Nck is as an adaptor protein previously implicated in actin cytoskeleton reorganization and by our laboratory, in translation and cell response to stress. Our primary objective was to determine the expression levels of Nck isotypes (Nck-1 and Nck-2) during cancer progression. We have performed western blot analysis of the Nck isotypes expression levels profile in various human cancer cell lines, at different stages of progression. Our data show significantly higher expression levels of Nck-2 protein in metastatic melanomas compared to non-metastatic melanomas and normal melanocytes. Using semi-quantitative RT-PCR, we demonstrated that this increase in Nck-2 expression can be also seen at the transcriptional level. The Ras/RAF/MEK/ERK pathway is often spontaneously activated in melanomas causing hyperactivation of ERK. By downregulating the expression of Nck-2 using siRNA, we have established a strong correlation between increased expression levels of Nck-2 and activated ERK. Furthermore, we have demonstrated the involvement of Nck-2 in cell proliferation and adhesion in metastatic melanomas, revealing that Nck-2 acts as a new player in this disease.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Research Doctorate - Doctor of Philosophy (PhD) / Psychotherapeutic interventions have been used to support people with cancer for more than half a century, with continuing debate as to whether there is any impact on the disease process itself. Very few of those studies have assessed physiological impact, and although art psychotherapyi has been employed to assess and enhance change in quality of life, reduction in distress and increased treatment compliance amongst adults and children with cancer, in the main these studies have been qualitative. To date few studies have been published reporting the use of art psychotherapy in the exploration of the human being from a systems perspective, using both biological and psychosocial means of assessment of efficacy, and at the time of research, none reporting immunological changes. The advantage of art psychotherapy over purely verbal psychotherapy is in the readily accessed unconscious content, and that a relaxed meditative state is entered in making images: it gives the body voice while creating a concrete record of the process. This study, therefore, addresses a gap in both psychoneuroimmunological and psycho-oncology research. The objective was to assess if engaging in group art psychotherapy would have a positive effect on general quality of life with a reduction of ‘stress’ and if this would be reflected in immunological, psychosocial, image and narrative data. The specific parameters under investigation were salivary immunoglobulin isotype A (S-IgA), salivary interferon gamma (SIFN- γ), delayed type hypersensitivity test response, psychosocial questionnaire results, images created during art psychotherapy and the narrative of that group process. The study utilised a mixed methods approach. The quantitative arm of the study was framed within the positivist paradigm required for the assessment of discrete physiological and psychosocial parameters, while the qualitative arm of the study was framed in the interpretive paradigm suited to the investigation of subjective experience. The dominant philosophy informing the methodology for the qualitative work was hermeneutic phenomenology. Analysis of salivary immunoglobulin alpha (S-IgA) results indicated that there was a general upwards trend in the group mean post-vs-pre-session levels, positively correlating to sessional emotional tone. The salivary interferon gamma (S-IFN-γ) results showed no such increase, although there was a correlation to emotional content in individual sessions and an overall upward trend. There was an observable lessening of anxiety and improvement in interpersonal dynamics and participants’ self report over time, not reflected in the psychosocial questionnaire results. The study suggested that involvement in art psychotherapy does have a positive impact on immunological function, and has contributed to the understanding of a systems approach to healing, which may broaden understanding of the value of psychological support to people with cancer. This may be extrapolated to other chronic illnesses where immunological function must be optimised. The study has also demonstrated that it is possible to involve art psychotherapy in the scientific dialogue, without losing its integrity.

Thesis (M.Eng.). / Written for the Dept. of Biomedical Engineering. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Biochemistry." Includes bibliographical references (p. 123-133).

Thesis (doctoral)--København, 1981.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.