ATM is a large and complex gene, identified as the recessive gene mutated in individuals with the childhood syndrome ataxia telangiectasia. While AT patients are rare and the disease has many severe symptoms, including cerebellar degeneration, immunodeficiency and a very high cancer risk, the relatively common carriers of one mutation are clinically normal. However, there is some evidence that these heterozygotes, too, have a high relative risk of cancer, especially breast cancer in women. The cellular radio sensitivity of carriers is intermediate between that of normal individuals and that of AT patients, and this may suggest the cells are predisposed to tumorigenic transformation. ATM is believed to be involved in DNA damage response, but the mechanisms by which it works are not fully understood. In this study, DNA from 412 Scottish women with breast cancers were screened for two ATM mutations, known to be relatively common in Celtic populations. In attempting to estimate the burden in Scotland of breast cancers in AT heterozygotes, it is shown here that even these two most common ATM mutations in the UK account for only a small proportion of all ATM mutations. None of these mutations were found, suggesting that these particular mutations do not confer a predisposition to breast cancer. Also presented are the results of various immunological studies. The ATM protein is shown to be localised to specific structures within the nucleus of a normal cell. It appears to be found in a number of different forms, varying in size and state of glycosylation. It sediments at a relatively low speed, suggesting that it is normally bound in a large protein complex. This work indicates that a large-scale screening study will be required to establish whether or not AT carriers are at an increased risk of cancer, and reveals that the ATM product may be present in more than one form, depending on whether the cell' stimulated to divide.

This dissertation examined injury-based and emotional adjustment factors that may influence an individual's self-awareness of neurobehavioral deficits following traumatic brain injury (TBI). Two studies were completed to examine these issues in acute and post-acute rehabilitation settings using the reports of TBI patients and their significant-others. In Study 1, the Patient Competency Rating Scale (PCRS) and Self-Awareness of Deficits Interview were used to assess the patients' awareness of deficit, while the Profile of Mood States and the Grief Experiences Inventory were used to assess emotional adjustment. Six patients and significant-others were followed weekly during the course of inpatient rehabilitation, and were seen approximately one month after their discharge from hospital. While individual variability was observed, most patients reported minor changes in their level of competence and limited emotional distress. The individual perceptions of patients and of significant-others were generally consistent over the course of inpatient care, and variations in patients' emotional adjustment appeared to be reasonable reactions to circumstantial factors. The emotional adjustment of significant-others varied considerably among the individuals assessed, and this variability likely influenced their ratings of the patient. Staff ratings of the patients were also collected, and identified improvements in functional abilities over time. These results suggest that patient awareness is not a prerequisite for rehabilitation success. A lack of applied or practical experiences may also influence patients' ability to accurately rate their self-competence during the acute phase following TBI. Study 2 examined 166 individuals referred for post-acute rehabilitation, using the PCRS and the Katz Adjustment Scale (KAS-R) to assess awareness and emotional adjustment. Patients with a history of moderate and severe TBI showed good awareness of their abilities, based on PCRS Discrepancy Scores, while patients with mild TBI were likely to report greater impairments than observed by significant-others. TBI patients showed significant emotional adjustment difficulties on the KAS-R, regardless of the severity of their injury, and there was a strong positive association between patients' acknowledgement of neurobehavioral problems and ratings of their emotional adjustment. General intellectual ability was also strongly related to patients' report of difficulties, such that low IQ and poor emotional adjustment were associated with low ratings of self-competence. On the other hand, the general location of cerebral trauma was not strongly associated with deficits in awareness. Thus, the nature and severity of TBI appeared to be less important than IQ and emotional adjustment in the post-acute rehabilitation patients, although mildly injured patients are more likely to report neurobehavioral deficits than moderate or severely injured patients. Strengths and weakness of the self-other discrepancy approach to measuring self-awareness were considered, and a robust approach to awareness assessment, based on multiple measures, is recommended. Available options include structured interviews, self-report, clinical observation, or objective testing. Furthermore, the emotional adjustment of the patient appeared to become increasingly salient in the assessment of awareness during the post-acute phase, compared to the acute phase of recovery from TBI, where significant-other adjustment may be quite relevant. / Graduate

Cells of ataxia telangiectasia (AT) individuals are hypersensitive to a variety of DNA damaging agents such as ionizing radiation and bleomycin, presumed to be due to an intrinsic defect in repair of DNA damage. The nature of the DNA lesion(s) to which AT cells are abnormally sensitive, and the defect in DNA repair are presently unclear. The major part of this project aimed at investigating the sensitivity of AT cells to DNA double-strand breaks (dsb) generated by restriction endonucleases (RE), thereby verifying the hypothesis that AT cells are deficient in the processing of dsb. AT lymphoblastoid cell lines (AT-PA and AT-KM) used in this study were initially characterized and found to be approximately 3 times more sensitive to ionizing radiation in the induction of micronuclei (Mn) and chromosomal aberrations (CA) compared with a normal lymphoblastoid cell line (N-SW). Other cellular characteristics were observed in AT-PA cells following-irradiation such as normal induction and rejoining of dsb and reduced inhibition of DNA synthesis. By using SLO poration, RE were introduced into the AT and normal cell lines and the yield of CA resulting from RE-induced dsb were subsequently investigated. The frequencies of CA induced by Pvu II were 2 - 4 fold higher in AT-PA than in N-SW cells at both 5 h and 24 h sampling times. The enhanced frequency of CA in AT cells treated with Pvu II was principally a result of an increase of chromatid aberrations, rather than chromosome aberrations at 24 h. higher frequencies of chromatid exchanges appeared in AT-PA than in N-SW cells. The results suggest that AT cells are characterized by a defect in dsb processing that converts a higher number of dsb into CA than in the normal cell line. With respect to the different end-structures of RE-induced dsb, cohesive-ended dsb generated by BamH I and Pst I were found to induce lower frequencies of CA than blunt-ended dsb generated by Pvu II and EcoR V in both the AT cell lines and the normal cell line. The results support the previous observations that cohesive-ended dsb are less clastogenic than blunt-ended dsb (Bryant 1984). Although inducing lower frequencies of CA than Pvu II and EcoR V, BamH I and Pst I induced higher number of CA in both AT-PA and AT-KM cells when compared with N-SW cells, again indicating a defect in processing cohesive-ended dsb exists in AT cells. A potent DNA repair inhibitor, Ara A, was found to potentiate the production of CA by RE in AT and normal cells. The enhancement ratios (by ara A) for CA induced by Pvu II and Pst 1 were higher in N-SW cells than in AT-PA and AT-KM cells. Ara A appeared to have no effect on the frequencies of CA induced by BamH I in any of the cell lines tested. Based on these findings, a mechanism for the rejoining of RE-induced dsb in which DNA repair synthesis may be involved is proposed, and it is postulated that dsb in AT cells are subjected to greater end degradation. Inhibition of DNA synthesis was observed in normal cells after treatment with Pvu II and EcoR V, while EcoR I and BamH I had only minor effect. AT-PA cells were found to be resistant to RE-induced inhibition of DNA synthesis, as in the case of ionizing radiation. This result suggests that RE-induced blunt-ended dsb mimic radiation-induced lesions in supressing DNA synthesis in normal cells and that AT cells respond to RE-induced dsb in a similar way to damage induced by ionizing radiation. Finally, when a nuclear extract from N-SW cells was introduced into Pvu Il-treated AT-PA cells, it was able to confer a normal frequency of CA. In contrast, neither whole cell nor nuclear extracts from normal cells influenced the production of CA induced by y-rays. These findings provide evidence for the presence of factor(s) in normal nuclear extract which complements the defect in processing of RE-induced dsb in AT cells.

Mutations are generally thought to be targeted events. The distribution of mutations is based upon the initial original deposition of DNA damage and the fidelity and efficiency of repair of this damage. These factors are dependent on the primary site of DNA modification and the surrounding nucleotides (i.e., mutation is “context sensitive"). To better understand mutagenesis, I measured DNA damage and/or mutation at the DNA sequence level, then considered the impact of mutation location and the surrounding nucleotide environment. The selected mutagens, ultraviolet light (UV) and benzo(a)pyrene [B(a)P], were chosen because they produce well-characterized lesions and are environmentally relevant. UVC (254nm) light-induced DNA damage is well documented. UVC produces a characteristic spectrum of mutations. The predominant UV-induced mutations are C → T transitions occurring at TC or CC sites, as well as CC → TT tandem transitions. The latter class of mutation is considered the hallmark of UV mutagenesis. Quantitatively speaking, the primary types of UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) and the 6-4 pyrimidine/pyrimidone (64PyPy). These are also the suspected predominant pre-mutagenic lesions. Each lesion was independently measured at the DNA sequence level in a defined region of DNA. The pattern of UVC-induced DNA damage revealed a complex induction pattern. The flanking DNA nucleotides partially influenced the pattern of damage deposition. Sites where C → T transitions and CC → TT transitions were recovered at high frequencies were also frequently damaged. Thus, at these sites, mutation fixation was potentially more influenced by initial DNA damage than the rate of DNA repair. Two other components of the UV spectrum [UVB (290-320 nm) and UVA (320-400 nm)] are more environmentally relevant than UVC since UVB and UVA reach the surface of the earth.The results of UVC experiments were used as a guide to interpret the results obtained using UVB since direct light absorption by DNA has been shown to be one of the main biological effect at both wavelengths. The model that was chosen for the studies was an in vivo transgenic rodent mutagenesis assay. The research presented in the thesis represents one of the first studies to characterize UV-induced mutation at the DNA sequence level in rodent skin. The backs of female C57B1/6 lacl transgenic mice were shaved and exposed to either UVB or UVA light. UVB was found to be significantly more mutagenic than UVA. The UVB-induced mutation spectrum was characterized by C → T transitions at dipyrimidine sites, implicating CPD and/or 64PyPy lesions as premutational DNA lesions. The majority of UVA-induced mutations was C → T transitions at dipyrimidines sites and hence, as with UVB-induced mutation, attributed to CPDs and 64PyPy. In the UVA-dose response experiments, the induced mutant frequency was lower than expected at higher doses. A statistically significant increase in putative clonal expansion suggested that skin cells might have undergone cell killing followed by repopulation. In a final study, C57Bl/6 lacI transgenic male mice were intraperitoneally injected with the mutagen B(a)P at doses of 0, 62.5, 125, 250, and 500 mg/kg. This resulted in a linear increase in mutation frequency (4.8 to 53 × 10⁻⁵). All mutations increased at GC basepairs and not AT basepairs following B(a)P treatment. This was consistent with models suggesting guanosine adducts to be mutagenic lesions. In conclusion, the transgenic lacI mouse mutagenesis model was a sensitive target for in vivo mutagenesis from UVB, UVA and benzo(a)pyrene exposures. The system detected class-specific mutation frequency differences and increases in cell proliferation after mutagen exposure. With a further refinement of techniques, the correlation of DNA damage and mutation will allow even more exquisite studies. / Graduate

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Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / It has been demonstrated that oligonucleotides homologous to the 3' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H202 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H20 2 showed no H20 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated β-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of β-galactosidase positive cells by 72 hours (<30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms. / 2031-01-01

Bibliography: pages 130-139. / The utility of the National Adult Reading Test (NART) as a predictor of premorbid intellectual functioning is dependent on its ability to adequately predict IQ from irregular word reading ability. The primary aim of this study is the replication, on a South African sample, of the findings reported by Nelson (1977 unpub. manuscript) in her standardisation study. A total of 234 subjects are divided into groups according to language usage and availability/type of IQ score and utilised in a correlational study which investigates the psychometric characteristics of the NART and the degree of correspondence between predicted and observed I.Q. values. The research yields regression formulae for the prediction of IQ from performance on the NART. While correlation coefficients obtained for English speaking subjects do not differ significantly from those derived from Nelson's data (1977 unpub. manuscript), it is concluded that the use of the test for Afrikaans subjects is not justified. Satisfactory reliability and validity characteristics reported for the sample utilised in this study suggest that the instrument can be usefully applied under local conditions. An investigation of 24 potential additional items for the test does not yield encouraging results. The lack of an adequate model explaining the mechanisms underlying the functioning of the test is addressed, and a number of areas of interest for further research are identified.