Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 4 uppsatser.

DNA methylation is an epigenetic mechanism that regulates the gene transcription. Folate is used in cellular synthesis of methyl groups, nucleic acids and amino acids. In complex diseases like cancer and neural tube defects (NTD), various genetic and epigenetic alterations can be found that disrupt the normal cell function. The main goals of this thesis were to analyze whether the genes responsible for the folate transport (FOLR1, PCFT, and RFC1) could be regulated by DNA methylation in placenta, blood leukocytes and colorectal cancer. We also addressed the genome-wide DNA methylation changes in colorectal cancer andcervical cancer.We found that changes in the methylated fraction of the RFC1 gene were dependent on the RFC1 80G>A polymorphism in placental specimens with NTDs and blood leukocytes from subjects with high homocysteine (Paper I). In colorectal cancer, the greatest difference in DNA methylation was observed in the RFC1 gene and was related to a lower protein expression (Paper II).In Paper III and IV we studied the DNA methylated fraction using a high-density array. Paper III was focused on genes in the DNA repair pathway and frequently mutated in colorectal cancer. We found that aberrant methylation in the DNA mismatch repair genes was not a frequent event in colorectal cancer and we identified five candidate biomarker genes in colorectal cancer, among them the GPC6 and DCLRE1C genes. In Paper IV, we found hypomethylation of genes involved in the immune system in cervical cancer specimens compared to healthy cervical scrapes and we identified twenty four candidate genes for further evaluation ofclinical value.In conclusion, the work of this thesis filled a relevant knowledge gap regarding the role of differential methylation of the folate transport genes in NTD and colorectal cancer. This thesis work also provided insights into the functional role of DNA methylation in cancer specific pathways and identified potential novel biomarker genes.

Made available in DSpace on 2015-07-22T20:49:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-18. Added 1 bitstream(s) on 2015-08-11T03:26:15Z : No. of bitstreams: 1 Publico-12957a.pdf: 1243731 bytes, checksum: 878db06d2872246e99cbd4358ee4a1d0 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:15Z : No. of bitstreams: 2 Publico-12957a.pdf: 1243731 bytes, checksum: 878db06d2872246e99cbd4358ee4a1d0 (MD5) Publico-12957b.pdf: 1376768 bytes, checksum: 4cdb6afbfd0e66e1d0fb5c0615f48744 (MD5) / A sepse apresenta crescente incidência com elevada morbidade e mortalidade, sendo a principal causa de óbito nas unidades de terapia intensiva. O controle da infecção depende do adequado reconhecimento dos microrganismos pelas células do hospedeiro e de resposta efetora competente. Os linfócitos, monócitos e neutrófilos são as principais populações celulares do sangue periférico envolvidas nesse processo. A apoptose representa um importante mecanismo de controle da resposta imune, mas em condições críticas, a apoptose desregulada dessas células pode levar o hospedeiro a imunossupressão, dificultando o controle da sepse. Este trabalho avaliou-se a porcentagem de apoptose em linfócitos monócitos e neutrófilos do sangue periférico de pacientes sépticos pela exposição de fosfatidilserina na superfície celular e pela detecção intracelular de caspase-3, também foi avaliado o número absoluto e percentual de linfócitos e suas subpopulações por citometria de fluxo. Foram incluídos 40 pacientes, sendo 4 em sepse, 9 em sepse grave e 27 em choque séptico, classificados de acordo com as definições do consenso de 1992. Quinze voluntários sadios foram incluídos para comparação. Nos ensaios de detecção de apoptose foi feita a separação dos leucócitos totais seguida de lise hipotônica das hemácias e marcação com anticorpos de superfície para identificação das populações leucocitarias. Para verificação da apoptose as amostras foram marcadas com anexina-V e coradas com Iodeto de Propídeo (PI) em tampão apropriado ou incubadas com anticorpo anti-caspase-3 após fixação e permeabilização. A contagem de linfócitos foi realizada em tubos TruCOUNT após a marcação com anticorpos anti-CD45, CD3, CD4, CD8, CD16/56 e CD19. Não houve diferença no percentual de linfócitos totais em apoptose, porém observou-se menor apoptose tardia em linfócitos T nos pacientes sépticos quando comparados aos sadios ( P<0,05). Foi observada menor contagem absoluta de linfócitos totais, linfócitos T, TCD4+, TCD8+ e NK de pacientes sépticos (p<0,01), embora o percentual destas populações celulares tenha se mantido inalterado. Os resultados mostram que o menor número de linfócitos circulantes observados durante o quadro de sepse não pôde ser explicado pela presença de apoptose. A diminuição da contagem destas células na periferia pode significar uma migração para os tecidos ou órgãos linfóides desencadeada pela infecção. Nos neutrófilos não foi observada diferença no percentual de células em apoptose precoce entre pacientes e indivíduos sadios. Houve queda no percentual de apoptose tardia e conseqüente aumento no percentual de células viáveis dos pacientes em comparação aos sadios (p<0,05). Não se observou diferença significativa na mensuração intracelular de caspase 3 em neutrófilos de pacientes comparado a indivíduos sadios. A análise da apoptose durante a evolução da sepse não mostrou diferença entre os grupos D0, D7 e D14. Não houve diferença na proporção de células em apoptose entre pacientes com sepse que sobreviveram e pacientes que evoluíram a óbito até o vigésimo oitavo dia após o diagnóstico de sepse. A apoptose tardia em neutrófilos parece estar diminuída. O possível efeito biológico da redução de apoptose tardia seria uma resposta adaptada à lesão, talvez prolongando a meia vida e atividade dos neutrófilos, entretanto, essa apoptose diminuída pode contribuir para a lesão inflamatória sistêmica e predispor o desenvolvimento da síndrome de disfunção de múltiplos órgãos. Os monócitos foram avaliados unicamente pela detecção intracelular de caspase-3, todavia, não foi encontrado diferença entre pacientes sépticos e indivíduos sadios. O mesmo ocorreu em amostras obtidas na admissão, 7º e 14º dia de seguimento. O percentual de monócitos com positividade para caspase-3 nos dias 0, 7 e 14 de sepse foi relacionado com a evolução dos pacientes em sobreviventes e óbitos, após 28 dias do diagnóstico. A análise realizada com amostras do D0 não indicou diferença na porcentagem de células apoptóticas entre os pacientes sobreviventes e que evoluíram a óbito. Entretanto, o percentual de apoptose observado no D7 mostrou que pacientes que evoluíram a óbito apresentaram menor porcentagem de células positivas para caspase-3 após 28 dias, e essa associação persistiu no seguimento de 60 e 180 dias. O papel da apoptose em monócitos parece ser muito importante na sepse, todavia são necessários novos estudos para elucidar a correlação entre apoptose de monócitos e sobrevida. / Sepsis incidence continues to rise with significant morbidity and mortality and is the leading cause of death in intensive care units. Infection control depends on proper recognition of the microorganisms by immune cells and an adequade effector immune response. Lymphocytes, monocytes and neutrophils are the major peripheral blood cell populations involved in this process. Apoptosis is an important mechanism for controlling the immune response, however, in critical conditions, deregulated apoptosis can lead to immune suppression, difficulting the control of sepsis. This study evaluated the percentage of apoptosis in peripheral blood derived lymphocytes, monocytes and neutrophils by means of exposure of phosphatidylserine on the cell surface and detection of intracellular caspase-3 and evaluated the absolute number and percentage of lymphocytes and their subpopulations by flow cytometry. Forty septic patients were included, of whom, 4 were septic, 9 presented severe sepsis and 27 were in septic shock, classified according to the definitions of the 1992´s consensus. Fifteen healthy volunteers’ were included for comparison. For apoptosis assays, total leucocytes were isolated from the whole blood by hypotonic lysis of the red blood cells and stained with surface antibodies in order to identify the leukocyte subpopulations. For apoptosis evaluation samples were stained with annexin-V and propidium iodide (PI) in an appropriate buffer or labeled with anti-caspase-3 after fixation and permeabilization. Lymphocyte counts were performed in TruCOUNT tubes after labeling the cells with anti-CD45, CD3, CD4, CD8, CD16/56 and CD19. There were no statistical differences in the percentage of apoptosis in total leukocytes, however, lower late apoptosis was observed in T lymphocytes of septic patients when compared to healthy subjects (P<0.05). Septic patients showed a smaller absolute count of total lymphocytes, T lymphocytes, CD4+, CD8+ and NK cells when compared to the healthy individuals (P<0.01), although the percentage of these cell populations has remained unchanged. These results demonstrate that the smallest number of circulating lymphocytes observed during sepsis could not be explained by the presence of apoptosis. The decrease of peripheral blood cell counts could be a result of the cell migration to the lymphoid tissues or organs triggered by infection. There were no differences in the percentage of early apoptosis in neutrophils of patients and healthy individuals. There was a decrease in the percentage of late apoptosis and a consequent increase in the percentage of viable cells in the patients when compared to the healthy subjects (P<0.05). There was no significant differences in the measurement of intracellular caspase-3 in neutrophils of patients compared to the healthy individuals. Analysis of apoptosis during the development os sepsis did not differ among the D0, D7 an D14 groups. There was no difference in the proportion of cells undergoing apoptosis between patients with sepsis who survived and those who died by the twenty-eight (D28) after the diagnosis of sepsis. Late apoptosis in neutrophils appears to be diminished. The possible biological effect of the reduction of apoptosis would be an adapted response to the injury, perhaps extending the half life and activity of neutrophils, however, this decreased apoptosis may contibute to inflammatory injury and predispose to the development of the multiple organ dysfunction syndrome. Only intracellular caspase-3 was evaluated in monocyte population, and no differences were found between this marker on septic patients and healthy individuals. The same results were observed in samples from adminssion, 7 and 14 days of follow-up. The percentage of monocytes with caspase-3 activity on days 0, 7 and 14 of sepsis was related to patient outcome as regards of survival and non-survival, after 28 days of diagnosis. The analysis performed on samples from D0 did not indicate differences in the percentage of apoptotic cells among the survivors and non-survivors. To counterpoint, when this analysis was performed on D7, the percentage of cells positive for caspase-3 were lower in those who died when compared with the survivors after 28 days; besides, this association persisted on 60 and 180 days follow-up. The role of apoptosis in monocytes seems to be important in sepsis, however, further research is needed to elucidate the correlation between monocyte apoptosis and survival. / TEDE