Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University, 17/03/2005.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.Grant to Dr. Predrag Slijepcevic from the Department of Health (RRX97), UK; EC Euratom (Fig h-CT-2002-0021 7); Laboratory of Anatomy, Embryology, Histology and Medial Physics, Faculty of Medicine, University of Ghent, Belgiu

Relationships Among Biomass, Carbon, and Microfibril Angle in Young Shorea spp. (Dipterocarpaceae) in Indonesia

Indonesia, particularly the island of Java, is dominated by a number of Shorea tree species (Dipterocarpaceae). Trees of the genus have been utilized for various practices, and they play a fundamental role in managing the stability of tropical forests. This study was carried out to understand the relationships between biomass and microfibril angle in Shorea spp. growing in West Java, where Shorea spp., are abundant. A total of 35 young trees belonging to 5 species were studied. The average age of these trees was 9 years, but in general there was a wide variation in tree diameter and total height. On average, biomass was the highest in S. leprosura and the lowest in S. palembanica. The lowest average microfibril angles (MFAs) were found in S. leprosura and S. mecistopteryx. The regression relationship between biomass and diameter was strong with an R2 value of 0.85, while the strength of the relationship between MFA and diameter was weaker (R2 = 0.195). In general, the MFA degree decreased with increased biomass accumulation Shorea species, which affects tree resistance to environmental variables and competitiveness in Indonesian tropical forests.

KONSEP DAN PERSEPSI MASYARAKAT ETNIS MEYAH TENTANG HUTAN ADAT DI KAMPUNG MEREJEMEG, KABUPATEN MANOKWARI

The study was aimed to obtain the concept of indigenous forest according to the Meyah ethnic community, the Meyah ethnic community's perception of indigenous forest, and the factors that influence the use of the indigenous forest by the Meyah ethnic community. Descriptive methods with observation and interview techniques are used in this study. Respondents were selected by purposive sampling, as many as 30 families. The results show that the concept of indigenous forest according to the Meyah ethnic community is a forest area given by God the Creator of the Universe as human property rights to live for generations, reproduce and adapt to their environment, and utilize the resources in the forest. Ownership of indigenous forests is controlled by individuals or clan groups for generations in certain areas marked by natural boundaries. The first activity in utilizing the forest as a source of life was by clearing land for houses and gardening as well as collecting forest products, which were used as the basis for determining the boundaries of land and forest rights. The Meyah ethnic community has a strong perception of indigenous forests, both perceptions of indigenous forest ownership, indigenous forest sustainability, and indigenous forest use. The factors that influence the Meyah ethnic community in the use of indigenous forests consist of a) determining factors, namely: customs and way of life about the forest; b) supporting factors, namely: livelihood and length of stay, and c) driving factors, namely: the role of traditional and religious leaders.

Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours

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.EThOS - Electronic Theses Online ServiceGrant to Dr. Predrag Slijepcevic from the Department of Health (RRX97)EC Euratom (Fig h-CT-2002-0021 7)my, Embryology, Histology and Medial Physics, Faculty of Medicine, University of Ghent, BelgiumGBUnited Kingdo

Analysis of alternative lengthening of telomere markers in BRCA1 defective cells

Telomeres are specialized structures responsible for the chromosome end protection. Previous studies have revealed that defective BRCA1 may lead to elevated telomere fusions and accelerated telomere shortening. In addition, BRCA1 associates with promyelocytic leukemia (PML) bodies in alternative lengthening of telomeres (ALTs) positive cells. We report here elevated recombination rates at telomeres in cells from human BRCA1 mutation carriers and in mouse embryonic stem cells lacking both copies of functional Brca1. An increased recombination rate at telomeres is one of the signs of ALT. To investigate this possibility further we employed the C-circle assay that identifies ALT unequivocally. Our results revealed elevated levels of ALT activity in Brca1 defective mouse cells. Similar results were obtained when the same cells were assayed for the presence of another ALT marker, namely the frequency of PML bodies. These results suggest that BRCA1 may act as a repressor of ALT.We acknowledge Dr Amir Hassan-Khani from Bent-Al-Hoda Hospital Mashhad, Iran, for partly funding Parisa K Kargaran. Supported in part by a grant from the DoReMi consortium, EC

IMPLEMENTASI STRATEGI PEMERINTAH DAERAH KABUPATEN SORONG DALAM PENGEMBANGAN WISATA ALAM DI TAMAN WISATA ALAM KLAMONO, PROVINSI PAPUA BARAT

Kabupaten Sorong banyak menyimpan sumber daya alam yang mempunyai kelebihan untuk dijadikan suatu obyek wisata alam, seperti Taman Wisata Alam Klamono (TWAK). Namun dalam pembangunan wisata alam memunculkan berbagai tantangan dan kendala, sehingga perlu dilakukan upaya pengembangan wisata alam dengan serius dan strategi yang tepat. Hasil penelitian menunjukkan bahwa terdapat 3 strategi yang diterapkan pemerintah Kabupaten Sorong, yaitu meningkatkan sumber daya finansial, sarana dan prasasaran serta sumber daya manusia, meningkatkan peran para stakeholder untuk membangun kerjasama yang sinergis dan bermanfaat, serta meningkatkan kemampuan masyarakat lokal untuk meningkatkan daya tarik wisata alam sesuai potensi TWAK. Beberapa faktor kendala yang mempengaruhi implementasi strategi dalam mengembangkan wisata alam di TWAK, yaitu ketersediaan anggaran daerah yang belum memadai, kualitas dan kuantitas sumber daya manusia yang masih rendah, sarana dan prasarana wisata alam yang belum memadai, kondisi sosial masyarakat yang masih mempermasalahkan hak ulayat, serta kerjasama yang kurang maksimal antar para stakeholder.

Autoroute A16 (Pas-de-Calais)

Date de l'opération : 1991 (PR) Inventeur(s) : Dien E. ; Mahin E. ; Martial Emmanuelle ; Cabuy Yves ; Ducrocq Thierry L’autoroute A 16, reliant Paris à Boulogne-sur-Mer, via Amiens, incise le département du Pas-de-Calais sur environ 46 km de long, selon un axe nord-sud entre Boulogne et l’Authie. À l’instar des grands travaux, les interventions archéologiques furent planifiées par une convention avec l’aménageur (SANEF). Pour la section Amiens-Boulogne, les modalités d’exécution des prospecti..

Primary laminopathy fibroblasts display altered genome organization and apoptosis

A number of diseases associated with specific tissue degeneration and premature aging have mutations in the nuclear envelope proteins A-type lamins or emerin. Those diseases with A-type lamin mutation are inclusively termed laminopathies. Due to various hypothetical roles of nuclear envelope proteins in genome function we investigated whether alterations to normal genomic behaviour are apparent in cells with mutations in A-type lamins and emerin. Even though the distributions of these proteins in proliferating laminopathy fibroblasts appear normal, there is abnormal nuclear positioning of both chromosome 18 and 13 territories, from the nuclear periphery to the interior. This genomic organization mimics that found in normal nonproliferating quiescent or senescent cells. This finding is supported by distributions of modified pRb in the laminopathy cells. All laminopathy cell lines tested and an X-linked Emery-Dreifuss muscular dystrophy cell line also demonstrate increased incidences of apoptosis. The most extreme cases of apoptosis occur in cells derived from diseases with mutations in the tail region of the LMNA gene, such as Dunningan-type familial partial lipodystrophy and mandibuloacral dysplasia, and this correlates with a significant level of micronucleation in these cells

Telomere Length Shows No Association with BRCA1 and BRCA2 Mutation Status

This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrolment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrolment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don't support the previous finding of association between hereditary breast cancer and shorter TL