Genregulation beeinflusst Phänotypen im Kontext von Gesundheit und Krankheit. In Krebszellen regulieren genetische und epigenetische Faktoren die Genexpression in cis. Das Neuroblastom ist eine Krebserkrankung, die häufig im Kindesalter auftritt. Es ist gekennzeichnet durch eine geringe Anzahl exonischer Mutationen und durch häufige Veränderungen der somatischen Kopienzahl, einschließlich Genamplifikationen auf extrachromosomaler zirkulärer DNA. Bisher ist wenig darüber bekannt, wie lokale genetische und epigenetische Faktoren Gene im Neuroblastom regulieren. In dieser Arbeit kombiniere ich die allelspezifische Analyse ganzer Genome (WGS), Transkriptome und zirkulärer DNA von Neuroblastom-Patienten, um genetische und cis-regulatorische Effekte zu charakterisieren. Ich zeige, dass somatische Dosis-Effekte der Kopienzahl andere lokale genetische Effekte dominieren und wichtige Signalwege regulieren. Genamplifikationen zeigen starke Dosis-Effekte und befinden sich häufig auf großen extrachromosomalen zirkulären DNAs. Die vorgestellte Analyse zeigt, dass der Verlust von 11q zu einer Hochregulation von Histonvarianten H3.3 und H2A in Tumoren mit alternativer Verlängerung der Telomere (ALT) führt, und dass erhöhte somatische Kopienzahl die Expression der TERT Gens verstärken können. Weitere Erkenntnisse sind, dass 17p-Ungleichgewichte und die damit verbundene Herunterregulierung neuronaler Gene sowie die Hochregulierung des genomisch geprägten Gens RTL1 durch Kopienzahl-unabhängige allelische Dosis-Effekte mit einer ungünstigen Prognose verbunden sind. Die cis-QTL-Analyse bestätigt eine zuvor beschriebene Regulation des LMO1 Gens durch einen Enhancer-Polymorphismus und charakterisiert das regulatorische Potenzial weiterer GWAS-Risiko-Loci. Die Arbeit unterstreicht die Bedeutung von Dosis-Effekten im Neuroblastom und liefert eine detaillierte Übersicht regulatorischer Varianten, die in dieser Krankheit aktiv sind. / Gene regulation controls phenotypes in health and disease. In cancer, the interplay between germline variation, genetic aberrations and epigenetic factors modulate gene expression in cis. The childhood cancer neuroblastoma originates from progenitor cells of the sympathetic nervous system. It is characterized by a sparsity of recurrent exonic mutations but frequent somatic copy-number alterations, including gene amplifications on extrachromosomal circular DNA. So far, little is known on how local genetic and epigenetic factors regulate genes in neuroblastoma to establish disease phenotypes. I here combine allele-specific analysis of whole genomes, transcriptomes and circular DNA from neuroblastoma patients to characterize genetic and cis-regulatory effects, and prioritize germline regulatory variants by cis-QTLs mapping and chromatin profiles. The results show that somatic copy-number dosage dominates local genetic effects and regulates pathways involved in telomere maintenance, genomic stability and neuronal processes. Gene amplifications show strong dosage effects and are frequently located on large but not small extrachromosomal circular DNAs. My analysis implicates 11q loss in the upregulation of histone variants H3.3 and H2A in tumors with alternative lengthening of telomeres and cooperative effects of somatic rearrangements and somatic copy-number gains in the upregulation of TERT. Both 17p copy-number imbalances and associated downregulation of neuronal genes as well as upregulation of the imprinted gene RTL1 by copy-number-independent allelic dosage effects is associated with an unfavorable prognosis. cis-QTL analysis confirms the previously reported regulation of the LMO1 gene by a super-enhancer risk polymorphism and characterizes the regulatory potential of additional GWAS risk loci. My work highlights the importance of dosage effects in neuroblastoma and provides a detailed map of regulatory variation active in this disease.

The dose-response relationship between physical activity (PA), disease and mortality has primarily been obtained from self-report questionnaires in Western populations. A major limitation of self-reported PA is the likelihood of measurement error and these recordings cannot account for all 24-h activities, thus negating the influence of sedentary time and daily light intensity activity. Modern-day studies using objective measures of PA are highly controversial in the description of PA, as well as reliable wear time of these objective devices to accurately assess PA behaviour. The aim of the research presented in this thesis was to ascertain the associations between seven-day objectively measured PA (expressed as time spent in four different metabolic equivalent of task (MET) categories), cardiovascular disease risk factors (24-h ambulatory blood pressure and central obesity), chronic stress (General Health Questionnaire total score and serum cortisol) and DNA damage (leukocyte telomere length) in a cohort of African and Caucasian school teachers recruited from the Dr Kenneth Kaunda Education District in the North West Province of South Africa. All parameters were objectively measured (the GHQ was only added for thoroughness on measures of cognitive perceived stress) in the study population. The Africans (n=96) were younger than the Caucasians (n=107) (48.33 versus 51.06 years, p=0.024), but presented with slightly higher waist circumferences, significantly higher 24-h ambulatory systolic blood pressure (SBP, p≤0.000), diastolic blood pressure (DBP, p≤0.000) and mean arterial pressure (MAP, p≤0.000); significantly higher perceived stress scores (GHQ total scores, p=0.001) and significantly shorter telomeres (p≤0.000). The hypertensive participants in the total group (Africans and Caucasians combined) recorded 2.2 hours (12.4%) more daily awake sedentary time than the normotensive participants (p=0.004) and sedentary time was also a slightly better predictor of hypertension than moderate and vigorous activity time (Odds ratio=1.00, p=0.006). Irrespective of race and sex, 24-h SBP and DBP measurements were respectively associated with daily awake sedentary time (ß=0.17, p=0.018 and ß=0.18, p=0.020), light activity time (ß=-0.15, p=0.043 and ß=-0.16, p=0.041), waist circumference (ß=0.45, p≤0.000 and ß=0.33, p≤0.000) and log serum gamma glutamyl transferase (γ-GT, alcohol use) (ß=0.18, p=0.018 and ß=0.24, p=0.004). An older age (ß=-0.28, p≤0.000), higher alcohol consumption (ß=-0.21, p=0.003) and increased central obesity (ß=-0.17, p=0.017) were associated with shorter telomeres. Attenuated cortisol levels (ß=-0.12, p=0.068) showed a tendency towards associations with longer telomeres that may indicate possible cortisol down regulation to protect against DNA damage. Time spent in the different MET-categories showed no direct associations with either cortisol or telomere length. However, a sensitivity analysis indicated that daily light intensity activity time was significantly correlated with lower waist circumference (r=-0.21, p=0.004); a parameter associated with both cortisol (ß=-0.22, p=0.003) and telomere length (ß=-0.17, p=0.017). The thorough recording of PA during the true awake time of 24-h cycles over a period of seven days ensured that the beneficial effect of light intensity activities, as well as the detrimental effect of sedentary time, was highlighted by this study. The average awake time of all ethnic and sex groups were around 17 hours per day, which was more than most previous studies using objective measures of PA. The exclusion of participants who did not comply through wearing the Actiheart for a full seven days (n=143, 40%) did, however, have a negative impact on sample size that may have affected the statistical power for uncovering some significant associations and the high participant burden of the Actiheart device became clear. Therefore, the researchers used the data of the full seven-day recordings to also determine the minimum number of consecutive days the Actiheart device could be worn to accurately estimate energy expenditure and PA. The two-day combination of Wednesday-to-Thursday did not differ from the weekly average TEE, as well as for all MET-categories in all ethnic and sex groups. This two-day combination is practically convenient and would lessen participant burden. Future researchers are urged to test this combination in other populations to standardize Actiheart wear time. It can be concluded from the findings in this study that less daily awake sedentary time, more light intensity activity time, as well as lower alcohol consumption favour improved health as it is beneficial to 24-h ambulatory blood pressure and helps to maintain a healthy waist circumference, which ultimately influence telomere shortening. Furthermore, the two-day combination of Wednesday-to-Thursday seems to be sufficient to accurately estimate weekly energy expenditure and habitual PA with the Actiheart apparatus. / PhD (Human Movement Science), North-West University, Potchefstroom Campus, 2015

The dose-response relationship between physical activity (PA), disease and mortality has primarily been obtained from self-report questionnaires in Western populations. A major limitation of self-reported PA is the likelihood of measurement error and these recordings cannot account for all 24-h activities, thus negating the influence of sedentary time and daily light intensity activity. Modern-day studies using objective measures of PA are highly controversial in the description of PA, as well as reliable wear time of these objective devices to accurately assess PA behaviour. The aim of the research presented in this thesis was to ascertain the associations between seven-day objectively measured PA (expressed as time spent in four different metabolic equivalent of task (MET) categories), cardiovascular disease risk factors (24-h ambulatory blood pressure and central obesity), chronic stress (General Health Questionnaire total score and serum cortisol) and DNA damage (leukocyte telomere length) in a cohort of African and Caucasian school teachers recruited from the Dr Kenneth Kaunda Education District in the North West Province of South Africa. All parameters were objectively measured (the GHQ was only added for thoroughness on measures of cognitive perceived stress) in the study population. The Africans (n=96) were younger than the Caucasians (n=107) (48.33 versus 51.06 years, p=0.024), but presented with slightly higher waist circumferences, significantly higher 24-h ambulatory systolic blood pressure (SBP, p≤0.000), diastolic blood pressure (DBP, p≤0.000) and mean arterial pressure (MAP, p≤0.000); significantly higher perceived stress scores (GHQ total scores, p=0.001) and significantly shorter telomeres (p≤0.000). The hypertensive participants in the total group (Africans and Caucasians combined) recorded 2.2 hours (12.4%) more daily awake sedentary time than the normotensive participants (p=0.004) and sedentary time was also a slightly better predictor of hypertension than moderate and vigorous activity time (Odds ratio=1.00, p=0.006). Irrespective of race and sex, 24-h SBP and DBP measurements were respectively associated with daily awake sedentary time (ß=0.17, p=0.018 and ß=0.18, p=0.020), light activity time (ß=-0.15, p=0.043 and ß=-0.16, p=0.041), waist circumference (ß=0.45, p≤0.000 and ß=0.33, p≤0.000) and log serum gamma glutamyl transferase (γ-GT, alcohol use) (ß=0.18, p=0.018 and ß=0.24, p=0.004). An older age (ß=-0.28, p≤0.000), higher alcohol consumption (ß=-0.21, p=0.003) and increased central obesity (ß=-0.17, p=0.017) were associated with shorter telomeres. Attenuated cortisol levels (ß=-0.12, p=0.068) showed a tendency towards associations with longer telomeres that may indicate possible cortisol down regulation to protect against DNA damage. Time spent in the different MET-categories showed no direct associations with either cortisol or telomere length. However, a sensitivity analysis indicated that daily light intensity activity time was significantly correlated with lower waist circumference (r=-0.21, p=0.004); a parameter associated with both cortisol (ß=-0.22, p=0.003) and telomere length (ß=-0.17, p=0.017). The thorough recording of PA during the true awake time of 24-h cycles over a period of seven days ensured that the beneficial effect of light intensity activities, as well as the detrimental effect of sedentary time, was highlighted by this study. The average awake time of all ethnic and sex groups were around 17 hours per day, which was more than most previous studies using objective measures of PA. The exclusion of participants who did not comply through wearing the Actiheart for a full seven days (n=143, 40%) did, however, have a negative impact on sample size that may have affected the statistical power for uncovering some significant associations and the high participant burden of the Actiheart device became clear. Therefore, the researchers used the data of the full seven-day recordings to also determine the minimum number of consecutive days the Actiheart device could be worn to accurately estimate energy expenditure and PA. The two-day combination of Wednesday-to-Thursday did not differ from the weekly average TEE, as well as for all MET-categories in all ethnic and sex groups. This two-day combination is practically convenient and would lessen participant burden. Future researchers are urged to test this combination in other populations to standardize Actiheart wear time. It can be concluded from the findings in this study that less daily awake sedentary time, more light intensity activity time, as well as lower alcohol consumption favour improved health as it is beneficial to 24-h ambulatory blood pressure and helps to maintain a healthy waist circumference, which ultimately influence telomere shortening. Furthermore, the two-day combination of Wednesday-to-Thursday seems to be sufficient to accurately estimate weekly energy expenditure and habitual PA with the Actiheart apparatus. / PhD (Human Movement Science), North-West University, Potchefstroom Campus, 2015

Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

Les cassures double-brins d’ADN (CDBs) constituent une menace pour la viabilité cellulaire et l’intégrité du génome puisque l’absence de la réparation d’une CDB pourrait conduire à la mort cellulaire. En plus de la réparation par jonction d’extrémités nonhomologues (NHEJ) en phase G1 et de la recombinaison homologue (RH) en phase S et G2, les CDBs peuvent être réparées par l’ajout de télomères par l’action de la télomérase; un phénomène qui s’appelle l’ajout de télomères de novo. Ce phénomène pourrait mettre en danger la stabilité génomique parce qu’il engendre, dans la plupart des cas, une perte du bras chromosomique du fragment non-centromérique. En conséquence, ceci engendre soit une perte de l’hétérozygotie (LOH) dans les cellules diploïdes ou la mort cellulaire dans les cellules haploïdes. Dans le but d’empêcher la formation de télomères de novo, la cellule possède des mécanismes et des voies qui préviennent l’action inappropriée de la télomérase à des CDBs. Une des principales questions dans le domaine est de comprendre comment la cellule inhibe l’ajout de télomères de novo par la télomérase en favorisant la réparation des CDBs par les autres voies (NHEJ et la RH).Dans ce projet, nous utilisons la technique d’hybridation in situ en fluorescence (FISH) sur le facteur limitant de la télomérase, l’ARN TLC1 de la levure S. cerevisiae. Nous avons pu montrer que l’ARN TLC1 fait un trafic intranucléaire durant le cycle cellulaire des cellules sauvages. En phase G1/S, l’ARN TLC1 adopte une localisation nucléoplasmique avec les télomères, alors qu’il s’accumule au nucléole en phase G2/M. Nous avons fait l’hypothèse que l’accumulation de l’ARN TLC1 au nucléole en G2/M pourrait réduire la compétition entre la RH, qui est exclusivement nucléoplasmique, et la télomérase pour la réparation des CDBs. Pour tester cette hypothèse, nous avons employé la bléomycine (blm), un composé chimique générant des CDBs, pour traiter des cellules sauvages ou déficientes de la RH par la délétion du gène RAD52. Nous avons observé que l’ARN TLC1 conserve une localisation nucléolaire dans les cellules sauvages traitées par la blm en phase G2/M, alors que dans lescellules délétées de RAD52 exposées à la blm, l’ARN TLC1 se localise maintenant au nucléoplasme et s’associe partiellement aux sites de cassures. De plus, nous avons trouvé que l’accumulation nucléoplasmique de l’ARN TLC1 dans les cellules délétéées de RAD52 traitées à la blm, dépend de la voie de dommage à l’ADN (MRX, ATM/Tel1 et ATR/Mec1) et de la sumoylation par la SUMO E3ligase, Siz1. Plus particulièrement, l’association de la télomérase à des CDBs dépend de son interaction avec Cdc13, une protéine qui recrute la télomérase aux télomères. D’une manière surprenante, nous avons observé une accumulation rapide de Cdc13 à des CDBs en absence de Rad52, bien que nos résultats suggèrent que Rad52 empêche l’accumulation de l’ARN TLC1 au nucléoplasme par l’inhibition de l’accumulation de Cdc13 aux sites de cassures. L’ensemble de nos résultats ont mis en évidence que la télomérase est normalement exclue des sites de la réparation d’ADN. Cependant, en absence d’une voie fonctionnelle de la RH, la télomérase se localise du nucléole au nucléoplasme et s’accumule partiellement à des CDBs d’une manière dépendante de Cdc13 et Siz1. / DNA double-strand breaks (DSB) constitute a threat to genome integrity and cell survival if they are not repaired. In addition to canonical DNA repair systems such as nonhomologous end joining (NHEJ) in G1 and homologous recombination (HR) in S and G2 phases, DSBs can also be repaired by addition of new telomeres by telomerase. This phenomenon is referred to as telomere healing or de novo telomere addition. This process threatens genome stability since it results in chromosome arm loss, which could be lethal in haploid cells and lead to loss of heterozygosity (LOH) in diploid cells. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. One of the questions driving this field is to understand how telomere addition by telomerase is inhibited and DSBs repair can be efficiently performed by canonical DSB repair (NHEJ and HR). In this project, we used fluorescent in situ hybridization (FISH) to detect the endogenous TLC1 RNA, which is the limiting component of telomerase of the budding yeast. Using this technique, we found that TLC1 RNA traffics inside the nucleus during the cell cycle of wild-type cells. In G1 and S phases, TLC1 RNA adopts a nucleoplasmic localization, which is related to its function in telomere elongation, while it accumulates in the nucleolus in G2/M. We hypothesize that the nucleolar accumulation of TLC1 RNA in G2/M may reduce the possibility that telomerase interferes with HR to repair DNA DSB, since HR is excluded from the nucleolus and occurs only in the nucleoplasm. To test this hypothesis, we treated wild-type and rad52 (HR deficient cells) with bleomycin, a radiomimetic agent that generates preferentially DSBs. Our results show that after induction of DSB with bleomycin, TLC1 RNA remains nucleolar in wild-type cells in G2/M, but accumulates in the nucleoplasm and colocalizes partially with DSBs sites in rad52 cells, suggesting that RAD52 inhibits the nucleoplasmic accumulation of TLC1 RNA in the presence of DSBs. Nucleoplasmic accumulation of TLC1 RNA after DSB induction requires the DNA damage pathway (MRX, ATM/Tel1 and ATR/Mec1), and the SUMO ligase E3 Siz1. Interestingly, association of TLC1 RNA with DSBs depends on the single-strand telomeric binding protein Cdc13, which rapidly accumulates at sites of DNA damage, while Rad52 suppresses this process by inhibiting Cdc13 accumulation at DSBs. These results suggest that telomerase is normally excluded from sites of DNA repair. In the absence of functional homologous recombination, telomerase leaves the nucleolus and accumulates partially at DSB in the nucleoplasm in a Cdc13- and Siz1-dependent manner.

La sénescence cellulaire, ou l’arrêt irréversible de la prolifération, influence des processus physiologiques et pathologiques, comme le cancer. Parmi les caractéristiques de la sénescence, se retrouve le PSAS ou phénotype sécrétoire associé à la sénescence. Le PSAS est composé d’une variété de cytokines, facteurs de croissance et protéases. Ses actions pro- et anti-tumorale sont connues, mais l’on ignore laquelle prédomine. Mes travaux s’attardent aux effets du PSAS sur l’induction de la sénescence dans les cellules environnantes et à sa régulation. Nous avons démontré que le PSAS ne synergise pas avec la dysfonction télomérique chronique ou aigue, afin de causer un arrêt de croissance. Également, l’étude du mécanisme responsable de l’induction de la sénescence par stress à la chromatine, suggère que la kinase c-Abl n’est pas requise pour cette voie, contrairement à des publications antérieures. Mes travaux éclairent les mécanismes d’action et la régulation du PSAS dans la sénescence induite par dysfonction télomérique et par stress à la chromatine. / Cellular senescence, or irreversible proliferation arrest, is known for its influence on physiological and pathological processes, such as cancer. Among the features found in the senescent phenotype is the inflammatory secretome, also known as the senescence associated secretory phenotype (SASP). The SASP consists of a variety of factors such as cytokines, growth factors and proteases. It is widely recognized that SASP can have either a pro- or anti-tumor effect, but it is not clear which one predominates. My work focused on the SASP effects on the induction of senescence in surrounding cells and its regulation mechanisms. We demonstrated that the SASP does not synergize with chronic or induced telomere dysfunction to cause cellular proliferation arrest. Also, study of chromatin stress-induced senescence mechanism suggests that kinase c-Abl is not required for this pathway, contrary to what had been previously published. My work helps understand the regulatory and working mechanisms of the SASP in chromatin stress-induced and telomere dysfunction-induced senescence models.

INTRODUÇÃO: A Leucemia/Linfoma de células T do adulto (ATL) é uma doença linfoproliferativa crônica com transformação clonal predominantemente de linfócitos TCD4+, causada pelo vírus linfotrópico T humano do tipo I (HTLV-I). A ATL se desenvolve em 3-5% dos portadores do vírus HTLV-I, após longo período de latência clínica, acompanhado de expansão clonal dos linfócitos infectados. As células da ATL apresentam várias anormalidades cromossômicas, semelhantes àquelas resultantes de disfunção telomérica e a instabilidade genômica contribui para o desenvolvimento da ATL. Para entender o papel do encurtamento telomérico na oncogênese da ATL, avaliamos o comprimento dos telômeros de linfócitos TCD4 e TCD8 em portadores do vírus HTLV-I e em portadores de ATL. RESULTADOS: Não foi evidenciada diferença significativa no comprimento de telômero dos subtipos linfocitários TCD4+ e TCD8+ entre portadores do vírus HTLV-I e indivíduos saudáveis, assim como, entre portadores de ATL e indivíduos saudáveis. Entretanto, quando incluímos na análise a variável idade, evidenciamos redução significativa do comprimento do telômero com a idade em portadores do vírus HTLV-I e maior perda telomérica nos portadores do vírus HTLV-I e portadores de ATL em relação aos indivíduos saudáveis de mesma idade, embora a diferença entre os grupos não atinja o nível de significância estatística. Estes resultados podem ser explicados pelo fato de que as células dos indivíduos infectados pelo vírus HTLV-I apresentam maior taxa proliferativa devido à ação viral, mesmo em estado de latência clínica. A perda telomérica em função da idade nos portadores de ATL não demostrou-se significativa devido ao pequeno número de casos analisados em decorrência da raridade da doença. Entretanto, quando analisamos o comprimento telomérico nos subtipos linfocitários de portadores de ATL, evidenciamos acentuada perda telomérica na célula maligna e valores próximos ao limite superior esperado para a idade no subtipo linfocitário não transformado, demonstrando que a disfunção telomérica deve estar associada à transformação celular. Estabelecemos valores de referência de comprimento telomérico dos subtipos linfocitários TCD4+ e TCD8+ de indivíduos saudáveis, definidos por faixa etária. CONCLUSÃO: Nossos resultados demonstram que portadores do vírus HTLV-I apresentam maior perda telomérica em função da idade que indivíduos saudáveis, mas, sem refletir significância estatística e clínica. Entretanto, portadores de ATL apresentam perda acentuada de comprimento de telômero na célula maligna, demonstrando que a determinação do comprimento de telômero pode auxiliar futuramente o monitoramento dos indivíduos infectados pelo HTLV-I, indicando conversão à doença / INTRODUCTION: Adult T-cell Leukemia/Lymphoma (ATL) is a chronic lymphproliferative disease with clonal transformation predominantly of the TCD4+ lymphocytes, caused by the Human T lymphotropic virus type-I (HTLV-I). ATL develops itself in 3-5% of HTLV-I carriers after a long period of clinical latency accompanied by clonal expansion of the infected lymphocytes. The ATL cells present several chromosomic abnormalities, similar to those resulting from telomere dysfunction and the genomic instability contributes to the development of ATL. In order to understanding the role of telomeric shortening in the ATL oncogenesis, we assessed the length of telomeres of lymphocytes TCD4 and TCD8 in HTLV-I carriers and in ATL carriers. RESULTS: No significant difference was evidentiated in the telomere length of lymphocytary subtypes TCD4+ and TCD8+ between HTLV-I carriers and healthy subjects, as well as, between ATL carriers and healthy subjects. However, when the age variable was included in the analysis, we observed significant decrease of telomeric length with age progression in HTLV-I carriers and higher telomeric loss in HTLV-I carriers and ATL carriers when compared to healthy subjects of the same age, although the difference between groups does not reach the level of statistic relevance. These results may be explained by the fact that the cells of HTLV-I infected subjects present higher proliferative rate due to the viral action, even during clinical latency. Age-related telomeric loss in ATL carriers did not manifest itself as significant due to the small number of analyzed cases as a consequence of the diseases rareness. However, when the telomere length on the lymphocytary subtypes of ATL carriers was analyzed, we evidentiated accentuated telomeric loss in the malignant cell and values close to the age-expected upper limit in the nontransformed lymphocytary subtype, demonstrating that the telomere dysfunction may be associated to the cellular transformation. We have determined reference values of telomere length for lymphocytary subtypes TCD4+ and TCD8+ on healthy subjects, defined by age range. CONCLUSION: Our results demonstrate that HTLV-I carriers present higher telomeric loss due to age than healthy subjects, however, with no reflection in clinical and statistical significance. Nevertheless, ATL carriers present accentuated loss of telomere length in the malignant cell, demonstrating that the telomere length determination may, in the future, assist in the monitoring of HTLV-I infected subjects, indicating conversion to the disease

Les lymphocytes constituent un modèle original de cellules somatiques puisqu’ils sont capables de réactiver la télomérase lorsqu’ils sont stimulés. Nous avons montré que les lymphocytes, en culture prolongée et soumis à des stimulations itératives par la PHA, présentent une diminution progressive de l’activité télomérasique interrompue à chaque stimulation par une augmentation transitoire. Ces variations sont corrélées positivement aux variations de hTERT et de la longueur des télomères. Les foyers γ-H2AX et 53BP1 et leur localisation au niveau des télomères augmentent lors du vieillissement cellulaire. Nous montrons un dysfonctionnement des télomères au cours de la sénescence lymphocytaire in vitro résultant d’une érosion accrue des télomères et d’une diminution de l’expression des protéines qui les coiffent. Le mécanisme des variations précoces de l’expression de hTERT lors de l’activation lymphocytaire restaient à comprendre. Les conséquences du traitement des lymphocytes par différents immunosuppresseurs agissant tous de façon directe ou indirecte sur l’activation de NFAT suggéraient le rôle de NFAT dans la régulation transcriptionnelle de hTERT. Nous avons montré i) 5 éléments de réponse potentiels pour NFAT au niveau du promoteur de hTERT, ii) l’activation in vitro du promoteur de hTERT par NFAT essentiellement via un site consensus localisé dans le coeur du promoteur de hTERT en position -40 et une synergie fonctionnelle entre NFAT et SP1, iii) la liaison directe de NFAT sur le promoteur de hTERT via ce site consensus in vivo. Ainsi, NFAT1 régule la transcription de hTERT et est impliqué dans l’activation de la télomérase lors de la stimulation lymphocytaire / Lymphocytes are an example of somatic cells capable to induce telomerase activity when stimulated. We showed that lymphocytes, during long-term culture and repeated PHA stimulations, present a progressive drop in telomerase activity interrupted at each stimulation by a transitory increase. These variations are positively correlated with hTERT and telomere length variations. γ-H2AX and 53BP1 foci and their localization on telomeres increase with cell aging. We show a telomere dysfunction during in vitro lymphocyte senescence resulting from an excessive telomere shortening and a decrease in shelterin content. The mechanism involved in early variations of hTERT expression during lymphocyte activation remained to be understood. Consequences of lymphocyte treatment with different immunosuppressors, all acting directly or indirectly on NFAT activation, suggested a role for NFAT in the regulation of hTERT transcription. Five putative responsive elements for NFAT were identified in the hTERT promoter. We showed that NFAT activates in vitro the hTERT promoter mainly via a consensus site localized in the promoter core at position -40 and a functional synergy between NFAT and SP1. Furthermore, NFAT1 binds directly to the endogenous hTERT promoter via this consensus site in vivo. Thus, NFAT positively regulates the hTERT transcription and we propose its implication in telomerase activation during lymphocyte stimulation

INTRODUÇÃO: A Leucemia/Linfoma de células T do adulto (ATL) é uma doença linfoproliferativa crônica com transformação clonal predominantemente de linfócitos TCD4+, causada pelo vírus linfotrópico T humano do tipo I (HTLV-I). A ATL se desenvolve em 3-5% dos portadores do vírus HTLV-I, após longo período de latência clínica, acompanhado de expansão clonal dos linfócitos infectados. As células da ATL apresentam várias anormalidades cromossômicas, semelhantes àquelas resultantes de disfunção telomérica e a instabilidade genômica contribui para o desenvolvimento da ATL. Para entender o papel do encurtamento telomérico na oncogênese da ATL, avaliamos o comprimento dos telômeros de linfócitos TCD4 e TCD8 em portadores do vírus HTLV-I e em portadores de ATL. RESULTADOS: Não foi evidenciada diferença significativa no comprimento de telômero dos subtipos linfocitários TCD4+ e TCD8+ entre portadores do vírus HTLV-I e indivíduos saudáveis, assim como, entre portadores de ATL e indivíduos saudáveis. Entretanto, quando incluímos na análise a variável idade, evidenciamos redução significativa do comprimento do telômero com a idade em portadores do vírus HTLV-I e maior perda telomérica nos portadores do vírus HTLV-I e portadores de ATL em relação aos indivíduos saudáveis de mesma idade, embora a diferença entre os grupos não atinja o nível de significância estatística. Estes resultados podem ser explicados pelo fato de que as células dos indivíduos infectados pelo vírus HTLV-I apresentam maior taxa proliferativa devido à ação viral, mesmo em estado de latência clínica. A perda telomérica em função da idade nos portadores de ATL não demostrou-se significativa devido ao pequeno número de casos analisados em decorrência da raridade da doença. Entretanto, quando analisamos o comprimento telomérico nos subtipos linfocitários de portadores de ATL, evidenciamos acentuada perda telomérica na célula maligna e valores próximos ao limite superior esperado para a idade no subtipo linfocitário não transformado, demonstrando que a disfunção telomérica deve estar associada à transformação celular. Estabelecemos valores de referência de comprimento telomérico dos subtipos linfocitários TCD4+ e TCD8+ de indivíduos saudáveis, definidos por faixa etária. CONCLUSÃO: Nossos resultados demonstram que portadores do vírus HTLV-I apresentam maior perda telomérica em função da idade que indivíduos saudáveis, mas, sem refletir significância estatística e clínica. Entretanto, portadores de ATL apresentam perda acentuada de comprimento de telômero na célula maligna, demonstrando que a determinação do comprimento de telômero pode auxiliar futuramente o monitoramento dos indivíduos infectados pelo HTLV-I, indicando conversão à doença / INTRODUCTION: Adult T-cell Leukemia/Lymphoma (ATL) is a chronic lymphproliferative disease with clonal transformation predominantly of the TCD4+ lymphocytes, caused by the Human T lymphotropic virus type-I (HTLV-I). ATL develops itself in 3-5% of HTLV-I carriers after a long period of clinical latency accompanied by clonal expansion of the infected lymphocytes. The ATL cells present several chromosomic abnormalities, similar to those resulting from telomere dysfunction and the genomic instability contributes to the development of ATL. In order to understanding the role of telomeric shortening in the ATL oncogenesis, we assessed the length of telomeres of lymphocytes TCD4 and TCD8 in HTLV-I carriers and in ATL carriers. RESULTS: No significant difference was evidentiated in the telomere length of lymphocytary subtypes TCD4+ and TCD8+ between HTLV-I carriers and healthy subjects, as well as, between ATL carriers and healthy subjects. However, when the age variable was included in the analysis, we observed significant decrease of telomeric length with age progression in HTLV-I carriers and higher telomeric loss in HTLV-I carriers and ATL carriers when compared to healthy subjects of the same age, although the difference between groups does not reach the level of statistic relevance. These results may be explained by the fact that the cells of HTLV-I infected subjects present higher proliferative rate due to the viral action, even during clinical latency. Age-related telomeric loss in ATL carriers did not manifest itself as significant due to the small number of analyzed cases as a consequence of the diseases rareness. However, when the telomere length on the lymphocytary subtypes of ATL carriers was analyzed, we evidentiated accentuated telomeric loss in the malignant cell and values close to the age-expected upper limit in the nontransformed lymphocytary subtype, demonstrating that the telomere dysfunction may be associated to the cellular transformation. We have determined reference values of telomere length for lymphocytary subtypes TCD4+ and TCD8+ on healthy subjects, defined by age range. CONCLUSION: Our results demonstrate that HTLV-I carriers present higher telomeric loss due to age than healthy subjects, however, with no reflection in clinical and statistical significance. Nevertheless, ATL carriers present accentuated loss of telomere length in the malignant cell, demonstrating that the telomere length determination may, in the future, assist in the monitoring of HTLV-I infected subjects, indicating conversion to the disease

Abstract Vitamin D deficiency, obesity and short telomere length are reported to be associated with increased risk of metabolic diseases and all-cause mortality, through modulation of inflammatory pathways. The season of blood sampling, obesity and physical activity have been identified as determinants of 25-hydroxyvitamin D [25(OH)D], but their association with 25(OH)D2 (D2) and 25(OH)D3 (D3) is still poorly understood. In addition, relationships between 25(OH)D, body mass index (BMI), inflammation, and leukocyte telomere length (LTL) has not been previously established. A better understanding of the determinants, risk factors of vitamin D deficiency, and their relationship with BMI, inflammation, and LTL is needed. The study was based on the 31-year follow-up study of the Northern Finland Birth Cohort 1966 (N=4,758). Statistical analyses were used to 1) examine potential determinants of D2 and D3, and identify risk factors associated with hypovitaminosis D, 2) investigate the relationship of 25(OH)D and BMI with LTL and test whether it is independent of inflammatory pathways and, 3) assess how the association of BMI with inflammatory biomarkers might be mediated through 25(OH)D. Our results showed that D2 contributed 5% and D3 95% of the total 25(OH)D concentrations. When examined, the determinants for each isoform, periods of low sunlight exposure associated with increased D2, but with decreased D3. Oral contraceptives associated with increased concentrations of both. We confirmed the known risk factors of low vitamin D: low sunlight periods, residing in northern latitudes, and physical inactivity. Serum 25(OH)D was not an important determinant of LTL, and inflammation may partly mediate the BMI-LTL association. Higher serum 25(OH)D was inversely associated with inflammatory biomarkers, and the association between BMI and biomarkers was modestly mediated through lowered 25(OH)D. In conclusion, our results support the role of known risk factors in vitamin D deficiency and add information on specific determinants of D2 and D3. 25(OH)D did not associate with LTL in young adulthood. We have also provided new insights into a plausible role of vitamin D in BMI associated inflammation. An improved understanding of the role of vitamin D benefits public health in many ways (it can help prevent vitamin D deficiency by implementing lifestyle modification and supplementation). / Tiivistelmä D-vitamiinin puutos, lihavuus ja lyhyt telomeerien pituus liittyvät mahdollisesti lisääntyneeseen riskiin sairastua metabolisiin sairauksiin sekä yleisemmin kuolleisuuteen. Eräs selitys tälle voi löytyä tulehdustekijöistä. Lihavuuden, liikunnan puutteen ja verinäytteenoton ajankohdan tiedetään vaikuttavan 25-hydroksi-D-vitamiinin [25(OH)D]-pitoisuuteen, mutta niiden yhteys D-vitamiinin isomuotoihin (D2, D3) on vielä huonosti tunnettu. Aiemmin ei ole selvitetty 25(OH)D:n, painoindeksin (BMI), tulehduksen ja leukosyyttien telomeerien pituuden (LTL) välisiä yhteyksiä, ja siksi näistä tarvitaan lisätutkimusta. Tutkimusaineistona oli Pohjois-Suomen 1966 syntymäkohortin, 31-vuoden seurantaan osallistuneet henkilöt (N=4,758). Tutkimuksessa keskityttiin 1) selvittämään D2- ja D3-vitamiinipitoisuuksien määrittäviä tekijöitä ja tunnistamaan D-vitamiinin puutteeseen liittyviä riskitekijöitä, 2) tutkimaan 25(OH)D-pitoisuuden ja BMI:n suhdetta LTL:n kanssa sekä testaamaan, onko suhde riippumaton tulehduksellisista tekijöistä ja 3) arvioimaan ilmeneekö BMI:n ja tulehdussytokiinien välinen yhteys 25(OH)D-pitoisuuden kautta. Tutkimus osoitti, että D2-isomuodon osuus oli 5 % ja D3:n osuus 95 % koko 25(OH)D-pitoisuudesta. Näitä isomuotoja määrittäviä tekijöitä tutkittaessa havaittiin, että vähäisellä auringonvalolle altistumisella on todennäköisesti yhteys lisääntyneeseen D2-pitoisuuteen, mutta alhaisempaan D3-pitoisuuteen. Suun kautta otettavien ehkäisypillereiden käytöllä oli yhteys molempien muotojen lisääntyneisiin pitoisuuksiin. Tutkimus vahvisti alhaisten D-vitamiinipitoisuuksien tunnetut riskitekijät: lyhyt altistus auringon valolle sekä fyysinen passiivisuus. 25(OH)D-pitoisuus ei ollut yhteydessä LTL:ään mutta tulehdus näytti osittain vaikuttavan BMI-LTL-assosiaatioon. Korkeampi 25(OH)D-pitoisuus yhdistyi matalampiin tulehdussytokiinipitoisuuksiin, kun taas matala 25(OH)D-pitoisuus muokkasi BMI:n ja biomarkkereiden välisisiä yhteyksiä, tosin heikosti. Yhteenvetona voidaan todeta, että tulokset tukevat tunnettujen riskitekijöiden merkitystä D-vitamiinin puutoksessa ja tuovat lisää tietoa eri isomuotoihin vaikuttavista tekijöistä. Tutkimus antaa myös uusia näkemyksiä D-vitamiinin roolista lihavuuteen liittyvässä matala-asteisessa tulehduksessa. D-vitamiinin vaikutuksien tarkempi tunteminen on merkityksellistä myös kansanterveyden kannalta.