeiros de unidades de internamento. Nesses, verificou-se uma associao positiva entre trabalhar em Servios onde o nmero de doentes falecidos foi muito elevado e a presena de stress, medido pelo mtodo da triangulao usando a entrevista (p=0,039), usando o GHQ12 (p=0,019), usando a escala de exausto emocional do MBI-HSS (p=0,012) e pela combinao dos trs mtodos (p=0,014). Verificou-se tambm uma associao positiva entre a presena de stress, identificada pelo mtodo da triangulao usando a escala de exausto emocional do MBI-HSS, e o trabalho em servios de internamento onde a percentagem de doentes idosos (p=0,025) e a taxa de letalidade (p=0,036) foram elevadas. Contudo, no se encontrou associao entre a exposio muito frequente ao sofrimento e morte de doentes e a reduo do ttulo de ac AH1 em T6. Possivelmente, a exposio a esse factor indutor de stress, apesar de estar relacionada com a presena de stress nos enfermeiros de servios de internamento, no foi suficientemente intenso para, por si s, estar associada reduo do ttulo de ac AH1 em T6. A associao encontrada entre a presena de stress crnico e a reduo do ttulo de anticorpos AH1 em T6 vem apoiar a resposta questo de investigao inicialmente colocada de que o stress poder influenciar negativamente a manuteno dos ttulos de anticorpos, mesmo em indivduos adultos no idosos. Assim, o risco de um enfermeiro com stress apresentar reduo do ttulo de anticorpos dirigidos hemaglutinina da estirpe AH1N1 A/Solomon Islands/3/2006 ao fim dos seis meses do estudo, foi 3,6, 2,7 ou 5,2 vezes superior ao de um enfermeiro sem stress, consoante o critrio de stress ter sido determinado, respectivamente, pelo mtodo da triangulao usando a entrevista, pelo mtodo da triangulao utilizando o GHQ12 ou pela combinao dos trs critrios. Summary: Health workers may be exposed to various factors causing chronic stress namely those related directly to their activity, in particular the decrease in the capacity of the response of antibodies after the administration of the vaccines, amongst others the Influenza vaccine. Since health workers are exposed to factors causing stress and at the same time biological agents, whose effects may be prevented through vaccination, it is important to study the influence of stress in the immunity response to the Influenza vaccine on nurses. The aims of this study are: (1) to examine the relation between chronic stress in hospital nurses and the insufficient immunity response to the Influenza vaccine, assessed at one month after vaccination (T1); (2) to examine the relation between chronic stress in hospital nurses and the decrease of the hemagglutinin titles of antibodies six months after vaccination (T6); (3) to identify some characteristics of internment units and the work of the participants that may be related to the presence of chronic stress and to study its possible relation with the immunity response to the Influenza vaccine. A control-case study, integrated in a coortes study, was carried out and the sample under analysis consisted of 136 healthy nurses (83,8% female; average age 33 years old) from a university hospital. Several individual interviews were conducted and the portuguese versions of General Health Questionnaire (GHQ12) and Maslach Burnout Inventory Human Services Survey (MBI-HSS) was applied in order to determine the presence of chronic stress, using the triangulation method at the beginning of the study (T0). Data concerning the particular features of the internment units was collected. The Influenza vaccine was administered and the titles of hemagglutinin antibodies of each strain composing the Influenza vaccine used in 2007, before vaccination, and a month and six months after vaccination, were determined. There was no statistically relevant (5%) relation between stress and the insufficient immune response to the Influenza vaccine, according to the rate of individuals that showed, after a month, a level of antibodies concentration lower than four times the level prior to the vaccination. Nevertheless, there was a greater number of individuals with stress in the group of participants in which there was a decrease of the hemagglutinin titles of antibodies AH1 (ac AH1) in T6, when compared to the control group under study. The difference between groups was statistically relevant when assessing the presence of stress by triangulation method using the interview (p=0,006), by triangulation method using the GHQ12 (p=0,045) and by the combination of the three criteria (p=0,001). After multivariate analysis, it was verified that the XXVIII relation between the presence of stress and the reduction of the ac AH1 in T6 was statistically relevant (respectively, p= 0,010, p= 0,042 and p=0,002) and the odds ratio were, according to each of the methods used to assess the presence of stress, 3,643, 2,733 and 5,223. Overwork was the most emphasised stress-causing factor (58,8% of the sample and 61,8% of the nurses working in the Internment Units), followed by conflicts arousing among co-workers. Witnessing the suffering and death of patients was ranked as the fourth cause of stress, but the second by the nurses of the internment units. The former revealed a positive connection between working in the services, where there was a high rate of deaths, and the presence of stress, when assessing the presence of stress by triangulation method using the interview (p=0,039), the GHQ12 (p=0,019), the MBI-HSS emotional exhaustion scale (p=0,012) and by the combination of the three criteria (p=0,014).There was also a connection between the presence of stress, identified by the method of triangulation using the MBI-HSS emotional exhaustion scale, and working in the internment units, where the percentage of elderly people (p=0,025) and the mortality rate (p=0,036) were high. However, there was no connection between frequent exposure to suffering and death in patients and the reduction of ac AH1 titles, in T6. Although one can establish a connection between stress in nurses working in the internment units and the aforementioned stress-causing factor, the exposure to that factor was not, per se, intense enough to reduce the ac AH1 title in T6. The relation found between the presence of chronic stress and the reduction of AH1 antibodies titles in T6, corroborates the hypothesis that stress can negatively influence the title of antibodies, even in non-elderly adults. Thus, and according to the criteria used to define stress, by the triangulation method using the interview, by the triangulation method using the GHQ12 or the combination of the three criteria respectively, the risk of a nurse suffering from stress showing a reduction in the title of hemagglutinin antibodies for the strain AH1N1 A/Solomon Islands/3/2006 six-month after Influenza vaccine was 3,6, 2,7 or 5,2 times greater than on a nurse suffering from no stress at all. Rsum: Les professionnels de la sant peuvent tre exposs diffrents facteurs inducteurs de stress chronique de nature professionnelle. On remarque, parmi les effets possibles, une baisse de la rponse des anticorps aprs ladministration de vaccins, comme en particulier, le vaccin de la grippe. Lorsque les professionnels de la sant ont t exposs des facteurs inducteurs de stress, et de manire simultane, des agents biologiques dont les effets pourront tre prvenus par la vaccination, il est pertinent dtudier linfluence du stress dans la rponse immunitaire au vaccin de la grippe chez les infirmiers. Ils ont constitu des objectifs dtudes et de discussion : (1) tudier lassociation entre la prsence de stress chronique chez les infirmiers, en milieu hospitalier, et la insuffisant rponse immunitaire au vaccin de la grippe, vrifie un mois aprs la vaccination (T1); (2) tudier lassociation entre la prsence de stress chronique chez les infirmiers, en milieu hospitalier, et la rduction de la teneur des anticorps dirig la hmaglutinina six mois aprs la vaccination (T6) (3) identifier certaines caractristiques des units dinternement, et tudier les aspects du travail des participants, qui puissent tre associe la prsence de stress chronique et tudier sa possible association avec la rponse immunitaire au vaccin de la grippe. Une tude cas-contrle incorpore dans une tude de groupe a t ralise et un chantillon, pour tude, a t constitu par 136 infirmiers sains (83,8% de sexe fminin, ge moyen 33 ans) travaillant dans un hpital universitaire. Des entretiens individuels ont t raliss et les versions portugaises des questionnaires de General Health Questionnaire (GHQ12) et Maslach Burnout Inventory- Human Service Survey (MBI-HSS) ont t utiliss pour dterminer la prsence de stress chronique grce la mthode de triangulation, au dbut de ltude (T0) et un relev de donnes relatives la caractrisation dlments de travail dans les units dinternement a t fait. Le vaccin de la grippe a t administr et les teneurs en anticorps dirigs aux hmaglutininas de chaque composant du vaccin de la grippe pour 2007 ont t dtermins, avant la vaccination et un mois et six mois aprs. On na pas trouv dassociation, un niveau significatif de au moins 5%, entre la prsence de stress et la insuffisant rponse au vaccin de la grippe, value par le taux dindividus qui ont prsent une augmentation, la fin du mois, infrieur quatre fois la teneur des anticorps par rapport avant la vaccination. Cependant , on a trouv une plus grande proportion dindividus victimes de stress dans le groupe des participants o il y a eu une baisse de la teneur des anticorps dirig la hmaglutinina AH1 (ac AH1) en T6, aprs comparaison avec le respectif groupe de contrle. La diffrence entre les groupes a t statistiquement significative lorsquon a vrifi la prsence de stress grce la mthode de triangulation, en utilisant lentretien (p=0,006), par la mthode de triangulation en utilisant le GHQ12 (p=0,045) et en utilisant aussi la combinaison des trois critres (p=0,001). Aprs une analyse XXX multivarie, on a vrifi que lassociation entre la prsence de stress et la rduction des ac AH1 en T6 a conserv un signifi statistique (respectivement, p=0,010, p=0,042 et p=0,002) et a prsent des odds ratio ajusts, en fonction de chacune des mthodes de vrification de la prsence de stress de 3,643, de 2,733 et de 5,223. La quantit de travail perue comme une surcharge constitue le facteur inducteur de stress le plus souvent cit (58,8% de lchantillon et 61,8% des infirmiers des units dinternement), suivi par les conflits entre professionnels. Le contact avec la souffrance et la mort des patients a t plac en quatrime position par lchantillon, mais en deuxime position par les infirmiers des units dinternement. Dans ces cas, on a vrifi une association vidente entre le fait de travailler dans des services o le nombre de patients dcds a t trs lev et la prsence de stress, identifie par la mthode de triangulation, en utilisant lentretien (p=0,039), le GHQ12 (p=0,019), lchelle de fatigue motionnelle du MBI-HSS (p=0,012) et en utilisant aussi la combinaison des trois critres (p=0,014). On a aussi vrifi une association positive entre la prsence de stress, identifie par la mthode de triangulation, en utilisant lchelle de fatigue motionnelle du MBI-HSS et le travail dans des services dinternement o le pourcentage de malade gs (p=0,025) et le taux de mortalit ont t levs (p=0,036). Malgr tout, on na pas trouv dassociation entre lexposition trs frquente la souffrance et la mort des patients et la rduction de la teneur de ac AH1 en T6. Probablement lexposition ce facteur inducteur de stress, bien quelle soit lie la prsence de stress chez les infirmiers des services dinternement, na pas t suffisamment intense pour, en elle-mme, tre associe la rduction de la teneur ac AH1 enT6. Lassociation trouve entre la prsence de stress chronique et la rduction de la teneur des anticorps AH1 en T6 vient renforcer lhypothse que le stress pourra influencer ngativement la manutention des teneurs en anticorps mme chez les individus adultes jeunes. Donc le risque quun infirmier stress prsente une rduction de la teneur en anticorps dirigs la hmaglutinina de le composant AH1N1-A/Solomon Island/3/2006 la fin des six mois dtudes a t 3,6, 2,7 ou 5,2 fois suprieure celui dun infirmier sans stress, aprs avoir dtermin le critre de stress, respectivement par la mthode de triangulation utilisant lentretien, par la mthode de triangulation utilisant le GHQ12 ou par la combinaison des trois critres.
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Dissertao para obteno do Grau de Mestre em Biotecnologia
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RESUMO: A sfilis uma infeco causada por T. pallidum que pode ser transmitida por via vertical, por contacto sexual ou por sangue, e cujo diagnstico se baseia na associao entre manifestaes clinicas e testes serolgicos. Neste estudo utilizaram-se os testes serolgicos RPR, TPHA, FTA-Abs, um teste rpido no comercializado (Signal-Spirolipin) que pesquisa anticorpos treponmicos (CDC-T) e no treponmicos (CDC-2) em simultneo no mesmo dispositivo e uma tcnica de PCR-multiplex para a pesquisa de ADN de T. pallidum. Da comparao das tcnicas serolgicas constatou-se que a sensibilidade dos testes RPR e TPHA foi de 84,5% e 98% respectivamente. A especificidade foi de 77,3% para o teste RPR e de 87,5% para o teste TPHA. Na avaliao do teste rpido a comparao do teste CDC-2 com o teste RPR resultou numa taxa de concordncia de 93,1% enquanto que, a do teste CDC-T com o teste TPHA foi de 94,8%. A sensibilidade e especificidade obtidas pelos testes CDC-2 e CDC-T foram de 88,7% e 65% e de 94,9% e 91,3% respectivamente. Considerando o diagnstico de sfilis activa com base na reactividade simultnea dos testes RPR e TPHA, avaliou-se a sensibilidade do teste rpido utilizando esse mesmo critrio. A sensibilidade obtida foi de 98,4% para o teste rpido e de 97,2% para a associao dos testes RPR/TPHA. A tcnica de PCR-multiplex apresentou fraca sensibilidade j que em apenas 33% dos casos de sfilis foi identificado ADN de T. pallidum. O teste rpido avaliado apresentou um comportamento idntico aos testes geralmente utilizados na rotina laboratorial tais como os utilizados neste estudo. Apresenta a vantagem de pesquisar anticorpos treponmicos e no treponmicos, ao contrrio dos testes rpidos comercializados que pesquisam apenas anticorpos treponmicos. No sendo necessrio para a sua execuo equipamento laboratorial especializado poder ser de grande utilidade para o clinico a exercer em locais sem laboratrio.------------- ABSTRACT: Syphilis is an infection caused by T. pallidum. Transmission may be vertical, through sexual contact or blood. The diagnosis is based in both serology and clinical manifestations. In this study, we used the serological tests RPR, TPHA, FTA-Abs and a non -commercialized rapid test (Signal-Spirolipin) to detect both treponemal (CDC-T) and non treponemal antibodies (CDC-2) in the same device. T. pallidum DNA was identified with a multiplex PCR technique. In this study, the RPR and TPHA tests sensitivity was 84,5% and 98%, respectively, and the specificity 77,3% for the RPR test and 87,5% for TPHA test, respectively. The concordance rate was 93,1% in the comparison between the RPR and the CDC2 tests and 94,8% between the CDC-T with the TPHA tests. Sensitivity and specificity of the CDC-2 and CDC-T tests were 88,7% and 65% and 94,9% e 91,3%, respectively. Taking into account that the diagnosis of active syphilis is made when both the RPR and TPHA tests are reactive, we evaluated the sensitivity of the rapid test (CDC2 and CDCT) in the diagnosis of active syphilis, using the above described criteria. The sensitivity was 98,4 % for the rapid test and 97,2 % for the association of reactivity in both RPA and TPHA tests. The multiplex PCR technique presented a low sensitivity, with T. pallidum DNA being identified in only 33% of the syphilis cases. The rapid test evaluated in this study presented identical results to the tests generally used in the routine laboratory diagnosis, such as those used here. However, it does have the advantage of detecting both treponemal and non treponemal antibodies what is not the case of the commercialized rapid test. Furthermore, there is no need of specialized laboratory equipment, which makes it of great utility in regions where such conditions do not exist, as in developing countries.
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The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.
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Grapevine (Vitis vinifera) is one of most agro-economically important fruit crops worldwide, with a special relevance in Portugal where over 300 varieties are used for wine production. Due to global warming, temperature stress is currently a serious issue affecting crop production especially in temperate climates. Mobile genetic elements such as retrotransposons have been shown to be involved in environmental stress induced genetic and epigenetic modifications. In this study, sequences related to Grapevine Retrotransposon 1 (Gret1) were utilized to determine heat induced genomic and transcriptomic modifications in Touriga Nacional, a traditional Portuguese grapevine variety. For this purpose, growing canes were treated to 42 oC for four hours and leaf genomic DNA and RNA was utilized for various techniques to observe possible genomic alterations and variation in transcription levels of coding and non-coding sequences between non-treated plants and treated plants immediately after heat stress (HS-0 h) or after a 24 hour recovery period (HS-24 h). Heat stress was found to induce a significant decrease in Gret1 related sequences in HS-24 h leaves, indicating an effect of heat stress on genomic structure. In order to identify putative heat induced DNA modifications, genome wide approaches such as Amplified Fragment Length Polymorphism were utilized. This resulted in the identification of a polymorphic DNA fragment in HS-0 h and HS-24 h leaves whose sequence mapped to a genomic region flanking a house keeping gene (NADH) that is represented in multiple copies in the Vitis vinifera genome. Heat stress was also found to affect the transcript levels of various non-coding and gene coding sequences. Accordingly, quantitative real time PCR results established that Gret1 related sequences are up regulated immediately after heat stress whereas the level of transcript of genes involved in identification and repair of double strand breaks are significantly down regulated in HS-0 h plants. Taken together, the results of this work demonstrated heat stress affects both genomic integrity and transcription levels.
