Dissertations / Theses on the topic 'Telomeres maintenance mechanism'

Le complexe Shelterin, composé de 6 protéines (POT1 / TRF1 / TRF2 / TIN2 / RAP1 et ACD) joue un rôle majeur au niveau des télomères. Ainsi, il permet la protection de l’extrémité simple brin par la formation de la D-loop, la régulation de la signalisation des voies de dommages à l’ADN ; il participe à la réplication des télomères et contrôle l’accessibilité et la processivité de la télomérase, unique enzyme permettant l’allongement des télomères. Au cours de cette thèse, mon travail s’est organisé autour de 2 principaux axes, le premier, fondamental s’est intéressé aux effets extra-télomériques de la protéine ACD (anciennement appelée TPP1). Le deuxième, plus transversal s’est attardé sur les processus de maintenance des télomères dans le cas des gliomes. Concernant le premier aspect, il est maintenant connu que la protéine ACD fait le lien entre TIN2 et TERT (sous unité catalytique de la télomérase) aux télomères. Ces deux protéines peuvent aussi partiellement se localiser à la mitochondrie et y possèdent alors divers effets sur le métabolisme, la régulation du stress oxydant ou encore la mitophagie. Ainsi, et suite à des prédictions in silico de potentiel MTS pour ACD, nous avons émis l’hypothèse qu’ACD pourrait être le partenaire manquant de TIN2 et TERT à la mitochondrie. Dans ce cas il restait alors à identifier ses fonctions mitochondriales. Après avoir démontré la localisation partielle d’ACD à la mitochondrie par différentes méthodes, nous avons pu mettre en évidence son influence dans la protection contre le stress oxydatif. Ainsi la surexpression d’ACD réduit la production secondaire de radicaux oxygénés mitochondriaux et la perte d’ADN mitochondrial. Le stress oxydatif causant la réduction des foci mitochondriaux d’ACD. Dans un second temps, nous nous sommes intéressés aux mécanismes de maintenance des télomères (TMM) que les cellules cancéreuses acquièrent afin d’outrepasser la sénescence réplicative. Dans ce sens, les tumeurs peuvent réactiver la télomérase (95% des cas) ou utiliser un processus alternatif (ALT) basé sur la recombinaison homologue (5% des cas). Pour les gliomes, jusqu’à 25% des tumeurs utilisent le processus ALT, associé à la perte d’ATRX, les autres gliomes utilisent la télomérase et présentent classiquement une mutation du promoteur de TERT (TERTmt). Ces deux marqueurs moléculaires ont par ailleurs une valeur diagnostique et pronostique et font parties des critères de classification histo-moléculaire de l’OMS . Or, de 4 à 28% des gliomes (selon les sous-types) ne possèdent ni altération d’ATRX ni mutation de TERT suggérant une activation de l’un des TMM par d’autres altérations voire d’autres voies. Dans ce sens, nous avons développé un test mesurant le TMM vrai en nous basant sur la recherche des c-circle (un marqueur du ALT) et proposé un algorithme breveté (TeloDiag) prenant en compte ce TMM, les mutations d’IDH et le grading histologique. Le TeloDiag permet de re-classer 38% des gliomes atypiques (au niveau moléculaire). Il a généré une nouvelle catégorie de tumeurs de haut grade IDHwt et ALT+, n’existant pas dans la classification OMS et montrant une tendance à un meilleur pronostic que les glioblastomes IDHwt (TERTmt). Enfin, nous avons apporté la preuve de concept de la faisabilité de ce test en circulant, pour les astrocytomes IDHmt
The Shelterin complex, made of 6 proteins (POT1 / TRF1 / TRF2 / TIN2 / RAP1 and ACD) plays a major role in telomeres. Thus, it allows the protection of the telomeric single-stranded end by the formation of the D-loop, the regulation of DNA damage signaling pathways; it participates in telomere replication and controls the accessibility and processivity of the telomerase, the unique enzyme allowing telomere lengthening. During this thesis, my work was organized in 2 main axes, the first, fundamental, was interested in the extra-telomeric effects of the ACD protein (also called TPP1). The second, more transversal, focused on the processes of telomere maintenance in gliomas. Concerning the first aspect, it is now known that the ACD protein makes the link between TIN2 and TERT (catalytic subunit of telomerase) in the telomeres. These two proteins can also partially localize to the mitochondria and then have various effects on mitochondrial metabolism, on the oxidative stress regulation or on the mitophagy process. Thus, and following in silico predictions of a putative MTS for ACD, we hypothesized that ACD could be the missing partner of TIN2 and TERT in the mitochondria. In this case, it then remained to identify its mitochondrial functions. After demonstrating the partial localization of ACD in the mitochondria by different methods, we were able to demonstrate its influence in the protection against oxidative stress. Thus overexpression of ACD reduces secondary production of mitochondrial oxygen radicals and loss of mitochondrial DNA. Oxidative stress causing reduction of ACD mitochondrial foci. Secondly, we looked at the telomere maintenance mechanisms (TMM) that cancer cells acquire in order to override replicative senescence. In this sense, tumors can reactivate telomerase (95% of cancer) or use an alternative process (ALT) based on homologous recombination (5% of cancer). In the case of gliomas, up to 25% of tumors use the ALT process, associated with the loss of ATRX, the other gliomas use telomerase and typically have a mutation of the TERT promoter (TERTmt). These two molecular markers also have diagnostic and prognostic value and are part of the WHO histo-molecular classification criteria. But, 4 to 28% of gliomas (depending on the subtypes) do not have an ATRX alteration or TERT mutation suggesting activation of one of the TMM by other alterations or even other pathways. In this sense, we have developed a test measuring the true TMM based on the detection of c-circles (a marker of ALT) and proposed a patented algorithm (TeloDiag) taking into account this TMM, IDH mutations and the histological grading. The TeloDiag makes it possible to re-classify 38% of atypical gliomas (at the molecular level). It generated a new category of high grade IDHwt and ALT + tumors, not found in the WHO classification and showing a tendency for a better prognosis than IDHwt glioblastomas (TERTmt). Finally, we provided the proof of concept of the feasibility of this circulating test for IDHmt astrocytomas

Certaines cellules cancéreuses peuvent utiliser un mécanisme indépendant de la télomérase, connu sous le nom ALT (Alternative Lengthening of Telomeres) pour allonger leurs télomères. Les cellules ALT sont caractérisées par des télomères hétérogènes extrêmement longs et d’autres très courts voire indétectables qui co-localisent avec les corps PML pour former des structures nucléaires appelées APB (ALT-associated PML Bodies), et une fréquence élevée d'échange entre les télomères des chromatides sœurs appelées T- SCE (Telomeric Sister Chromatid Exchange). Bien qu'il soit concevable que la recombinaison homologue soit le mécanisme clé pour le maintien des télomères par la voie ALT, les acteurs moléculaires ne sont pas très bien connus. Nous avons identifié de nouveaux régulateurs potentiellement impliqués dans le mécanisme ALT: PCAF (P300/CBP-associated factor) et GCN5 (General Control Non-derepressible 5), deux lysines acétyl transférases homologues. Elles représentent généralement des facteurs de transcription, cependant, elles peuvent aussi acétyler des protéines non histones. Elles sont mutuellement exclusives dans de multiples complexes y compris le complexe SAGA. Nous avons montré que l’inhibition de ces deux protéines induit des effets opposés sur le phénotype ALT. Bien que l’absence de GCN5 augmentait l'instabilité des télomères et la fréquence des T-SCE et, la sous-expression de PCAF diminuait les T-SCE, la formation des APB et l'instabilité des télomères. Nos résultats suggèrent que dans les cellules ALT GCN5 est présent au niveau de l’ADN télomérique il inhibe la recombinaison entre les télomères et n’affecte pas la formation des APB, contrairement à PCAF qui peut indirectement les favoriser et stimuler aussi la formation des APB. Ensuite, nous avons cherché les mécanismes par lesquels PCAF et GCN5 contribuent au maintien des télomères dans les cellules ALT. Nous avons proposé que la participation de ces deux protéines consiste à réguler le turnover de la protéine télomérique TRF1 via USP22, une déubiquitinase identifiée pour la première fois comme un constituant des APB. En outre, l'intérêt de cibler l’activité de ces lysines acétyl transférase dans les cellules ALT a été testé in vitro en utilisant des inhibiteurs seuls ou combinés à l’irradiation. Nous avons montré que les cellules ALT sont particulièrement sensibles à l'inhibition de l'activité lysine acétyl transférase par l'acide anacardique (AA). Le traitement par cette molécule récapitule l'effet de la sous-expression de PCAF sur le phénotype ALT, suggérant que l’AA défavorise le mécanisme ALT en inhibant l'activité lysine acétyl transférase de PCAF, et non pas celle du GCN5. De plus, l'AA sensibilise spécifiquement les cellules ALT humaines à l’irradiation en comparant aux cellules télomérase-positives, prouvant que l'inhibition de l'activité des lysines acétyl transférases peut être un outil pour traiter les cellules ALT en augmentant l'efficacité de la radiothérapie
Some cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. ALT cells present unusual characteristics: extremely long and heterogeneous telomeres that colocalize with PML bodies to form nuclear structures called ALT-associated PML Bodies (APB), and high frequency of exchange events between sisters chromatid telomere referred to as Telomeric Sister Chromatid Exchange (T-SCE). Although it is agreed that homologous recombination is the key mechanism allowing the maintenance of the telomeres of ALT cells, the molecular actors involved are not yet known. We identified new actors potentially involved in the ALT mechanism: general control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF). Although they represent transcription factors, they can also acetylate non-histone proteins. They are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. While GCN5 knockdown increased T-SCE and telomere instability, PCAF knockdown decreased T-SCE, APBs formation and telomere instability. GCN5 and PCAF knockdowns had thus differential effects on ALT, up-regulating it or down-regulating it respectively. Our results suggest that in ALT cells GCN5 is present at telomeres and opposes telomere recombination and does not affect the formation of APBs, unlike PCAF which may indirectly favour them and stimulate the APB formation. Then we evaluate the mechanisms by which PCAF and GCN5 contribute to the maintenance of telomeres in ALT cells. We have proposed that the participation of these two proteins should involve regulating the turnover of the telomeric protein TRF1 via USP22, a deubiquitinase identified for the first time as a component of APBs. In addition, the interest of targeting lysine acetyl transferase activities in ALT cells to oppose the maintenance of telomeres was subsequently tested in vitro using inhibitors alone or combined to irradiation. We have shown that ALT cells are particularly sensitive to the inhibition of acetyltransferases activities using Anacardic Acid (AA). AA treatment recapitulates the effect of PCAF knockdown on several ALT features, suggesting that AA decreased the ALT mechanism through the inhibition of lysine transferase activity of PCAF, but not that of GCN5. Furthermore, AA specifically sensitizes human ALT cells to radiation as compared to telomerase-positive cells suggesting that the inhibition of lysine acetyltransferases activity may be used to increase the radiotherapy efficiency against ALT cancers

An SGA approach to discover cdc13-1ts supressors. Telomeres, the DNA-protein complexes at the end of eukaryotic chromosomes, are essential for chromosomal stability. In yeast, the telomeric single-strand binding protein Cdc13p has multiple important roles related to telomere maintenance: (1) telomeric"capping"--protection of telomeres by forming complexes with yKu70/80 and with Stn1p/Ten1p; (2) positive regulation of telomere replication via interaction with Est1p, which is a part of telomerase; (3) negative regulation of telomerase by the recruitment of telomere elongation suppressors Stn1p and Ten1p. In an attempt to identify genes that are involved in the deleterious outcome of an absence of Cdc13p, we screened the yeast gene knock-out library for genes that could suppress the growth defect of cdc13-1 cells at 33ê C. For this purpose, we performed an SGA array experiment. We scored for the ability of double mutant haploids to grow at 33ê C. Eventually, we hoped to find the elusive genes involved in telomere 5'-end processing (exonucleases). Based on the comparative analysis of growth properties of the strains (23ê C vs 33ê C), the initial screen identified up to 111 genes that displayed an apparent growth at 33ê C. In order to verify these results, diploids were regenerated, sporulated, microdissected, and haploid double mutants cdc13-1 yfg[deletion] were isolated from 38 potential cdc13-1 suppressors. Unfortunately, this verification failed to reproduce a suppression of the growth defect by any of the selected genes at any temperature. While disappointing, the results reemphasize that careful re-examination of large scale SGA approaches are indispensable before going on to more involved experimentation. Similarities and differences between adaptation to DNA double-strand break and to telomere uncapping in yeast Saccharomyces cerevisiae. It was previously shown that a certain proportion of telomerase negative survivor cells (both type I and type II cells) is able to survive in the absence of the telomere capping protein Cdc13p. These strains (named [deletion]13s) were characterized in great detail and one of their discovered features was a striking ability to continuously inactivate DNA-damage checkpoints. Based on structural similarities between DNA double strand breaks (DSB) and unprotected telomeres, we attempted to verify if the molecular mechanisms regulating adaptation to a single irreparable DSB also regulate adaptation to a loss of Cdc13p. For this purpose we created three tlc1[deletion] cdc13[deletion] strains also harboring DSB adaptation related mutations tid1[deletion], ptc2[deletion] and rfa1-t11. After deprotection of their telomeres, mutant survivor cells showed similar cell cycle progression patterns as compared to the cells where a single irreparable DSB was introduced. Adaptation defective mutants tid1[deletion] and ptc2[deletion] demonstrated an inability to adapt to telomere uncapping and to resume cell cycle. Interestingly, cells harboring the rfa1-t11 allele, which was reported to suppress adaptation defects of other mutations, did not show any distinguishable phenotype in terms of initial adaptation to telomere deprotection; i.e. rfa1-t11 mutant survivors do escape the G2/M arrest and re-enter the cell cycle. However, all three mutant survivor strains failed to produce viable [deletion]13 capping independent cells, which is consistent with the hypothesis that adaptation to loss of Cdc13p depends on the same pathway as the previously reported adaptation phenomenon. Finally, we report the surprising finding that if cells had once experienced an adapted [deletion]13 state, they will re-produce capping negative survivors much more readily. Thus, while a culture of type II survivor cells generates [deletion]13s at a rate of about 1×10 -5 events per division, cells that had been [deletion]13s and re-transformed with a Cdc13p carrying plasmid will produce capping independent cells at about 1×10-2 events per division. We are currently examining why these cells re-generate [deletion]13 cell lines more readily and suspect structural differences in telomere terminal sequence arrangements.

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.

The overall survival for patients with glioblastoma multiforme (GBM) has not improved in two decades. A better understanding of the molecular basis for gliomagenesis would aid therapeutic advances. Recombinational based alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) distinct from telomerase, which serves as a prognostic factor in GBM. In this thesis, I have compared components of the p53 axis, namely p53, p21[WAF-1] and the paired box-containing transcription factors (PAX) PAX2, 5, and 8, with TMM in gliomas. Analysis of TP53 status in relation to TMM in 110 gliomas revealed that activation of ALT during tumorigenesis possibly requires loss of normal TP53 function (P < 0.0001). Overexpression of p21[WAF-1] was also found to correlate with telomerase-positive gliomas (P = 0.0002). Moreover, high p21[WAF-1] expression is a poor prognostic factor in patients under 56 years (P = 0.015). Telomere length (TL) was also found as a prognostic factor, such that short TL (< 5 kb) is a poor prognostic factor in the group without defined TMM ("None")(P = 0.0160). Pax2,5 and 8 belong to Group II of the PAX family. They are expressed at the midbrain-hindbrain boundary (MHB) and in the neural tube of the vertebrate embryo, whereas their expression levels are low in the adult brain. To explore their roles in glioma pathology, I analyzed mRNA levels in 54 gliomas and 16 established glioma cell lines. Increased levels of PAX8 mRNA were detected in 74.1% of gliomas and 62.5% of established glioma cell lines by real time PCR. Sixty-six percent of glioma specimens expressed high levels of active PAX2, 5, and 8 by immunohistochemistry. There were more males than females having high PAX2 expression (P = 0.0408). Suppression of PAX8 by small interfering RNA induced glioma cell death, independent of TP53 status. These findings identify PAX8 as a survival factor for GBM, and PAX2, 5, and 8 expression as contributing to the aggressive behavior of gliomas. The mRNA level of PAX8 showed a positive correlation with telomerase activity in glioma biopsies (r� = 0.75, P < 0.001). The relationship was explored and I found that PAX2 and PAX8 are able to activate the reporter constructs of both the catalytic subunit (hTERT) and the RNA component (hTR) of telomerase. PAX8 had a stronger effect than PAX2 on the activation of the hTERT and hTR promoters. By electrophoretic mobility shift assay, Western blotting and telomerase activity assay, I showed that PAX8 bound directly to hTERT and hTR promoters, and upregulated hTERT protein and telomerase activity. Moreover, gliomas carrying wild type TP53 had higher levels of PAX8 expression compared to those with mutant TP53 (P = 0.0075) suggesting that PAX8 is significant only in some GBMs during gliomagenesis. These results show that the oncofetal proteins, PAX2 and PAX8, may have roles in telomerase regulation. Taken together, molecular markers examined in this thesis suggest gliomas with different TMMs are derived from different pathways.

Telomeres are fundamental structures found at the end of all eukaryotic chromosomes that function to protect the end of chromosomes from end-to-end fusion, erosion and subsequent telomere dysfunction. Telomerase and alternative lengthening of telomere (ALT) mechanisms maintain the telomeres by compensating natural telomeric loss. ALT is found to be present in 15% of human tumours lines and it may be expressed at low levels in the normal mouse tissues. However, the exact mechanism behind ALT depression and/or activation in the mammalian cells is not fully understood. Previous studies have highlighted the role of BRCA1 in telomere dysfunction. Also, it has recently been shown that BRCA1 co-localises at telomeres in the ALT + human cells through BLM and Rad50. However, it is still unclear whether BRCA1 plays a direct role on telomere length maintenance and integrity. The aim of this project was to examine the role of BRCA1 in telomere maintenance associate with ALT in BRCA1 defective mammalian cells. Therefore to achieve this, we have set up series of experiments to look at, (a) hallmarks of ALT activity at the cytological level, (b) measuring of ALT activity using biochemical and immunocytochemistry techniques and (c) understanding the role of BRCA1 in DNA damage response mechanism and telomere dysfunction. Firstly, we found elevated levels of recombination at telomeres in the two human BRCA1 carrier cell lines and mouse embryonic stem cell with deficiency in Brca1-/-. Secondly, our data showed that human and mouse BRCA1 defective cells are significantly more sensitive to ionizing radiation in line with the DNA repair function of BRCA1. Moreover, we found persistent DNA damage at telomeres in the BRCA1 defective environment when after exposure of cells to ionizing radiation. Thirdly, we found evidence of ALT activity in some mouse cell lines, and elevated ALT in mouse cells defective in Brca1. Finally, we examined some other ALT markers using immunofluorescence. Our data indicate differences between human and mouse cells in regulating ALT. Taken together data presented in this thesis revealed that (i) BRCA1 plays a major role in telomere maintenance and defective BRCA1 mammalian cells show evidence of telomere dysfunction and telomere length shortening in line with previous publish data, (ii) BRCA1 defective mouse cells have elevated levels of ALT, (iii) the mouse lymphoblastoid LY-S cells have complete absence of ALT.

Molecular mechanisms, which cause changes of the ends of chromosomes, the telomeres, play an important role in the generation of genomic instability. In normal somatic cells, the length of the telomeres are shortened during cell division due to the inability of the cells to replicate their chromosome ends completely. Telomeres below a critical length are dysfunctional and undergo apoptosis or permanent growth arrest referred to as replicative senescence. One possibility to bypass telomere dysfunction and maintenance of stable telomere length is the activation of a telomere maintenance mechanism. The currently known telomere maintenance mechanisms in humans are telomerase activity (TA-activity) and the alternative lengthening of telomeres (ALT-Mechanism). The preliminary goal of the thesis was to assess the frequency distribution of the activation of telomerase and of the ALT-mechanism in the major soft tissue sarcomas subtypes, which had been genetically well characterized in preceding studies. A first step of the thesis was the establishment of a method to assess the ALT-mechanism. One hallmark of ALT positive tumors/cells is the presence of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are characterized by promyelocytic leukemia (PML) bodies, which colocalize with telomeric DNA or telomere-specic binding proteins. A further marker of ALT is their highly heterogeneous telomere length with some exceptionally long telomeres. In order to assess ALT in human soft tissue sarcoma subtypes by combined telomere uorescence in situ hybridization and PML immunouorescence, confocal laser scanning microscope was used. 3D images were acquired, which visualize telomere spots in the rst channel, PML bodies in the second channel and DAPI stained nuclei in the third channel. The central task of image analysis was to automatically detect and classify APBs within the cell nucleus as well as to detect and quantify very large telomeres. Telomerase activity was evaluated by the TRAP assay (telomeric repeat amplication protocol). Afterwards, these data were correlated with the number of chromosomal imbalances detected by comparative genomic hybridization (CGH). This study contributed to the understanding of the frequency distribution of telomere maintenance mechanisms in soft tissue sarcoma subgroups and whether these mechanisms are associated with specic chromosomal imbalances. The most important results based on this thesis are: 1. The study demonstrates that the occurrence of the telomere maintenance mechanism is characteristic for the subtype of soft tissue sarcoma. The presence of telomerase activity ranges from 100% in synovial sarcoma to 46% in pleomorphic liposarcomas. The frequency of the marker for the ALT mechanism depends on the subtype of soft tissue sarcoma. While the telomeres detected in synovial sarcomas were equal in length, 92% of all cases of pleomorphic liposarcomas showed exceptionally long telomere signals. Furthermore, the correlation of both telomere maintenance mechanisms has shown that in tumors with high telomerase activity, the markers for the ALT-mechanism are significantly reduced. 2. In order to investigated an abundant number of healthy tissue with regard to markers for the ALT mechanism automatic quantication were performed. Healthy tissue, as well as our investigated tumor samples, show the appearance of ALT-associated PML bodies, whereas no telomeres with heterogeneous size and exceptionally long telomeres could be detected in healthy tissue. Therefore an important questions remains: how tightly are APBs linked to the ALT mechanism? 3. Comparative genomic hybridization (CGH) allows for a genome-wide screen for chromosomal imbalances in tumor samples. The correlation of the CGH data and the telomere maintenance mechanisms revealed that tumors with telomerase activity show a dierent cytogenetic prole compared to those tumors with markers for ALT. In respect to the frequency of chromosomal imbalances, such as gains and losses, there were no dierences between both telomere maintenance mechanisms. 4. A new approach for an automated quantication of telomere and PML spots as well as colocalization in multi-channel 3D microscopy images to assess the ALT mechanism were developed. To discriminate between spots inside and outside the cell nucleus, DAPI staining was used for the nuclear segmentation. With our program more than 500 confocal images were successfully analyzed. The characterization of tumors is highly relevant for tumor diagnostics and treatment planning. Due to the appearances of APBs in healthy tissue, one important question remains. How tightly are these structures linked to the ALT-mechanism? Further investigation is needed to clarify if in healthy tissue APBs are induced by the ALT mechanism or other nuclear processes besides ALT. Therefore, the characterization and understanding of telomere maintenance mechanism in tumorigenesis could have important implications in the development new treatments for these malignancies.

- Extended longevity and high fecundity are two phenomena typical for reproductive castes (queens, eventually kings) of eusocial insects. In my thesis, we explore the hypothesis that the longevity of reproductives in the termite Prorhinotermes simplex is linked with the activation of the telomerase enzyme complex. Telomerase is well known for its life-extension functions, due especially to its capacity to prolong the telomeric ends of chromosomes. Therefore, we studied here the gene expression of: (1) the gene TERT coding for the catalytic subunit of the telomerase and (2) the genes of the main endocrine regulatory pathways, known to be responsible for the control of reproduction and longevity in insects. Expression dynamics of these genes were measured in sterile and reproductive castes of P. simplex during their development and sexual maturation. Based on our results obtained from the TERT expression analyses and their comparison with telomerase enzyme activities, we assume that the telomerase action in the long-lived reproductive individuals is regulated at a post-transcription level. Furthermore, we observed in reproductive castes a simultaneous upregulation of some transcription variants of vitellogenin and the genes for insulin signalling pathways. We can, therefore, conclude that in...