Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

Ovarian cancer is the deadliest gynaecologic cancer in New Zealand. Its high mortality rate is due to the fact that it is usually diagnosed at an advanced stage. Advanced ovarian cancer is less responsive to current cytotoxic treatment. Thus, there is an urgent need for novel anti-cancer drugs that can improve patient longevity and quality of life. One of the clinical features of advanced ovarian cancer is the growth of secondary tumours due to the highly metastatic nature of the disease. Cancer cells disseminate from the ovary, some form cell clusters that travel through the abdominal cavity by physiological movement of body fluid and then deposit on the abdominal wall and internal organs to generate secondary tumours. The exact mechanisms of how these cells metastasize are unclear, but prognosis typically worsens if levels of vascular endothelial growth factor (VEGF) are elevated. This study investigated the anti-tumour activities of naturally occurring food compounds resveratrol, acetyl resveratrol and (-)-Epicatechin-3-gallate (EGCG), in cell spheroids/clusters of ovarian cancer. It also examined the protein expression of various proteins involved in the NF-κB signalling pathway. This pathway has been suggested to mediate the secretion of VEGF and is a possible target for the naturally occurring compounds. Results show that resveratrol and acetyl resveratrol reduce cell growth and cellular metabolism in a dose-, time- and cell line- dependent fashion. In addition, the reduction of VEGF is also dose-, time- and cell line- dependent. Paradoxically, another angiogenic protein interleukin-8 (IL-8) secretion is increased. Resveratrol and acetyl resveratrol attenuate the expression of NF-κB but this effect is cell line specific. EGCG has limited effect on cell growth, cellular metabolism and the secretion of VEGF and IL-8. These findings suggest that resveratrol and its derivative may have the ability to supress the angiogenic activity of ovarian cancer cells and warrant further in vivo study.

Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

Thesis (Ph. D.)--University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Includes bibliographical references (p. 65-80).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22738号 / 医博第4656号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Tumor-distal immune suppression promotes tumor growth by preventing the recruitment of leukocytes to the tumor-proximal microenvironment. Tumor necrosis factor (TNF)-a is both secreted by and expressed on the cell-surface (mTNF-a) of macrophages. When stimulated with LPS, tumor-burdened host (TBH) macrophages secrete more TNF-a than normal host (NH) macrophages. In this study, I showed that mTNF-a is elevated both in freshly isolated and stimulated TBH macrophages. Additionally, I analyzed the expression of Fas and FasL on freshly isolated and LPS-stimulated macrophages and found no differences between TBH and NH macrophages. Fas and Fas ligand (FasL) cell-surface expression was analyzed on NH and TBH T-cells. While no difference was observed in freshly isolated cells, cell-surface expression of both proteins remained higher in TBH T-cells than NH T-cells after mitogenic stimulation. Fas and FasL analysis was also extended to the MethKDE fibrosarcoma and I found that these tumor cells express high levels of FasL. Because past observations show increased TNF-a mRNA expression in TBH macrophages relative to NH macrophages, I hypothesized that NF-kB activation may be increased as well. NF-kB is a transcription factor whose activation is required for TNF-a transcription. I observed increased NF-kB activation in both splenic and peritoneal TBH macrophages. Interestingly, electrophoretic mobility shift analysis (EMSA) suggests that different species of NF-kB were found in each distinct population of macrophages. Together, these data demonstrate that cell-surface tumoricidal molecules and NF-kB are dysregulated in the tumor-burdened host. / Master of Science

No description available.

No description available.

No description available.