Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

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Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

The cardiac wall has complex three-dimensional fiber structures and mechanical properties that enable the heart to efficiently pump the blood through the body. By studying the myocardial strains induced during diastole, information about the pumping performance of the heart and what mechanisms that are responsible for this effective blood filling, can be achieved. Two different computation methods for myocardial strain, both based on data acquired from marker technique, were compared using a theoretical cylinder model. The non-homogeneous polynomial fitting method yielded higher accuracy than a homogeneous tetrahedron method, and was further used to investigate cardiac and fiber strains at different wall depths and myocardial regions in normal and ischemic ovine hearts. Large spatial and regional variations were found, as well as alterations, conveyed by ischemic conditions, of fiber mechanisms responsible for the circumferential expansion and wall thinning during diastole.

<p>Hypoxic ischemic encephalopathy (HIE) is a serious birth complication of full term infants; 40-60% of affected infants die by 2 years or have severe disabilities. Infants with HIE often have a normal gestation and parents anticipate a healthy birth. HIE can be managed with aggressively with moderate hypothermia < 6 hours of life, cardiopulmonary support, and seizure management. Experimental interventions such as moderate hypothermia > 6 hours of life and umbilical cord stem cell transplant are also available. Additional decision-making for these infants may include long-term developmental therapy, nutritional support, and respiratory support. However, who makes these decisions, what factors influence decision-making and the long-term impact of decision-making on parents and health care providers remains unknown. Therefore, the purpose of this study was to explore parental and health care provider decision-making for infants with HIE.</p><p>A longitudinal case study design was used to study 11 cases of infants with HIE. Each case included the infant, the parent, and the infant's providers. Infant medical record data, interviews and questionnaires were used to collect data from infant birth through 6 months of age. Content analysis was used to analyze the interviews. Descriptive statistics were used with the questionnaires. Visualization techniques were used to search for patterns and trends in the assembled data. </p><p>All infants required resuscitation and their treatment plans included aggressive care or aggressive and experimental care. The level of parental participation varied with in the first week of life depending on whether the infant was enrolled in experimental interventions plus aggressive care or only aggressive care. Parental hopefulness was lower in parents of infants who received experimental interventions, but the infants receiving experimental interventions were less critically ill than infants who received aggressive care only. Parental stress was also lower among parents of infants who received experimental interventions over the first 2 months of life. </p><p>Parents were concerned about the short and long-term impact of HIE, few parents understood that even though their infant had appropriate developmental outcomes at 6-months that did mean that neurological damage occurred. However in one case of an infant, the neurological development became central to the parental decision-making for the infant. Parents became less hopeful as diagnostic examinations continued find more complex conditions that were individually not problematic for the parents, but when the complexity of the infant's illnesses continued to unfold, parents feared that too many complications existed for their daughter to have an acceptable quality of life. Yet, when parents broached the topic of transitioning from aggressive care to palliative care with providers, they were told that withholding/withdrawing treatment was not appropriate for the infant. Not discussing withholding or withdrawing treatment ultimately created conflict between parents and providers due to differences in opinions about the predicted neurological outcomes for the infant. The conflict led to distrust and parents regretted most decisions they made for their infant. </p><p>Parental and provider decision-making is complex and many of the decisions within the 6-month trajectory were made within the first 6 hours of birth. Parents felt that the decision-making was appropriate in most cases, but the extent of the infant's injury remains unknown. How parents will evaluate the decision-making when the infant begins to miss developmental milestones is unknown. Results from this dissertation suggest that decision-making is a trajectory and decisions are not made in isolation. Implications for practice include discussing and educating parents during the first 6 months and later about developmental milestones and the importance of continuing therapy, even when the infant appears normal. Providers can also acknowledge to parents, up front, that the extent of the neurological injury is unknown and different providers may have different opinions about the long-term effects. By acknowledging these differences, providers can begin discussing the treatment options with parents and educating them about the specific needs of their infant.</p> / Dissertation

Stroke is an important public health problem and the third leading cause of death after coronary heart diesase and all cancers in all over the world. Free radicals and oxidative stress play important role in the pathogenesis of several diseases including atherosclerosis, stroke, cancer, neurodegenerative diseases such as Alzheimer&#039 / s dementia. The activity of paraoxonase (PON1) aganist phenylacetate is known as arylesterase (ARE). Paraoxonase is an esterase associated with high-density lipoprotein (HDL) and contributes to the protective role of this lipoprotein on low-density lipoprotein (LDL) oxidation. Oxidized LDL is known to play a central role in early events in the progression of atherosclerosis which is a risk factor for stroke. Glutathione S-transferases (GSTs) catalyze the conjugation of nonpolar compounds to reduced glutathione (GSH) and detoxify toxic metabolites produced within the cell by oxidative stress to protect cells from oxidant injury. v The maximum ARE enzyme activity was detected at 10 mM Tris-HCl buffer, pH 8.0 and at 45 &deg / C. ARE enzyme was saturated with its substrate phenylacetate around 20 mM concentration. The apparent Km and Vmax values of human blood serum ARE for phenylacetate were found as 1.66 mM and 3300 nmol/min/mg, respectively. The maximum GST enzyme activity was detected at 2 mM potassium phosphate buffer, pH 5.5 and at 65 &deg / C. GST enzyme was saturated with its substrate, CDNB around 4.5 mM concentration and with its cofactor, GSH around 8 mM concentration. The apparent Km and Vmax values of human blood serum GST for CDNB substrate were found as 2.8 mM and 0.43 nmol/min/mg and for GSH were found as 4.11 mM and 0.23 nmol/min/mg, respectively. In addition, effects of three different heavy metal ions, Cd+2, Hg+2 and Ni+2, on human blood serum ARE and GST activity were studied and half maximal inhibitory concentrations (IC50) were determined. The main objective of this study was to investigate the human blood serum ARE and GST activities in patient and control groups using the optimized conditions. For this purpose, blood samples were collected from 172 ischemic stroke patients and 105 controls. Then serum obtained from blood samples were used to determine ARE and GST activities. The mean of ARE activity in patient group (n=172, 109.9 &plusmn / 32.5 U/mL ) was insignificantly lower than the mean of ARE activity in control group (n=105, 113.5 &plusmn / 33.1 U/mL, P=0.284). GST activity of the patients (10.8 &plusmn / 4.4 U/L) was insignificantly higher than that of controls (10.5 &plusmn / 4.2 U/L, P=0.483 ). In addition, statistical analysis showed hypertension, diabetes and HDL as significant risk factors of stroke.

ANALYSIS OF CYTOCHROME P4501A1 GENETIC POLYMORPHISMS IN PATIENTS WITH ISCHEMIC STROKE Adali, Ayse &Ccedil / inar M.Sc., Department of Biochemistry Supervisor: Prof. Dr. Orhan Adali Co-Supervisor: Dr. Birsen Can Demird&ouml / gen January 2011, 179 pages Stroke is the third leading cause of death worldwide and results in serious disabilities. Cytochrome P450 1A1 gene (CYP1A1) is a highly polymorphic gene encoding its corresponding xenobiotic metabolizing enzyme which is responsible from the metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs) that are engaged with the formation of free radicals. Atherosclerosis is a major cause of ischemic stroke and this pathology may be associated with the disruption of vascular homeostasis due to the formation of these chemicals. The main objective of this study was to investigate the coding region (A4889G) and non-coding region (T6235C) polymorphisms of the CYP1A1 gene as a risk factor for ischemic stroke. The study group in Turkish population consisted of 226 unrelated ischemic stroke patients and 113 control subjects. There was no statistically significant difference between the groups with respect to age and gender. Total blood samples were obtained from G&uuml / lhane Military Medical Academy Hospital, Neurology Department, Ankara. In stroke patients, hypertension, diabetes mellitus, smoking and obesity were at least 2 times more common and high density lipoprotein cholesterol (HDL-C) was significantly lower than controls. The frequency of mutant allele 4889G was 0.445 in patients and was nearly the same with controls. The frequency of mutant allele 6235C was 0.151 in patients and was significantly higher in controls (0.226, P=0.015). The risk of diabetic, smoker and obese individuals having ischemic stroke was significantly higher in 4889G allele carriers (AG+GG / Odds ratio / OR= 2.1, 2.4 and 3, respectively). The risk of hypertensive and diabetic individuals having ischemic stroke was higher in 6235TT genotypic people (OR= 3 and 2.2, respectively). On the contrary, the risk of smoker and obese individuals having ischemic stroke was significantly higher in 6235 C allele carriers (OR=5.3 and 3.7, respectively). Logistic regression analysis revealed that hypertension, smoking, levels of low density lipoprotein cholesterol (LDL-C) and HDL-C and 6235C allele were significant predictors of stroke. In this analysis, high level of LDL-C was found to be associated with almost 1.5-fold risk of ischemic stroke. On the other hand, HDL-C and having mutant 6235C allele decreased the risk of ischemic stroke 2.5 and 2-fold, respectively. This is the first study investigating the relation between A4889G polymorphism and stroke risk. Additionally, in Turkish population A4889G and T6235C polymorphisms were analyzed for the first time in terms of its relation to ischemic stroke. The present study demonstrated that the frequency of mutant 4889G allele was nearly the same in stroke patients and control subjects / whereas the frequency of mutant 6235 C allele was higher in control subjects than in stroke patients. Consequently, we decided that carrying mutant 4889 G allele does not constitute a risk for ischemic stroke and carrying mutant 6235C allele may have a protective effect against stroke.

Stroke is characterized with loss of one or more functions of the body resulted by inadequate blood supply to the brain. Most of the cases result from a blood clot forms on an atherosclerotic plaque in the brain which is called as ischemic stroke. Structure of the arteries and vascular tone are listed in major determinants in the development of the disorder. Soluble epoxide hydrolase (sEH, EPHX2) catalyzes conversion of epoxyeicosatrienoic acids to inactive diol metabolites. EETs are potent vasodilators that participate in the regulation of vascular tone and cerebral blood flow. Microsomal epoxide hydrolase (mEH, EPHX1) is a critical phase I enzyme that catalyzes the conversion of various xenobiotic epoxide substrates and polycyclic aromatic hydrocarbons (PAHs). Animal studies show that tobacco smoke mutagens such as PAHs and heterocyclic amines directly increase the development of atherosclerotic lesions. The main purpose of this study is evaluation of effect of Arg287Gln single nucleotide polymorphism of EPHX2 gene and Tyr113His and His139Arg single nucleotide polymorphisms of EPHX1 gene as a risk factor for ischemic stroke in Turkish population. Blood samples of 237 ischemic stroke patients and 120 controls were collected and all polymorphisms were determined by PCR-RFLP method. Mutant allele frequencies in terms of Arg287Gln polymorphism of EPHX2 gene (A) were found as 0.08 for patient group and 0.09 for controls. Tyr113His polymorphism of EPHX1 gene (C) were found as 0.27 for patient group and 0.31 for controls when, His139Arg polymorphism of EPHX1 gene (G) were 0.820 and 0.814 for patient and control groups, respectively. The differences between mutant allele frequencies of patients and controls were not found to be statistically significant. Subgroup analysis was used to investigate the effects of conventional vascular factors according to the genotypes in the stroke susceptibility. Smoking, diabetes, obesity and hypertension were found to significantly increase the risk of having stroke. More detailed analysis on these factors with respect to genotypes showed that the risk of hypertensive individuals having ischemic stroke was higher in wild type homozygous genotype groups of Tyr113His (TT) and His139Arg (AA) polymorphisms and heterozygous and mutant homozygous genotypes of Arg287Gln (GA+AA) polymorphism than their counterparts (OR= 3.21, 3.15 and 4.69, respectively). Smoker people within the heterozygous and mutant homozygous genotypes group of Arg287His (GA+AA) polymorphism and wild type homozygous group of His139Arg (AA) polymorphism were found to be more susceptible to have stroke (OR= 11.81 and 4.78 respectively). Finally, diabetes mellitus was found to double the risk of having stroke regardless of the genetic background. Logistic regression analyses were used to ascertain the effects of vascular factors, lipid parameters and genotypes in the stroke susceptibility. LDL-cholesterol (OR=1.46 / 95%CI, 1.12-1.89, P=0.00), smoking (OR=3.46 / 95%CI, 1.66-7.21, P=0.00) and hypertension (OR=3.19 / 95%CI, 1.92-5.30, P=0.00) were found to be significant risk factors for ischemic stroke, whereas HDL (OR=0.27 / 95%CI, 0.12-0.65, P=0.02) was found to be a protective factor in general population. In this study, the relation of Tyr113His and His139Arg polymorphisms of EPHX1 gene and risk of ischemic stroke is investigated for the first time in literature while, Arg287Gln polymorphism and ischemic stroke risk in Turkish population was studied for the first time.

Thesis (M.S.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Science." Title from title page of PDF document. Bibliography: p. 39-48.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on December 1, 2009). Department of Anatomy and Cell Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Zao C. Xu, Feng C. Zhou, Charles R. Yang, Theodore R. Cummins. Includes vitae. Includes bibliographical references (leaves 115-135).