Thesis (Ph. D.)--University OF MICHIGAN.

The recorded use of medical implants dates to before Christ. Synthetic cardiovascular implants, more specifically, have been widely used since the 1960s. While research and emerging technologies have achieved numerous advances in this latter field, the host body still recognizes many implants as "foreign" and initiates a healing response that can ultimately interfere with the long-term function of the prostheses. Particular to the area of synthetic vascular grafts, biological responses include platelet activation, thrombosis, and smooth muscle cell migration and proliferation. These reactions have been, and continue to be, characterized in relation to interpositional synthetic conduits. Translumenally placed endovascular grafts (EVGs), in contrast, are a relatively new treatment modality whose associated healing responses are, as yet, not well described. Endovascular implants are unique in that their surrounding environment can be quite different from that observed in association with open surgical procedures. The endovascular delivery process can preserve viable endothelium, reduce the surgical dissection of an artery, eliminate the placement of a foreign body deep in the arterial wall, and negate flow disturbances with straight inline arterial reconstruction. In addition, the apposition of the EVG to the native vessel is primarily an intimal as opposed to a medial or adventitial interface, potentially influencing the cells that initiate and impact all subsequent events in the healing response. To better characterize the cellular and angiogenic adaptations that occur in association with intimal approximations, experiments were performed to examine the healing responses related to both conventional grafts anastamosed with non-penetrating clips as well as to EVG placement. Research indicates that modulation of endothelial and smooth muscle cell (SMC) phenotypes during healing is a complex process that is likely regulated by multiple environmental cues. Soluble factor communication and cell substrate interactions are two likely signals that may ultimately influence the fate of the involved cells. Data demonstrate that endothelial cells are present and can be the initial cellular responders at the site of vascular intervention. In addition, these cells can directly contribute to neointimal thickening through cellular transmodulation. Alternatively, they can participate in process herein described as "differentiated vasculogenesis," whereby individual and/or groups of endothelial cells are liberated from the luminal monolayer into the provisional matrix that has been deposited around the vascular implants. Here they coalesce into non-patent vascular channels. Longer duration studies further specify that SMCs can be induced to re-attain a contractile state in association with low profile, highly porous endoluminal prostheses. Finally, experimental findings suggest that a preformed endothelial and SMC lining can be established in vitro within a tissue engineered vascular graft (TEVG). Implantation of the TEVGS, however, resulted in the dissolution and eventual re-population of the cellular constituents. Based on this work, it is evident that vascular wall responses to biomedical implants are far more complex than they have appeared in the past. Implant associated vascular wall healing can no longer be considered an ordered orchestration of SMC migration and proliferation followed by endothelial cell sheet migration over the neointimal lining. Future anti-stenosis and anti angiogenic therapies need to take into account the multi-factor/multi-cellular responses that can be involved.

Background. Several epidemiological studies have established an association between chlamydia pneumoniae (CP) and coronary artery disease. The aim of this study was to test the hypothesis that seropositivity to CP, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) is associated with decreased endothelial function in healthy young men. / Methodology. A convenience sample of 65 male volunteers, ages 20 to 45, with no known coronary atherosclerosis or risk factors for coronary artery disease were enrolled in a seroepidemiological cross-sectional study. / Conclusion. A lack of association between chronic infection to CP, CMV, and EBV with endothelial function was observed. This finding suggests that these infectious agents are not implicated as etiological triggers in the genesis of coronary artery disease but does not preclude active involvement at later stages of the pathophysiological process, such as acceleration of atherosclerosis and acute plaque rupture. (Abstract shortened by UMI.)

The impact of hepatitis C virus (HCV) coinfection on HIV related morbidity and mortality is debated, particularly in the era of highly active antiretroviral therapy (HAART). Using the Montreal Chest Institute HIV clinic database, a retrospective cohort spanning 1990--1999 was formed to determine the effect of HCV status on risk of opportunistic infection, death and hospitalisation before and after the introduction of HAART. While HCV- subjects experienced rate reductions for all outcomes after HAART, no such decrease was observed for HCV+ subjects. No effect of HCV status on outcomes was observed before HAART. In contrast, HCV infection was associated with an increased risk of death (RR, 1.80; 95% CI 0.88--3.65) and hospitalisation (RR, 1.90; 95% CI, 1.19--3.05) in the post HAART era after adjustment for age, duration of HIV infection, history of AIDS, antiretroviral use and time-updated CD4 cell and HIV viral load measures. In conclusion, HCV coinfection appears to be preventing the realisation of substantial health benefits associated with HAART.

The adult, competent patient has the ability to be involved in decisions regarding care and treatment. The critically ill patient, who is incompetent or incapacitated will be unable to speak for herself and yet, decisions will need to continue to be made. Decisions should reflect the values and beliefs of the patient. In health care we have determined that one of the best methods of preserving the autonomy of the patient through the reflection of values and beliefs is by involving a surrogate decision-maker. This thesis examines the many facets and factors that define the complex role of surrogacy. The role is described by reviewing literature on the current legal standards of decision-making, by analysing the data describing patient-surrogate preferences and the relevancy of such factors as culture and religion as facilitators or inhibitors in the decision-making process. The thesis suggests that a shared decision-making approach could provide the key to a partnership between the health care professional and the surrogates to assist in the preservation of patient autonomy.

Von Willebrand factor (vWF) is a large multimeric protein found in plasma, intracellular stores of platelets and endothelial cells, as well as in the extracellular matrix. VWF has been implicated in venous and arterial thrombosis. Its importance in the primary adhesion of platelets at sites of vascular injury, and for platelet aggregation at very high shear rates was clearly demonstrated by other investigators. We have investigated the role of vWF in the recruitment of platelets activated with low concentrations of agonist, such as may be found with partial ADP secretion or thrombin generation in vivo, at physiologic shear rates (G) ranging from 100--2000 s--1. / In the presence of ristocetin, soluble vWF bound to the glycoprotein Ib receptor (GPIb), and mediated shear-associated aggregation independently of the glycoprotein IIb-IIIa receptor (GPIIb-IIIa), with very few vWF monomer equivalents required to achieve high capture efficiencies (alphaG; reflecting initial rates of aggregation). Activation of washed platelets in the absence of soluble ligands, with low concentrations of the physiologic agonists, ADP or thrombin, resulted in good aggregation. Participation by GPIb was shearrate dependent, with the extent of contribution further varying with activation conditions. Inhibition of vWF-GPIb interactions in ADP and thrombin-induced aggregation, caused 25 and 50% inhibition of alpha G at G = 300 and 1000 s--1 respectively. alpha G's were similar for both agonists, and showed an absolute dependence on activated GPIIb-IIIa in each case. Further investigations using thrombin versus ADP as activator, however, revealed differences in participation by other surface-expressed ligands, in particular Fg. Thus, antibodies against TSP and Fg inhibited aggregation differentially, depending on shear rate and agonist activating conditions. Removal of catalytically-active thrombin from its receptors by antagonists hirudin and PPACK at ≥1 minute following activation, allowed normal secretion to occur from alpha-granules (monitored using P-selectin). However, the thrombin antagonists significantly decreased both platelet aggregation and surface-expression of vWF and TSP for platelets activated at low (0.05 U/ml), but not intermediate (0.2 U/ml) thrombin concentrations. In conclusion, all our studies demonstrated a consistently important cross-bridging role for vWF, but multi-ligand, multi-receptor participation was required for optimal shear-associated aggregation of platelet suspensions activated at very low agonist concentrations.

No description available.

No description available.

No description available.

According to the Canadian Cancer Statistics 2012 annual report, cancer remains a major cause of death, with an estimated 186 400 new cases, and 75 700 deaths. eIF4E is dysregulated in many cancers, including breast cancer. Our lab has considerable preliminary data to suggest that eIF4E, and specifically phosphorylated eIF4E, is involved in the Epithelial-Mesenchymal Transition (EMT) and metastasis. Moreover, Borden, K.L.B., et al., and our lab have recently published that the drug Ribavirin targets eIF4E and inhibits breast cancer cell growth. The main objective of this thesis project is to determine the impact of Ribavirin on EMT. Our data demonstrate that Ribavirin attenuates TGF-β1-induced EMT in NMuMG cells. At the morphological level, cells appear to adopt less of a mesenchymal phenotype, and this is associated with a reduction in expression of the mesenchymal marker, Fibronectin. Importantly, phenotypic changes associated with a TGF-β1-induced EMT could be inhibited by Ribavirin. Specifically, Pro-MMP9 levels are reduced in the presence of Ribavirin, and Ribavirin inhibits wound closure ability by 15%. Ribavirin also attenuates the induction of Snail by TGF-β1 stimulation. Interestingly, Ribavirin also reverts mesenchymal characteristics of mouse MT2186 and human MDA-MB-231 breast cancer lines. As well, Ribavirin inhibits cell invasion by 30% in MT2186 and by 65% in MDA-MB-231 cells. At the molecular level, we provide evidence that Ribavirin may exert its anti-metastatic activity by attenuating the expression of Snail. Snail is a transcription factor and EMT regulator, leading to increased invasiveness and decreased cell proliferation. When we overexpress Snail in MDA-MB-231 cells, this reduces their cell proliferation by 50% and antagonizes Ribavirin's ability to decrease cell growth. Our preliminary data show that Ribavirin can inhibit invasion of MT2186 and MDA-MB-231 cells. However, Ribavirin's ability to inhibit Matrigel invasion is abrogated by overexpressing Snail in MDA-MB-231 cells. In addition, we also provide evidence that Snail is regulated by phosphorylated eIF4E using MEF cells with a non-phosphorylated form of eIF4E, derived from eIF4E Serine 209 Alanine knock-in mice. Together, the results presented in this thesis warrant further investigation into the anti-metastatic activity of Ribavirin in breast cancer. To this end, our lab is planning on testing the efficacy of Ribaivirin in in vivo models of breast cancer metastasis. / Selon le rapport annuel 2012 de Statistiques Canadiennes sur le Cancer, le cancer demeure une cause majeure de décès, avec 186 400 nouveaux diagnostics et 75 700 décès. Il y a perturbation de la fonction de la protéine eIF4E dans plusieurs de ces cas, incluant dans le cancer du sein. Notre laboratoire a accumulé un nombre considérable d'évidences préliminaires qui suggèrent qu'eIF4E, et spécialement sa protéine phosphorylée, joue un rôle dans la transition épithélio-mésenchymateuse (EMT) et dans la formation des métastases. De plus, nous avons récemment publié que le Ribavirin cible eIF4E et inhibe la croissance des cellules du cancer du sein. L'objectif principal de ce projet de thèse de maîtrise consistait à déterminer les effets du Ribavirin sur l'EMT. Nos données montrent que le Ribavirin atténue l'induction par le TGF- β1 de l'EMT dans les cellules NMuMG. La morphologie des cellules s'éloigne alors du phénotype mésenchymial, et une réduction de l'expression du marqueur mésenchymial Fibronectin est observé. D'une façon importante, les changements de phénotype normalement associé à l'EMT engendrée par le TGF- β1 sont inhibés par le Ribavirin. Les niveaux de pro-MMP9 sont notamment diminués en présence de Ribavirin, alors que celui-ci diminue aussi la capacité de guérison d'une plaie de 15%, telle que mesurée par la migration cellulaire. Par ailleurs, alors que TGF- β1 augmentent les niveaux d'ARNm de Snail, Ribavirin les diminue. Aussi, le Ribavirin réussi à modifier les caractéristiques mésenchymiales des cellules murines MT2186 et humaines MDA-MB-231 en les ramenant vers un phénotype similaire à celui d'une cellule épithéliale. Aussi, le Ribavirin inhibe de 30% et de 65% l'invasion cellulaire dans les cellules MT2186 et MDA-MB-231, respectivement. Pour ce qui est de l'effet du Ribavirin au niveau moléculaire, nos observations nous portent à croire que celui-ci diminue la formation de métastases en atténuant l'expression de Snail. Snail est un facteur de transcription qui module l'EMT en augmentant l'invasion et la prolifération cellulaire. La surexpression de Snail induit d'ailleurs une diminution de 50% de la prolifération cellulaire dans les cellules MDA-MB-231 et empêche le Ribavirin de diminuer la croissance cellulaire. Nos données préliminaires montrent que le Ribavirin peut inhiber l'invasion cellulaire des cellules MT2186 et MDA-MB-231. Cependant, la surexpression de Snail dans les cellules MDA-MB-231 empêche le Ribavirin d'inhiber l'invasion des ces cellules sur Matrigel. En plus, nous avons démontrés que Snail est régulé par la forme phosphorylée d'eIF4E en employant un modèle cellulaire de MEF présentant une forme non phosphorylée d'eIF4E. Ce modèle est dérivé d'une souris knock-in pour eIF4E (Serine 209 Alanine). En conclusion, l'ensemble des résultats présentés démontrent le besoin d'études supplémentaires pour éclaircir l'activité anti-métastasique du Ribavirin dans le cancer du sein. Notre laboratoire planifie d'ailleurs d'évaluer son efficacité in vivo chez un modèle murin de cancer du sein métastasique.