Le but de ce travail était d'étudier l'activité anti-leucémique de UNBS1450, un cardénolide semi-synthétique appartenant à la famille des glycosides stéroïdes cardiaques, et qui a été démontré d'être doué d'un puissant potentiel inducteur d'autophagie dans les cellules cancéreuses issues de tumeurs solides. Par cette étude, nous démontrons pour la première fois que des concentrations nanomolaires de UNBS1450 exercent un effet pro-apoptotique sur des cellules leucémiques humaines. Par conséquent, nous avons élucidé les mécanismes moléculaires impliqués : nos résultats mettent en évidence une inhibition de la transactivation de NF-kB et une induction de l'apoptose par clivages des pro-caspases 8,9 et 3/7, en réprimant l'expression de Mcl-1 anti-apoptotique et en recrutant Bak et Bax, deux protéines pro-apoptotiques, aboutissant ainsi à une mort cellulaire apoptotique / The aim of this study was to investigate the anti-leukemic activity of UNBS1450, a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family, known to be a potent autophagy inducer in solid tumor cells. Interestingly, we hereby report for the first time that at low nanomolar concentrations, UNBS1450 induces apoptotic cell death in human leukemia cell lines. Subsequently, we have investigated the molecular mechanisms induced : Our results show that UNBS1450 inhibits NF-?B transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9 and 3/7, by decreasing the expression of anti-apoptotic Mcl-1 and by recruiting pro-apoptotic Bak and Bax eventually resulting in cell death

Il a été suggéré que les Desulfovibrio, qui sont des bactéries anaérobies sulfato-réductrices, pourraient être impliqués dans les parodontites. Nous avons évalué le pouvoir invasif de Desulfovibrio vis-à-vis de cellules épithéliales et leur capacité à induire la production de cytokines par ces cellules. Nous avons montré que Desulfovibrio desulfuricans et Desulfovibrio fairfieldensis sont capables d'envahir et de se multiplier dans les cellules épithéliales buccales (cellules KB). La localisation intracytoplasmique de ces deux bactéries a été confirmée par microscopie confocale et électronique à transmission. L'internalisation de ces souches était dépendante de la polymérisation des microtubules mais pas de celle de l'actine. L'infection avec Desulfovibrio était responsable d'une augmentation de la production d'IL-6 et d'IL-8 par les cellules KB. La capacité de D. desulfuricans et de D. fairfieldensis à survivre dans les cellules épithéliales et à moduler leur réponse immunitaire pourrait contribuer au développement des maladies parodontales. Desulfovibrio ainsi que d'autres parodontopathogènes peuvent produire des ß-lactamases et sont capables d'envahir les cellules épithéliales. Il a été suggéré que l'hydrolyse de l'amoxicilline pourrait être évitée grâce à l'utilisation d'un complexe amoxicilline-ß-cyclodextrine (ßCD) et que la diffusion intracellulaire d'agents antimicrobiens pourrait être améliorée après complexation avec des ßCD. Un complexe stable amoxicilline-ßCD, caractérisé après analyse spectrale et thermique, n'a ni amélioré l'activité de l'amoxicilline vis-à-vis de souches produisant des ß-lactamases ni augmenté la diffusion intracellulaire de ce composé. / It has been suggested that Desulfovibrio, which are sulfate-reducing anaerobic bacteria, may be involved in periodontitis. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. We showed that Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to invade and to multiply within oral epithelial cells (KB cells). Intracytoplasmic location of both bacteria was confirmed by confocal laser scanning and transmission microscopy. Invasion of these strains involved microtubule but not microfilament polymerization. Infection by Desulfovibrio resulted in an increase of the production of IL-6 and IL-8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases. Desulfovibrio as well as other periodontopathogens may produce ß-lactamases and have the capacity to invade epithelial cells. It has been suggested that the hydrolysis of amoxicillin might be prevented by using an amoxicillin-ß-cyclodextrin (ßCD) complex and that intracellular diffusion of antimicrobial agents might be enhanced after complexation with ßCDs. A stable [1:1] amoxicillin-ßCD complex, characterized by spectroscopic and thermal analysis, did neither improve the activity of amoxicillin against ß-lactamase producing strains nor enhance the intracellular diffusion of this compound.

Obesity is a known risk factor for many types of cancer including pancreatic. Calorie restriction (CR), an anti-obesity diet regimen, has potent anticancer effects that may be mediated through its ability to reduce serum metabolic hormones and protumorigenic cytokines such as insulin-like growth factor (IGF)-1. IGF-1 is a metabolic hormone responsive to nutrient status that activates the inflammatory, cancer-related pathway, nuclear factor (NF)-[kappa]B. For this report, we tested the hypothesis that CR, via regulation of IGF-1, inhibits pancreatic tumor cell growth through modulation of NF-kB activation and protumorigenic gene expression. Male athymic nude mice were randomized to either a control diet consumed ad libitum (n=15) or a 30% CR diet (n=15) for 17 weeks, at which time, mice were injected with human pancreatic cancer cells (MiaPaca) and tumor growth was monitored for 6 weeks. Translocation of p65, a regulatory element of NF-[kappa]B, and expression of its downstream gene targets were analyzed in excised tumors. CR mice weighed less, (p<0.05), and had smaller tumors (p=0.022) relative to controls. Tumors from CR mice, relative to controls, demonstrated significant decreases in NF-[kappa]B downstream genes CCND1, RELA, Survivin, VEGF, and XIAP. These findings parallel our previous studies in pancreatic tumors from mouse origin, and suggest that the inhibitory effects of CR on MiaPaca pancreatic tumor growth are associated with decreased NF-kB activation. / text

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