Altered apoptotic signaling in skeletal muscle has been observed in a number of disease states associated with skeletal muscle atrophy. Therefore, understanding the mechanisms that lead to increased skeletal muscle apoptosis may help to prevent the atrophy associated with various diseases. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is able to inhibit apoptosis mediated by both the death-receptor and mitochondrial pathways. In addition, ARC has a unique distribution pattern and is highly expressed in terminally differentiated tissue such as skeletal muscle. To characterize the role of ARC in skeletal muscle morphology and apoptosis, soleus and plantaris muscles of 18 week-old ARC-deficient mice were excised and compared to those of age-matched wild-type littermates. While no differences were seen in muscle weights between genotypes, in the ARC KO animals, the cross-sectional area (CSA) of the soleus was smaller, while the CSA of the plantaris was larger. With respect to fiber type distribution, both muscles demonstrated a shift towards a faster myosin heavy chain expression pattern. For example, soleus muscles of ARC KO animals had significantly less type I fibers and more IIa fibers, while plantaris muscles had significantly less type IIa fibers, and more IIb fibers. In ARC KO animals, type I and IIa fibers were significantly smaller in the soleus, while type IIb fibers were larger in the plantaris. DNA fragmentation (a hallmark of apoptosis) was increased in the soleus, but not plantaris muscles of ARC KO animals. Surprisingly, activity of the proteolytic enzymes caspase-2, -3, -8, and -9, as well as calpains, was not different in either soleus or plantaris muscles. To determine whether a lack of ARC protein affects apoptotic signaling in skeletal muscle, the total expression of pro- and anti-apoptotic proteins were also assessed. In the soleus, no changes were observed in whole tissue AIF, cytochrome c, EndoG, and Smac. In the plantaris, there was no change in total muscle AIF; however, there were trends towards decreased cytochrome c, and increased Smac, as well as a significant decrease in EndoG ARC KO animals. No changes were observed in Bcl-2 and XIAP in the soleus; however, there were significant reductions in FLIP(s) and HSP70 content. In the plantaris, no changes were observed in anti-apoptotic protein content. Subcellular fractionation of red quadriceps for ARC KO mice revealed an increased Bax:Bcl-2 ratio in the isolated mitochondrial fractions. Furthermore, in cytosolic fractions of red quadriceps, AIF protein content was significantly increased in ARC KO animals. Conversely, no changes in apoptotic-related protein content were observed in any fractions from white quadriceps between groups. In agreement with these findings, isolated mitochondria from ARC-deficient animals were more susceptible to calcium induced swelling, as well as membrane potential loss compared to controls. Taken together, these results suggest that in slow-oxidative skeletal muscle of ARC-deficient mice there is increased apoptosis due to caspase-independent, mitochondrial-mediated apoptotic signaling. Furthermore, this study is the first to show ARC plays an important role in skeletal muscle morphology, as ARC KO mice have an altered skeletal muscle phenotype and morphology.

Apoptosis plays important roles in host defences against virus infection. It is therefore not surprising that viruses have developed a vast array of modulators that block this process at different stages within the apoptotic pathways. Intrestingly, Orf virus (ORFV), a member of the Parapoxvirus genus, did not reveal any of the known poxviral inhibitors of apoptosis, but was found to express a unique anti-apoptotic protein, ORFV125. The aim of this PhD project was to determine the subcellular localisation of this protein and to further characterise its anti-apoptotic activity. This included exploring its ability to inhibit early, intermediate and late events of apoptosis and identifying the mechanism by which this viral protein functions to prevent cell death. Experiments revealed that ORFV125 was localised to the mitochondria through a C-terminal mitochondrial-targeting motif, and this specific location was necessary for the protein�s anti-apoptotic function. Furthermore, the viral protein inhibited UV-induced apoptotic events at and downstream of the mitochondria such as cytochrome c release, caspase activation and DNA fragmentation. However, it was not able to prevent UV-induced activation of the c-Jun-NH₂ kinase (JNK), an event occurring upstream of the mitochondria, consistent with its localisation to this organelle. The ability to prevent apoptosis was comparable with that of the cellular anti-apoptotic protein Bcl-2, which belongs to a family of mitochondrial regulators of apoptosis. Although standard BLAST analysis failed to detect homology to anti-apoptotic members of the Bcl-2 family, a manual alignment of the primary sequence of ORFV125 with these proteins revealed characteristic residues of Bcl-2 homology (BH) domains within ORFV125. These motifs are conserved within the Bcl-2 proteins and important for their structure and function. In addition, mutating amino acids within the ORFV125 BH domains led to a loss of the anti-apoptotic function of the mutated proteins, indicating the functional importance of these residues for the viral protein. These observations suggest that ORFV125 might be classified as a viral Bcl-2-like protein. To provide evidence for this hypothesis, it was investigated if ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. The viral protein was able to entirely block the activation of the pro-apoptotic Bcl-2 family members Bak and Bax, although it did not directly bind to these proteins. Instead, ORFV125 interacted with a subset of the pro-apoptotic BH3-only proteins, which can trigger the activation of Bax and Bak. Furthermore, this study demonstrated that ORFV125 could inhibit apoptosis induced by BH3-only proteins to which the viral protein could bind. On the other hand, ORFV125 was not able to prevent the activity of pro-apoptotic proteins that it failed to interact with. This shows that ORFV125�s mechanism of action is to inhibit the activity of BH3-only proteins by binding and neutralising their function. Overall, these results provided evidence that ORFV125 is potent anti-apoptotic protein that can prevent UV-induced cell death without the participation of other ORFV proteins. Furthermore, the viral protein shared primary sequence and secondary structure similarities to Bcl-2 family members and acted in a Bcl-2-like manner to inhibit apoptosis.

Angiogenesis is an essential physiological process involved in wound repair, endometrial growth and embryogenesis and is tightly regulated in normal tissues. However, angiogenesis is also associated with some pathological conditions; in these conditions, angiogenesis eludes regulation and presents as either unabated (as in diabetic retinopathy and neoplasia), or as an inefficient process (as in ischaemic coronary disease). Regardless of this, all angiogenic mechanisms have a common biological basis, ergo, the shift of anti-angiogenic research has been towards biologically-based angiogenesis inhibitors; it is well established that these anti-angiogenic paradigms can be applied to inhibit the growth of solid neoplasms. / Antagonists of tumour-associated angiogenesis from a diverse range of sources have been identified and analysed, including agents capable of eliciting an apoptotic response in the endothelial cells lining the tumour vasculature. Tumour-associated microvascular endothelial cells, although derived from normal, host endothelial cells, exhibit differential characteristics which are able to be exploited, in order to induce endothelial cell apoptosis in tumour vessels, while normal, host vessels remain unaffected. / Thesis (PhDBiomedicalScience)--University of South Australia, 2005.

Although exposed to similar environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. A model involving immunization and aerosol challenge with ovalbumin (OVA) was used to investigate factors that may confer strain-dependent resistance or susceptibility to pathology. BALB/c and C57BL/6 mice developed many features of human asthma including inflammation, mucus production and airway obstruction. In contrast, CBA/Ca mice were relatively resistant to development of disease. This was despite the presence of a robust systemic allergic response, as indicated by high levels of OVA-specific and total immunoglobulin and increases in circulating eosinophils comparable to those in BALB/c and C57BL/6 mice. In interleukin (IL)-5 transgenic (Tg) mice the strain specific susceptibility to lung mucus production and airway obstruction was maintained and pathology was greatly accentuated in C57BL/6 and BALB/c but not in CBA/Ca mice. Eosinophils recovered by bronchoalveolar lavage (BAL) from wt and IL-5 Tg CBA/Ca mice lost viability faster than BAL eosinophils from the other two strains and this phenomenon was lung-specific. This may result in less eosinophil accumulation in the lungs of CBA/Ca mice and resistance to asthma-like pathology. Fl hybrids of CBA/Ca mice crossed with either BALB/c or C57BL/6 mice had BAL leukocyte, eosinophil lifespan and cell-free protein profiles similar to those of the respective disease-susceptible parental strains. It is likely that eosinophil apoptosis was not mediated through the extrinsic or receptor mediated pathway. Bcl-2 and Bcl-xL, which both inhibit the intrinsic pathway of apoptosis were highest in BAL eosinophils from the BALB/c strain and this correlated with relatively high IL-5 levels in the lungs. Survivin inhibits apoptosis and expression was significantly higher in BALB/c and C57BL/6 BAL eosinophils than in cells from CBA/Ca mice. This suggests a possible mechanism whereby eosinophils from the asthma-susceptible C57BL/6 and BALB/c mice are more resistant to apoptosis and may account, in part, for the more extensive pathology in these strains. Using global gene expression analysis we identified groups of genes that were differentially regulated in the lungs of mice that are susceptible or resistant to development of asthma-like pathology. 242, 145 and 42 genes were differentially regulated in the lungs of the C57BL/6, BALB/C and CBA/Ca strains respectively. In C57BL/6 mice, transcripts were significantly enriched for adhesion molecules and we postulate that heightened expression of L-selectin, CD 18, PGSL-1 and LPAM-l on lung eosinophils is responsible for robust recruitment and therefore accumulation of these cells in C57BL/6 mice. 64 genes were differentially regulated only in the asthma-susceptible strains, several of which have not previously been associated witb asthma. The late expression of Chi313, Retnla and Mmp12 correlated with increased expression of IL-10 in the lungs and we hypothesise that this cytokine may be produced by alternatively activated macrophages as part of the resolution of disease. This study identifies several novel genes and mechanisms associated with the modulation of airway inflammation and pathology. The identification of factors that control allergic inflammation may provide novel therapeutic targets for disease intervention. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1366239 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Title page, table of contents and introduction only. The complete thesis in print form is available from the University of Adelaide Library. / Apoptosis and inflammation have been considered to be linked mechanisms. Defective apoptosis may result in cell accumulation and prolonged half life of inflammatory cells in rheumatoid arthritis (RA). A similar phenomenon is seen in malignancy. Therefore, defects in apoptosis pathways that have been proposed to contribute to the pathogenesis of malignancy may also be seen in RA. It has been reported by many studies that treatments that induce apoptosis of malignant cells are advantageous for cancer treatment and this suggests that targeting apoptosis pathways may be important in the management of a variety of pathologies that involve abnormalities in cell proliferation. While apoptosis induction has been a common mechanism in the treatment of cancer, it has only recently been seriously considered to be effective in regulating proliferative cells in inflammation. This thesis provides important information regarding the TRAIL mediated pathway of apoptosis and possible factors that modulate the ability of this pathway to induce apoptosis in RA. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1286764 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2007

In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.

ApoE is a polymorphic protein that has been found to play many different roles in biological processes including lipid transport, neurobiology and immunoregulation. ApoE occurs in the human population in three major isoforms; apoE2, apoE3 and apoE4. The apoE4 isoform has been identified as a major risk factor for several diseases including atherosclerosis and Alzheimer's disease, therefore a greater understanding of apoE biology is highly sought after. In my thesis, I have investigated several novel aspects of apoE biology. I have identified an association between increased apoE expression and apoptosis in a neuronal cell type and demonstrated that apoE becomes enriched within the neuronal apoptotic debris, consistent with a possible role for apoE in facilitating apoptotic debris clearance. A possible anti-apoptotic role of apoE in macrophages was assessed by reducing or eliminating apoE expression using siRNA and cells isolated from apoE knockout animals, respectively. The removal of apoE did not alter overall sensitivity to apoptosis, however, it did significantly increase staurosporine-induced caspase-3 activation. In other studies, the poorly understood accumulation of apoE within the nucleus was found to be enhanced during serum starvation and to localise in intra-nuclear structures that are distinct from inter-chromatin granule clusters. Analysis of apoE within the human brain revealed a correlation between fragmentation and the apoE3 isoform which was independent from AD status and brain region examined. Additionally, a portion of brain apoE3 was found to be present in the form of disulphide-linked dimers. Collectively, these studies have further expanded the current knowledge of apoE biology in terms of its association with apoptosis, nuclear localization and structural differences between the apoE3 and apoE4 isoforms in the human brain.

Angiogenesis is an essential physiological process involved in wound repair, endometrial growth and embryogenesis and is tightly regulated in normal tissues. However, angiogenesis is also associated with some pathological conditions; in these conditions, angiogenesis eludes regulation and presents as either unabated (as in diabetic retinopathy and neoplasia), or as an inefficient process (as in ischaemic coronary disease). Regardless of this, all angiogenic mechanisms have a common biological basis, ergo, the shift of anti-angiogenic research has been towards biologically-based angiogenesis inhibitors; it is well established that these anti-angiogenic paradigms can be applied to inhibit the growth of solid neoplasms. / Antagonists of tumour-associated angiogenesis from a diverse range of sources have been identified and analysed, including agents capable of eliciting an apoptotic response in the endothelial cells lining the tumour vasculature. Tumour-associated microvascular endothelial cells, although derived from normal, host endothelial cells, exhibit differential characteristics which are able to be exploited, in order to induce endothelial cell apoptosis in tumour vessels, while normal, host vessels remain unaffected. / Thesis (PhDBiomedicalScience)--University of South Australia, 2005.

Although exposed to similar environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. A model involving immunization and aerosol challenge with ovalbumin (OVA) was used to investigate factors that may confer strain-dependent resistance or susceptibility to pathology. BALB/c and C57BL/6 mice developed many features of human asthma including inflammation, mucus production and airway obstruction. In contrast, CBA/Ca mice were relatively resistant to development of disease. This was despite the presence of a robust systemic allergic response, as indicated by high levels of OVA-specific and total immunoglobulin and increases in circulating eosinophils comparable to those in BALB/c and C57BL/6 mice. In interleukin (IL)-5 transgenic (Tg) mice the strain specific susceptibility to lung mucus production and airway obstruction was maintained and pathology was greatly accentuated in C57BL/6 and BALB/c but not in CBA/Ca mice. Eosinophils recovered by bronchoalveolar lavage (BAL) from wt and IL-5 Tg CBA/Ca mice lost viability faster than BAL eosinophils from the other two strains and this phenomenon was lung-specific. This may result in less eosinophil accumulation in the lungs of CBA/Ca mice and resistance to asthma-like pathology. Fl hybrids of CBA/Ca mice crossed with either BALB/c or C57BL/6 mice had BAL leukocyte, eosinophil lifespan and cell-free protein profiles similar to those of the respective disease-susceptible parental strains. It is likely that eosinophil apoptosis was not mediated through the extrinsic or receptor mediated pathway. Bcl-2 and Bcl-xL, which both inhibit the intrinsic pathway of apoptosis were highest in BAL eosinophils from the BALB/c strain and this correlated with relatively high IL-5 levels in the lungs. Survivin inhibits apoptosis and expression was significantly higher in BALB/c and C57BL/6 BAL eosinophils than in cells from CBA/Ca mice. This suggests a possible mechanism whereby eosinophils from the asthma-susceptible C57BL/6 and BALB/c mice are more resistant to apoptosis and may account, in part, for the more extensive pathology in these strains. Using global gene expression analysis we identified groups of genes that were differentially regulated in the lungs of mice that are susceptible or resistant to development of asthma-like pathology. 242, 145 and 42 genes were differentially regulated in the lungs of the C57BL/6, BALB/C and CBA/Ca strains respectively. In C57BL/6 mice, transcripts were significantly enriched for adhesion molecules and we postulate that heightened expression of L-selectin, CD 18, PGSL-1 and LPAM-l on lung eosinophils is responsible for robust recruitment and therefore accumulation of these cells in C57BL/6 mice. 64 genes were differentially regulated only in the asthma-susceptible strains, several of which have not previously been associated witb asthma. The late expression of Chi313, Retnla and Mmp12 correlated with increased expression of IL-10 in the lungs and we hypothesise that this cytokine may be produced by alternatively activated macrophages as part of the resolution of disease. This study identifies several novel genes and mechanisms associated with the modulation of airway inflammation and pathology. The identification of factors that control allergic inflammation may provide novel therapeutic targets for disease intervention. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1366239 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Title page, table of contents and introduction only. The complete thesis in print form is available from the University of Adelaide Library. / Apoptosis and inflammation have been considered to be linked mechanisms. Defective apoptosis may result in cell accumulation and prolonged half life of inflammatory cells in rheumatoid arthritis (RA). A similar phenomenon is seen in malignancy. Therefore, defects in apoptosis pathways that have been proposed to contribute to the pathogenesis of malignancy may also be seen in RA. It has been reported by many studies that treatments that induce apoptosis of malignant cells are advantageous for cancer treatment and this suggests that targeting apoptosis pathways may be important in the management of a variety of pathologies that involve abnormalities in cell proliferation. While apoptosis induction has been a common mechanism in the treatment of cancer, it has only recently been seriously considered to be effective in regulating proliferative cells in inflammation. This thesis provides important information regarding the TRAIL mediated pathway of apoptosis and possible factors that modulate the ability of this pathway to induce apoptosis in RA. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1286764 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2007