Thesis (M.Phil.) - University of Queensland, 2006. / Includes bibliography.

A presente pesquisa pretende demonstrar evidências capazes de contribuir para o entendimento dos mecanismos envolvidos na ataxia-telangiectasia, bem como oferecer dados que auxiliem na implementação de técnicas complementares ao diagnóstico desta síndrome. Desde a descoberta do gene envolvido na ataxia-telangiectasia (o gene ATM), muito conhecimento tem sido acumulado, especialmente sobre os mecanismos moleculares envolvidos na síndrome, bem como nas respectivas doenças relacionadas. Este estudo teve como objetivo avaliar o dano de DNA, a capacidade de reparo e a sensibilidade à radiação ionizante comparada à bleomicina em indivíduos com ataxiatelangiectasia e em seus pais heterozigotos, relacionando-os com um grupo controle. O trabalho foi desenvolvido através de voluntários em acompanhamento no Hospital de Clínicas de Porto Alegre. As amostras foram submetidas às técnicas de micronúcleo e cometa, de maneira espontânea e com dano de DNA induzido. Os resultados demonstraram que a técnica de micronúcleo sem a indução de dano é o suficiente para a diferenciação de pacientes, pais e controles, incluindo níveis de micronúcleo (MN), pontes nucleoplasmáticas (PN) e BUDs nucleares. Não foi possível distinguir os três grupos com a técnica do cometa espontâneo, mas a monitoração da cinética de reparo de DNA através dessa técnica demonstrou que o grupo de pacientes apresenta um atraso no processo de reparo do DNA, em relação aos controles. Os achados nas duas técnicas são complementares, e a sua combinação é altamente recomendável na identificação de instabilidade cromossômica, com o objetivo de auxiliar no diagnóstico de pacientes AT.

Cells respond to the induction of DNA damage with activation of the DNA damage response (DDR), a complex signalling network which orchestrates cell cycle arrest and DNA repair in order to maintain genomic stability and cell viability. Activation of these signalling pathways enables cancer cells to survive DNA damaging chemo- or radiotherapy and contributes to the development of therapy resistance. Therefore, components of the DDR have become attractive targets for chemo- or radiosensitisation. Furthermore, cancer cells frequently exhibit defects in certain DDR components and may, as a consequence, become highly dependent on remaining DDR pathways to survive DNA damage. Two apical mediators of the DDR are the serine/threonine protein kinases ATM and ATR. ATM is frequently mutated in non-small cell lung cancer (NSCLC), and defects in ATM may render the tumour cells dependent on ATR signalling for survival. In this study, we characterised the functional consequences of ATM mutations in NSCLC cell lines and established an immunohistochemistry-based assay to identify patients with loss of ATM expression. As a single agent, pharmacological ATR inhibition (ATRi) was selectively cytotoxic for cells deficient in both ATM and p53. Furthermore, ATRi in combination with either ATM or PARP inhibition selectively killed tumour cells with mutant p53. We show that following ATR inhibition, ATM and p53 perform critical cell cycle checkpoint functions, independently of each other. Our results suggest that while retained function in any of these pathways is sufficient to maintain cell viability, functional loss of ATM, ATR and p53 results in premature mitotic entry, chromosome fragmentation and mitotic catastrophe. We conclude that in NSCLC the functional status of both ATM and p53 determines the cellular response to ATR inhibition, and propose that a combination of ATR inhibition with ATM or PARP inhibition may have broad utility for the treatment of p53-mutated NSCLC.

A presente pesquisa pretende demonstrar evidências capazes de contribuir para o entendimento dos mecanismos envolvidos na ataxia-telangiectasia, bem como oferecer dados que auxiliem na implementação de técnicas complementares ao diagnóstico desta síndrome. Desde a descoberta do gene envolvido na ataxia-telangiectasia (o gene ATM), muito conhecimento tem sido acumulado, especialmente sobre os mecanismos moleculares envolvidos na síndrome, bem como nas respectivas doenças relacionadas. Este estudo teve como objetivo avaliar o dano de DNA, a capacidade de reparo e a sensibilidade à radiação ionizante comparada à bleomicina em indivíduos com ataxiatelangiectasia e em seus pais heterozigotos, relacionando-os com um grupo controle. O trabalho foi desenvolvido através de voluntários em acompanhamento no Hospital de Clínicas de Porto Alegre. As amostras foram submetidas às técnicas de micronúcleo e cometa, de maneira espontânea e com dano de DNA induzido. Os resultados demonstraram que a técnica de micronúcleo sem a indução de dano é o suficiente para a diferenciação de pacientes, pais e controles, incluindo níveis de micronúcleo (MN), pontes nucleoplasmáticas (PN) e BUDs nucleares. Não foi possível distinguir os três grupos com a técnica do cometa espontâneo, mas a monitoração da cinética de reparo de DNA através dessa técnica demonstrou que o grupo de pacientes apresenta um atraso no processo de reparo do DNA, em relação aos controles. Os achados nas duas técnicas são complementares, e a sua combinação é altamente recomendável na identificação de instabilidade cromossômica, com o objetivo de auxiliar no diagnóstico de pacientes AT.

A presente pesquisa pretende demonstrar evidências capazes de contribuir para o entendimento dos mecanismos envolvidos na ataxia-telangiectasia, bem como oferecer dados que auxiliem na implementação de técnicas complementares ao diagnóstico desta síndrome. Desde a descoberta do gene envolvido na ataxia-telangiectasia (o gene ATM), muito conhecimento tem sido acumulado, especialmente sobre os mecanismos moleculares envolvidos na síndrome, bem como nas respectivas doenças relacionadas. Este estudo teve como objetivo avaliar o dano de DNA, a capacidade de reparo e a sensibilidade à radiação ionizante comparada à bleomicina em indivíduos com ataxiatelangiectasia e em seus pais heterozigotos, relacionando-os com um grupo controle. O trabalho foi desenvolvido através de voluntários em acompanhamento no Hospital de Clínicas de Porto Alegre. As amostras foram submetidas às técnicas de micronúcleo e cometa, de maneira espontânea e com dano de DNA induzido. Os resultados demonstraram que a técnica de micronúcleo sem a indução de dano é o suficiente para a diferenciação de pacientes, pais e controles, incluindo níveis de micronúcleo (MN), pontes nucleoplasmáticas (PN) e BUDs nucleares. Não foi possível distinguir os três grupos com a técnica do cometa espontâneo, mas a monitoração da cinética de reparo de DNA através dessa técnica demonstrou que o grupo de pacientes apresenta um atraso no processo de reparo do DNA, em relação aos controles. Os achados nas duas técnicas são complementares, e a sua combinação é altamente recomendável na identificação de instabilidade cromossômica, com o objetivo de auxiliar no diagnóstico de pacientes AT.

Ataxia-telangiectasia mutated gene product (ATM) is a 350 kDa Serine/Threonine kinase belonging to the family of Phosphatidylinositol-3 kinase like kinases. ATM functions as a key element in DNA Damage Response (DDR), a mechanism that maintains genomic integrity within the cells. ATM is activated after double stranded DNA damage and initiates signalling cascades that determine the process of decision-making of cell fate and involves the participation of multiple proteins. This vital protein acts first by sensing double stranded DNA breaks and second by transducing the signal and activating other downstream proteins of the repair pathway via its kinase function. This provides an important link between signals generated after DNA damage, the cell cycle pathway and apoptotic machinery. This function is crucial for mammalian cells which are constantly challenged by genotoxic agents from a variety of sources and therefore require a robust sensing and repair mechanism to maintain cell vitality. Cells lacking ATM are hypersensitive to cytotoxic insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. This thesis describes the discovery and characterisation of novel autoregulatory feedback loops of ATM kinase in human cells. Firstly, I have discovered that inhibition of ATM kinase activity causes induction of ATM protein expression followed by time dependent oscillations. This novel autoregulatory mechanism was demonstrated in cell cycle independent manner and both in the absence and presence of DNA damage. ATM promoter assay revealed that this autoregulation was governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM was also accompanied by a transient upregulation of P53, pATR and E2F1 levels. Elucidation of the underlying trafficking mechanism of ATM during such autoregulation and in DDR also revealed a novel ATM sub-cellular trafficking mechanism which was dependent on its own kinase activity. This trafficking mechanism involved DNA damage induced Golgi to nuclear transport of phosphorylated ATM S-1981 to elicit DDR. This was found to be a conserved pathway required during the initiation of DDR and was demonstrated in multiple cell lines. Further studies into the sub-cellular transport machinery revealed the involvement of β-COPI coatomer protein in this mechanism of ATM trafficking, which was found to be autoregulated by ATM kinase, and required 387-388 ATM di-Lysine motif. The discovery of these functionally important autoregulatory mechanisms of ATM were further utilised to develop Luciferase reporter based biosensor of DNA damage and single cell fluorescence based ATM inhibition assay to screen for ATM inhibitors. Finally, following the discovery and characterisation of these functional spatio-temporal autoregulations of ATM, quantitative estimations of the kinetics of signalling cascades initiated by it during DDR and its overall outcome on cellular fate were determined to study ATM pathway systematically for employing a quantitative systems biology approach. These novel findings have immensely increased our understanding of ATM regulation and function. Elucidation of the mechanisms of novel autoregulations of ATM provide new dimensions through which DDR pathway could be manipulated, and as such could be utilised for achieving targeted cellular sensitivity in therapeutic intervention of cancer.

The Ataxia-telangiectasia mutated (ATM) protein is regarded as the major regulator of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs, ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress, suggesting that ATM plays an important role in the regulation of reactive oxygen species (ROS). I show for the first time that ATM can be activated by oxidative stress directly in the form of exposure to H₂O₂. In vitro kinase assays with purified ATM suggest that the activation by H₂O₂ is independent of DSBs and the MRN complex. In 293T cells, H₂O₂ induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated by ATM after H₂O₂ treatment but not histone H2AX and heterochromatin protein Kap1, indicating that ATM activation by H₂O₂ in human cells is independent of DNA damage. I also show that the cysteine residue 2991 is critical for ATM activation by H₂O₂ in vitro. / text

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser + appendix.

Made available in DSpace on 2015-12-06T23:46:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2013 / Introdução: A ataxia telangiectasia (AT) e uma doenca neurodegenerativa, que cursa com imunodefiCiência em graus variaveis, disfuncao mitocondrial e exacerbacao do estresse oxidativo. Objetivo: avaliar o estado nutricional, perfil lipidico, peroxidacao lipidica e as concentracoes plasmaticas de vitaminas C e E e relaciona-las com biomarcadores associados ao risco de aterosclerose de pacientes com AT e controles. Metodos: Estudo transversal controlado envolvendo 13 pacientes e 22 controles saudaveis, pareados por genero e idade. Foram avaliados: estado nutricional, consumo alimentar, perfil lipidico e suas relacoes, concentracoes plasmaticas de vitaminas C e E, malondialdeido (MDA) e proteina C reativa ultrassensivel (PCRus). Resultados: A media de idade do grupo AT foi 14,6 anos; 4/13 (30,8%) eram desnutridos e 3/13 (23,1%) apresentavam baixa estatura para idade. Os pacientes apresentaram maior comprometimento de massa magra comparativamente aos controles. As concentracoes de triglicerides, colesterol total e de LDL-c foram significantemente mais elevadas nos pacientes e as de HDL-c, mais baixas. As relacoes associadas ao risco de aterosclerose (CT/HDL-c, LDL-c/HDL-c e Log TG/HDL-c) e o colesterol nao HDL (NHDL-c) foram significantemente superiores no grupo de pacientes em comparacao aos controles. Nao houve diferenca para as concentracoes de malondialdeido, proteina C reativa e de vitaminas C e E entre os dois grupos. As relacoes vitamina E/lipideos totais e vitamina E/triglicerides mostraram valores mais baixos no grupo de pacientes; correlacao significante e inversa entre estas relacoes e NHDL-c, CT/HDL-c, LDL-c/HDL-c e Log TG/HDL-c foi observada no grupo de pacientes. A alanina aminotransferase (ALT) correlacionou-se de forma direta e significante com NHDL-c, CT/HDL-c e LDL-c/HDL-c, no grupo de pacientes. A inGestão dietetica de energia, macronutrientes e de vitaminas C e E nao diferiu entre os grupos. Conclusao: O elevado risco aterosclerotico de pacientes com AT aliado ao comprometimento da defesa antioxidante e do estado nutricional pode complicar a evolucao da doenca e enfatiza a importancia da atencao multiprofissional com monitoramento de biomarcadores e orientacao nutricional apropriada / Introduction: Ataxia telangiectasia (AT) is a neurodegenerative disease that leads to immunodeficiency in varying degrees, mitochondrial dysfunction and oxidative stress. Objective: To evaluate the nutritional status, lipid profile, lipid peroxidation, and plasma concentrations of vitamins C and E and relate them with biomarkers associated with risk of atherosclerosis in patients with AT and controls. Methods: Cross sectional and controlled study involving 13 patients and 22 controls healthy, matched by gender and age. We evaluated: nutritional status, food intake, lipid profile and their relationships, plasma concentrations of vitamins C and E, malondialdehyde (MDA) and high-sensitivity C-reactive protein (hs CRP). Results: The mean age of the AT group was 14.6 years, 4/13 (30.8%) were malnourished and 3/13 (23.1%) had stunting. The patients showed greater impairment of lean body mass compared to controls. The concentrations of triglycerides, total cholesterol and LDL-c were significantly higher in patients and of HDL-c,lower. The ratios associated with the risk of atherosclerosis (TC/ HDL-c, and LDL-c/HDL-c Log TG / HDL-c) and non-HDL cholesterol (NHDL-c) were significantly higher in patients compared to controls. There was no difference in concentrations of malondialdehyde, C-reactive protein and vitamins C and E between the two groups. The ratios vitamin E / total lipids and vitamin E / triglycerides showed lower values in the group of patients; significant inverse between these ratiosand NHDL-c, TC/ HDL-c, and LDL-c/HDL-c Log TG / HDL -c was observed in the AT group. Alanine aminotransferase (ALT) correlated directly and significantly with NHDL-C, TC / HDL-c and LDL-c/HDL-c, in patients. Dietary intake of energy, macronutrients and vitamins C and E did not differ between groups. Conclusion: The high atherosclerotic risk of patients with AT coupled with impaired antioxidant defense and nutritional status may complicate the clinical course of the disease and emphasizes the importance of multidisciplinary care with monitoring of appropriate biomarkers and nutrition al guidance. / BV UNIFESP: Teses e dissertações

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Ataxia telangiectasia (AT) is a well-characterized neurodegenerative disease resulting from a genetic defect in the Atm gene causing an absence or very low expression of the ATM protein. As AT patients are prone to the development of insulin resistance and atherosclerosis, the aim or the current study was to investigate the importance of the ATM protein in the endothelium and its role in the signalling pathways of nitric oxide (NO) production. To accomplish this, the first objective was to establish an in-house endothelial cell isolation technique harvested from normal and insulin resistant animals. Unfortunately, these cultures, although staining positive with an endothelial cell specific stain, were not pure enough and did not express endothelial NO synthase (eNOS), the central enzyme in NO production. The remainder of the study utilized commercial aortic endothelial cells (AECs) and found that there was a significant increase in NO production when the ATM protein was inhibited by the specific inhibitor, Ku-60019. The beneficial impact of increased NO production includes maintaining vascular homeostasis, promoting angiogenesis, initiating DNA repair by activating p53 and inhibiting smooth muscle cell proliferation. On the other hand, reactive oxygen species (ROS) and reactive nitrogen species (RNS) also generated by high levels of NO, can exert both protective and harmful effects. Examples of these include cell death due to high concentrations of ROS. However, Ku-60019 did not result in increased cell death of AECs. We demonstrated for the first time, a relationship between endothelial ATM protein kinase and the generation of NO. The signalling pathways involved in NO production and glucose utilization form a network of interrelationships. Central to both pathways is the activity of two protein kinases, PKB/Akt and AMPK. Both these kinases are known to phosphorylate the eNOS enzyme to produce NO on the one hand and AS160 to induce GLUT 4 translocation and glucose uptake on the other hand. Activation of the ATM protein is postulated to be a prerequisite for PKB/Akt activation and it may also result in activation of AMPK. However, using insulin to stimulate ATM, we could not show that inhibition of ATM in endothelial cells affected expression or insulin-stimulated activation of PKB/Akt while the PI3-K inhibitor wortmannin, inhibited the latter. In addition, inhibition of ATM negatively regulated the phospho/total ratio of AMPK. We therefore postulate that the NO production elicited by inhibition of ATM, may not be as result of eNOS activity. A second important observation was that inhibition of ATM significantly enhanced phosphorylation of the p85 regulatory subunit of PI3-K. This would imply that ATM normally has an inhibitory effect on p85 phosphorylation and therefore PI3-K activation. We base this assumption on previous publications showing that Ku-60019 does not inhibit PI3K. This again indicates that ATM has a hitherto unexplored regulatory role in endothelial function. / AFRIKAANSE OPSOMMING: Ataxia telangiectasia (AT) is a goed-gekarakteriseerde neurodegeneratiewe siekte a.g.v. ‘n genetiese afwyking in the Atm geen wat lei tot ‘n afwesige of lae uitdrukking van die ATM proteïen. Aangesien AT pasiënte geneig is om insulienweerstandigheid en aterosklerose te ontwikkel, was die doel van hierdie studie om die belang van die ATM proteïen in die endoteel, en sy rol in die seintransduksiepaaie betrokke by stikstofoksied (NO) produksie, te ondersoek. Om dit te bereik, was die eerste mikpunt om ‘n eie endoteelsel isolasie-tegniek (ge-oes van normale en insulienweerstandige diere) te vestig. Ongelukkig was hierdie selkulture nie suiwer genoeg nie.Ten spyte daarvan dat hulle positief getoets het met ‘n endoteelsel-spesifieke kleurstof kon geen uitdrukking van eNOS, die sentrale ensiem verantwoordelik vir NO produksie, waargeneem word nie. Die res van die studie het van kommersiële aorta endoteelselle (AES) gebruik gemaak, en daar is gevind dat die inhibisie van die ATM proteïen met die spesifieke inhibitor, Ku-60019, tot ‘n beduidende toename in NO produksie gelei het. Die voordelige impak van verhoogde NO produksie sluit die handhawing van vaskulêre homeostase, bevordering van angiogenese, inisiëring van DNA herstel deur p53 aktivering en inhibisie van gladdespiersel proliferasie in. Reaktiewe suurstofspesies (ROS) en reaktiewe stikstofspesies (RNS) wat ook a.g.v.verhoogde NO gegenereer word, kan egter beide beskermende sowel as skadelike effekte uitoefen. Voorbeelde sluit seldood a.g.v. hoë ROS konsentrasies in. Ku-60019 het egter nie tot ‘n toename in seldood van die AES gelei nie. Hierdie studie het vir die eerste keer aangetoon dat daar ‘n verwantskap tussen die endoteel ATM proteïen kinase en die produksie van NO bestaan. Die seintransduksie paaie betrokke by NO produksie en glukose verbruik vorm ‘n interafhanklike netwerk. Die aktiwiteit van twee proteïen kinases, PKB/Akt en AMPK, is sentrale rolspelers in beide paaie. Albei hierdie kinases is daarvoor bekend dat hulle die eNOS ensiem fosforileer om NO te produseer, maar terselfdertyd ook lei tot AS160 fosforilering, wat tot GLUT 4 translokering en glukose opname lei. Dis is voorgestel dat aktivering van die ATM proteïen ‘n voorvereiste vir PKB/Akt aktivering mag wees en verder kan dit ook tot aktivering van AMPK lei. Ons kon nie aantoon dat inhibisie van ATM in endoteelselle die uitdrukking of insulien-geïnduseerde aktivering van PKB/Akt onderdruk nie, terwyl die PI3-K inhibitor, wortmannin, wel laasgenoemde geïnhibeer het. Verder het die inhibisie van ATM die fosfo/totale AMPK verhouding negatief gereguleer. Ons postuleer dus dat die NO produksie waargeneem tydens ATM inhibisie, moontlik nie die gevolg van eNOS aktiwiteit was nie. ‘n Tweede belangrike waarneming was dat die inhibisie van ATM die fosforilering van die p85 regulatoriese subeenheid van PI3-K beduidend laat toeneem het. Dit impliseer dat ATM normaalweg ‘n inhibitoriese effek op p85 fosforilering, en dus PI3-K aktivering, het. Hierdie aanname word gemaak n.a.v. vorige publikasies wat getoon het dat Ku-60019 nie PI3-K inhibeer nie. Dit dui weer eens daarop dat ATM ‘n tot nog toe onbekende regulatoriese rol in endoteelfunksie het.