Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Texas at Austin, 2009. / Title from PDF title page (University of Texas Digital Repository, viewed on Sept. 9, 2009). Vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2009. / Härtill 4 uppsatser.

Thesis (M.S.)--Brigham Young University. Dept. of Integrative Biology, 2005. / Includes bibliographical references (leaves 21-27).

Honors Project--Smith College, Northampton, Mass., 2010. / Includes bibliographical references (p. 18-21, 54-60).

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr. Publié aussi en version électronique.

No description available.

There is a large body of clinical and experimental evidence linking heart disease with impairment of myocardial energetics, particularly the creatine kinase (CK) system. The goal of the experiments described in this thesis was to develop and study models of increased CK phosphotransfer, by overexpressing the CK isoenzymes and/or augmenting intracellular creatine stores. Pilot experiments were performed in cultured cells, which were used to (a) study the effects of CK overexpression in vitro, and (b) validate constructs prior to generation of transgenic mice. Expression was verified at the protein level for all constructs in HL-1 and HEK293 cells, and enzymatic activity was confirmed. Mitochondrial CK (CKmt) was expressed in the mitochondria, as expected, and CKmt overexpression was associated with a significant reduction in cell death in a model of ischemia/reperfusion injury (68.1 ± 7.1% of control, p≤0.05). Transgenic mice overexpressing CKmt in the heart were generated by a targeted approach, using PhiC31 integration at the ROSA26 locus. Transgene expression was confirmed in vitro in embryonic stem cells, and in vivo at the mRNA and protein levels. There was only a modest increase in CKmt activity; therefore, homozygous transgenic mice were generated to increase expression levels, and had 27% higher CKmt activity than wild-types (p≤0.01). Mitochondrial localization of CKmt was confirmed by electron microscopy. Citrate synthase activity, a marker of mitochondrial volume, was ~10% lower in transgenic mice (p≤0.05). Baseline phenotyping studies found that CKmt-overexpressing mice have normal cardiac structure and function. These mice are currently being backcrossed onto a pure C57BL/6 background for further studies in models of heart disease. In addition to CKmt, transgenic mice overexpressing the cytosolic CK isoenzymes, CK-M and CK-B, were generated. Due to the modest level of expression observed at ROSA26, random-integration transgenesis was used, and multiple lines were generated for each construct (carrying 2 or 6 transgene copies in the CK-M line; 2, 3, or ~30 in CK-B). Transgene expression was validated at the mRNA, protein, and activity levels. These lines are currently being expanded for further validation and phenotyping studies. Previous experiments in our group have demonstrated that increasing intracellular creatine (Cr) reduces ischemia/reperfusion injury, and a series of in vitro experiments was performed to determine whether this effect may be mediated by inhibition of the mitochondrial permeability transition pore (mPTP). The mPTP plays a significant role in ischemia/reperfusion, and there is evidence linking the CK system to regulation of the mPTP. Therefore, a model was developed to test whether Cr affects mPTP opening in cardiac-derived HL-1 cells, as this mechanism may contribute to the protective effect observed in vivo. Cr incubation conditions were determined empirically, and 24-hour incubation with 5mM or 10mM Cr was found to significantly delay mPTP opening, to a similar degree to the established mPTP inhibitor, cyclosporin A. This provides evidence that Cr may exert protective effects in the heart by a variety of mechanisms, in addition to its traditional role in energy metabolism. In summary, the experiments conducted in this thesis have produced a range of tools for studying augmentation of the creatine kinase system as a therapeutic target in heart disease. The results of in vitro assays indicate that mitochondrial CK may be a particularly promising target, and that inhibition of the mitochondrial permeability transition pore may contribute to the cardioprotective effect of creatine. Finally, the transgenic models generated and validated over the course of this project will allow for a wide range of future studies into the potential benefits of CK overexpression in the mammalian heart.

Spinal Muscular Atrophy (SMA) is caused by mutation or deletion of the Survival Motor Neuron 1 (SMN1), which encodes cell-ubiquitous SMN protein. Although classified as a neuromuscular disease, a range of systemic pathologies is reported in SMA patients. Despite a clear understanding of the genetics, the role of SMN protein in SMA pathogenesis is somewhat unclear, especially in tissues outside the CNS. Here, we describe failed liver development in response to reduced SMN levels, in a Taiwanese mouse model of severe SMA. Molecular analysis revealed significant changes in proteins involved in cell cycling and blood homeostasis including coagulation prior to motor neuron pathology. With SMN being directly associated with some of these proteins, this indicates primary liver pathology in SMA. Study of livers obtained from two other mouse models of SMA; severe SMNΔ7 and intermediate 2B, which have slightly higher SMN levels than Taiwanese SMA mice, also revealed significant overlapping pathologies, suggestive of high intrinsic susceptibility of the liver to SMN decrease. Proteomic study of pre-symptomatic 2B/- liver revealed significant perturbations in mitochondrial bioenergetics, which could account for metabolic defects in SMA patients. Vascular changes can be observed in mouse models of SMA and even skeletal muscle of severe SMA patients. Although Taiwanese SMA liver showed no morphological changes to its vasculature, it does have impairments in several key vascular signaling molecules, including VEGF and Tie-2. Furthermore, we report for the first time significant vascular changes in a zebrafish model of SMA, that could be associated with defective neuronal-vascular signaling and is supported by preliminary findings in the Taiwanese SMA retina. This thesis uncovers perturbations in several clinically relevant signalling pathways directly linked to SMN decrease, independent of the motor neurone pathology. Taken together this work emphasises the importance of a systemic therapy in SMA.

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.