A single application of cyclic compression to bioengineered cartilage improves tissue formation through cell shape changes that are mediated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if this response is controlled by calcium, we investigated how calcium regulated cell shape changes, MT1-MMP and integrin activity in response to stimulation. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished with chelation of extracellular calcium, and reinstated with its re-introduction. Spreading and retraction were inhibited by blocking the stretch-activated and L-Type voltage-gated channels, respectively; channel blocking also inhibited MT1-MMP upregulation. Channels’ role was confirmed through treatment with calcium A23187 ionophore, which alleviated the effects of channel blocking. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through the kinase Src. Both calcium- and integrin-mediated pathways converged on activating ERK in response to stimulation. Understanding the molecular mechanisms regulating chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

Glucocorticoids (GCs) are commonly used in the clinic as a treatment for Chronic Lymphocytic Leukemia (CLL). The exact mechanism of GC action remains unclear and patients eventually develop resistance to this group of agents. Our findings show that GC-cytotoxicity in circulating CLL cells is caused by bioenergetic restriction resulting from the down-regulation of a key glycolytic enzyme, pyruvate kinase, muscle isozyme 2 (PKM2). Conversely, GCs were shown to promote fatty acid oxidation instead by up-regulating the expression of peroxisome proliferator activated receptor α (PPARα). These findings establish PPARα and fatty acid oxidation as novel mediators of GC resistance in CLL. Our findings also demonstrate that GCs enhance the cytotoxic effects of membrane-damaging agents such as ionophores and complement-mediated cytotoxicity. A clinically relevant agent known to intercalate in the cell membrane, Danazol was also found to have activity against CLL and can be combined safely with GCs for enhanced treatment efficacy.

The role of antioxidants in cancer has been controversial for a long time. Although the public’s belief is that antioxidants prevent and/or inhibit cancer, there is increasing evidence to suggest the opposite: that cancer cells require antioxidants to survive. We wanted to interrogate the role of antioxidants in cancer by investigating both upstream regulators and downstream effectors of antioxidant signaling. We have identified protein tyrosine phosphatase non-receptor type 12 (PTPN12) as a novel regulator of antioxidant signaling in cancer. PTPN12 reduces reactive oxygen species (ROS) levels by promoting activity of the forkhead box O (FOXO) family of antioxidant transcription factors. We have also elucidated the impact of glutathione (GSH), the most abundant antioxidant in the cell, on tumorigenesis. We have found that GSH is required for cancer initiation, yet dispensable once transformation has occurred due to compensation provided by the thioredoxin (TXN) antioxidant pathway. Together, these studies expand our knowledge of the role of antioxidants in cancer and provide numerous avenues of research for the future.

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

Cardiovascular diseases, such as stroke and heart attack, are one of the largest causes of death and morbidity in Canada. Atherosclerosis, or the thickening of the arterial wall, has been identified as the primary culprit lesion behind the end organ damage associated with cardiovascular diseases. Magnetic resonance imaging has taken a primary role in characterising the constituents of these atherosclerotic plaques. Of these components, MR detected intraplaque haemorrhage (IPH), or bleeding inside the vessel wall, appears to predispose a patient to future clinical events. This leads us to the conclusion that IPH is a secondary manifestation of plaque progression and complication, or somehow contributes to the complication of these atherosclerotic plaques. This thesis explores this second possibility, in line with the “iron hypothesis” of atherosclerosis which suggests that iron plays a significant, primary role in atherogenesis. In chapter two, the signal hyperintensity associated with IPH is correlated with the lipid oxidising potential of blood products in various oxidation states. It is found that the ferric paramagnetic species has a significantly greater ability to generate lipid oxide components and oxidise lipid surrogates. This finding suggests that it may be possible to alter the course of plaque progression by inhibiting the active ferric iron state. Chapter three explores an endogenous molecule, haptoglobin, whose purpose is to bind and inactivate free haemoglobin. It is shown that haptoglobin has the ability to modulate the MR signal intensity from IPH. Chapter four explores a mechanism to detect this imaging biomarker outside the MR environment using the electron paramagnetic resonance of the ferric haemoglobin. Results are shown in a custom made bench top system detecting ferric haemoglobin in an in vitro sample. This thesis provides more evidence for the iron hypothesis of atherosclerosis and explores methods of inhibiting and detecting this biomarker of disease.

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.