Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

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This study presents detailed phonetic and acoustic analyses of the speech characteristics of two new cases of Foreign Accent Syndrome (FAS). Participants include a 48-year-old female who began speaking with an "Eastern European" accent following a traumatic brain injury, and a 45-year-old male who presented with a "British" accent following a subcortical cerebral vascular accident (CVA). Identical samples of the participants' pre- and post-morbid speech were obtained, thus affording a new level of control in the study of Foreign Accent Syndrome. The speech tasks consisted of oral readings of the Grandfather Passage and 18 real words comprised of the stop consonants /p/, /t/, /k/, /b/, /d/, /g/ combined with the peripheral vowels /i/, /a/ and /u/ and ending in a voiceless stop. Computer-based acoustic measures included: 1) voice onset time (VOT), 2) vowel durations, 3) whole word durations, 4) first, second and third formant frequencies, and 5) fundamental frequency. Formant frequencies were measured at three points in the vowel duration: a) 20%, b) 50%, and c) 80% to assess differences in vowel 'onglides' and 'offglides'. The phonetic analysis provided perceptual identification of the major phonetic features associated with the foreign quality of participant's FAS speech, while acoustic measures allowed precise quantification of these features. Results indicated evidence of backing of consonant and vowel productions for both participants. The implications for future research and clinical applications are also considered.

The present study is the first to examine the effect of first versus second language (L1 versus L2) speech production on specific temporal parameters of speech in bilingual speakers with neurogenic speech disorders. Three persons with apraxia of speech (AOS), three with phonemic paraphasia (PP) and five normal speaking participants were included as subjects in the study. Subjects were required to read phonemically similar L1 and L2 target utterances in a carrier phrase, five times each, at a normal and fast speaking rate, respectively. This rendered four speaking contexts that included speech production in L1 at either a normal (L1NR) or fast speaking rate (L1FR) and speech production in L2 at either a normal (L2NR) or fast speaking rate (L2FR). Acoustic analysis of on-target productions involved measurement of utterance onset duration, vowel duration, utterance duration and voice onset time. Results revealed that in normal speakers, speech production in L2 results in greater token-to-token variability than in L1. However, token-to-token variability in the experimental subjects did not tend to increase whilst speaking in L2, most probably because these subjects generally decreased their speaking rate in this context, resulting in more consistent production. The subjects with AOS and PP seemed to be influenced by the increased processing demands of speaking in L2 to a greater extent than the normal speakers, in that they more frequently experienced difficulty with durational adjustments (decreasing duration in the fast speaking rate) in L2 than in L1. Furthermore, the subjects with AOS or PP also exhibited a greater extent of durational adjustment in L1 than in L2. The durations of most of the subjects with either AOS or PP tended to differ from those of the normal group to a greater extent in L2FR that was hypothesized to be the most demanding speaking context for these subjects. The longer than normal durations and greater than normal token-to-token variability in the subjects with either AOS or PP imply the presence of a motor control deficit. The extent of the motor control deficit appears to be more severe in AOS than in PP as is evident from the finding that the subjects with AOS generally exhibited longer durations and greater token-to-token variability than the subjects with PP. The pattern of breakdown in respect of different parameters and utterance groups also differed between subjects with AOS and PP. The nature of the disorder in AOS and PP thus appears to be both quantitatively and qualitatively different. Regarding measurement of the different temporal parameters, voice onset time appears to be less subject to the influence of L2 than the other measured temporal parameters. The results of this study imply that bilingual AOS is as much a reality as bilingual aphasia. Furthermore, the results underscore the importance of taking contextual factors, specifically L1 versus L2, into account when compiling assessment and treatment procedures for persons with either AOS or PP, since speech production in L2 appears to be motorically more difficult than in L1 for persons with neurogenic involvement. The significance of the results is discussed with reference to the influence of speech production in L2 on temporal control and the underlying nature of AOS and PP with regard to theories of speech sensorimotor control. Copyright / Dissertation (DPhil (Communication Pathology))--University of Pretoria, 2004. / Speech-Language Pathology and Audiology / unrestricted