Browsing by Subject "Therapeutics"
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Item A Study to Determine Improved Compliance of Biophosphonate Treatment in Subjects with Osteoporosis(2005-02-01) Enard, April T.; Gwirtz, Patricia A.; Jimenez-Williams, Cynthia; Rubin, BernardEnard, April T. A Study to Determine Improved Compliance of Bisphosphonate Treatment in Subjects with Osteoporosis. Master of Science, February 11, 2005. The ability of patients to adhere to treatment regimens is very poor and continues to impede optimal therapy of osteoporosis. The shortcomings in treating osteoporosis are: a) noncompliance and/or lack of continued persistence of therapy, b) efficacy of therapy on bone turnover marker levels and fracture prevalence, and c) tolerability of therapy to patients. Studies have shown that interventions such as education and awareness of bone mineral density promote patient usage compliance. The slightest improvement in compliance allows further understanding of accurate efficacy of medication therapy to fractures, bone marker levels, and overall improvement of bone mass. Increased compliance/persistence allows accurate comparison of bisphosphonates to one another for effectiveness on osteoporosis patients and allows improvement opportunity in treatment modalities that can positively influence the course of osteoporosis. This phase IV study targets compliancy/persistence in bisphosphonate therapy in treatment of osteoporosis.Item Advanced Problem Solving in the Biotherapeutics Industry: Parameters influencing the delivery of a novel cell therapy product and exploration of a new method for determining activity of Clostridium histolyticum collagenase, a wound debridement enzyme(2015-05-01) Harris, Melanie A.; Jerry W. Simecka; Patricia A. GwirtzBiotechnology is a multi-faceted industry with many unique challenges that require knowledge in a broad range of topics. When working in the wound care field it is necessary to not only create a product in the laboratory, but also effectively bring it to the patient. This task requires many skilled people who can test it for efficacy, design and conduct clinical trials, confirm quality and consistency, design packaging, consider transportation issues and so on. The following investigation focuses on the testing of a cellular product and its accompanying device under various conditions as well as the exploration of a new assay capable of the activity of a wound debridement enzyme. The results of the product/device testing have generally confirmed the comparability of the cellular product devices as well as their resistance to various temperatures encountered in the clinical environment. A new modified assay for the testing of collagenase has been established as precise and comparable to current methods, though it requires more testing to confirm robustness.Item Analysis of the Clinical Research Methodologies Employed During a Phrase Three Efficacy Study for Ultracet as a Post-Herniorrhaphy Analgesic(2001-08-01) Aguilar-Zanatta, Jorge; Rustin Reeves; Don Peska; Della WeisThe history of pain management stems back many thousands of years. However, not until recent times have significant advancements in biochemistry and pharmacology allowed analgesics to be incorporated in clinical interventions and everyday life. Due to these advancement, attempts to refine pharmacological action on receptors in terms of specificity would render medications with fewer side effects. The technology is present, but the application and development of modern analgesics in post-surgical settings is substandard. According to C.L. Ireson and R.W. Schwartz, (2001), the outcomes of ailment interventions in the United States are “…no better and in numerous situations worse that those achieved in other countries,” even though the United States has the most expensive healthcare in the world. Furthermore, a study performed by Carr et al. (1998), has identified the United States as demonstrating consistent inadequacies in postoperative pain management. Several factors have been identified as being contributors of poor post-surgical pain control in America. Lack of awareness of the available strategies in acute pain control and its implementation in post surgical care are labeled as being problematic observations (Puid et al., 2001). In response to these conditions as well as the managed health care time and cost limitations, new and efficacious pharmaceuticals must be made available to a broad spectrum of socio-economic strata. Currently, there is a great debate over the use of laparoscopic herniorrhaphy versus open tension free approaches. In terms of cost, the laparoscopic herniorrhaphy versus open tension free approaches. In terms of cost, the laparoscopic herniorrhaphy versus open tension free approaches. In terms of cost, the laparoscopic procedure is more expensive and yields less postoperative pain, however the open tension free approaches are less expensive and yield more postoperative pain (Sarli et al., 2001, Medical Research Council Laparoscopic Groin Hernia Trial Group, 2001, Parviz et al., 1995). There are advantages and disadvantages to both procedures. Assuming that efficacious postoperative analgesics were available, the open tension free repair would be more feasible in terms of cost and hernia recurrence rates (Sarli et al., 2001). In terms of pharmaceutical development, the laws and guidelines by the regulatory agencies such as the Food and Drug Administration, institutional review boards, and pharmaceutical sponsor protocols must be followed. Along with good clinical practice standards, interdisciplinary collaboration in pain studies produce results that are statistically and clinically salient. The patient’s well-being and comfort is the ultimate goal in clinical pain studies and in medicine in general, therefore postoperative pain should be aggressively managed.Item Anti-Tumor Immune Responses Against MTLn3 Mammary Adenocarcinoma(2014-05-01) Carter, KiahRae J.; Hodge, Lisa M.Lymphatic pump treatment (LPT) is used as a lymph enhancing therapy to treat edema. In animals, LPT enhanced lymphatic flow, released leukocytes and inflammatory mediators into lymph, and inhibited pulmonary tumor formation. Therefore, we hypothesized the administration of LPT would enhance immunity and inhibit primary breast tumor growth. Rats were subcutaneously injected with MTLn3 and divided into MTLn3, MTLn3+Sham-LPT and MTLn3+LTP group. Sham group received light touch under anesthesia and LPT group received treatment under anesthesia. There were no changes in tumor growth between groups. Administration of Sham-LPT resulted in an increase in tumor-adjacent lymph node weight. Collectively, our data suggests LPT did not enhance primary tumor growth and may also protect against the pathogenesis exhibited by sham-LPT.Item Antioxidants, Exercise, APOE Genotype and Brain Function(2014-12-01) Chaudhari, Kiran; Nathalie Sumien; Michael J. Forster; Eric B. GonzalesApolipoprotein E4 (APOE4) is a well-established and extensively prevalent genetic risk factor for the development of Alzheimer’s disease (AD). The presence of APOE4 allele accelerates the pathophysiology and symptomology of AD. A large set (36%) of the population suffering from AD expresses APOE4. Being a chronic progressive disease with very few pharmaco-therapeutic agents approved by FDA, non-drug lifestyle modifications have been an important part of management of AD. People often eat healthy diet rich in antioxidants and focus on healthy living habits such as exercise. Health care providers frequently suggest combining antioxidants with physical activity for higher benefits. Antioxidants have been beneficial in counteracting oxidative stress and improving learning and memory. Similarly, different regimens of exercise also improved cognition and delayed development of AD. However, the nature of the interaction between antioxidants and exercise remain elusive and complicated. While some studies reported additive effects, others have also shown a concerning antagonistic action of the antioxidants on the beneficial effects of exercise. In the context of APOE genotype, we set our study to determine the nature of such interaction between antioxidants and exercise. Using vitamins C and E and a treadmill-based forced exercise in a genetically modified mouse model expressing human APOE3 and APOE4 (GFAP-APOE3, GFAP-APOE4), we explored the nature of that interaction on functional and biochemical outcomes. We examined the mice for spatial learning and memory, working memory and executive function, coordinated running performance, muscular reflexes, spontaneous locomotor activity, anxiety and muscle strength. Interestingly, we observed that the young adult mice expressing E4 allele performed better on higher brain functions including spatial learning and memory and short term memory in contrast to middle age mice, which developed a cognitive deficit as expected. Motor functions, reflexes and coordination were poor among all the mice carrying E4 allele irrespective of age. Antioxidants and exercise interventions led to outcomes that were dependent on genotype, age and the brain function under consideration. There was additive beneficial effect of combination of antioxidants and exercise on cognitive outcomes but not on motor outcomes in middle age groups. However, in young adults, an antagonistic interaction was observed on motor outcomes but no such interaction was observed on cognitive outcomes. Hence we can conclude that, combination of antioxidants and exercise is not a “fit for all” approach and needs to be tailored base on individual’s age and genotype.Item Barriers to Completion of a Family Health History Tool and Increasing Subject Retention(2016-12-01) Bennett, Margarett A.; Robert T. MalletFamily Health Histories (FHH) are crucial for identifying disease risk factors, and such information is paramount to the diagnosis, treatment, and long term care of patients. Many if not most human diseases include a hereditary component. Despite the recognized importance of health history information, the FHH of many Americans remains uncaptured. This study examined impediments to FHH completion and evaluated a strategy for increasing completion. Participants were givenreminder prompts, offers of assistance, and surveyed to identify barriers and effective methods to improve FHH completion. The most frequently cited barrier was that the participant did not remember being part of the study or anything about the study. Although the reminder phone prompts produced a modest increase in participation, this strategy was time consuming and inefficient. The possibilities that providing additional information during the recruitment process and earlier reminder phone calls after registration may improve FHH participation and completion rates warrants further investigation.Item Comparative assessment of IV acetaminophen and conscious sedation for pain relief during invasive cardiac procedures(2016-05-01) Alam, Zainab I.; Jerry W. Simecka; Patricia A. Gwirtz; Ranajit ChakrabortyAlthough current consensus in the field of Interventional Cardiology allows for pain medications to be administered at the discretion of the operator, oftentimes, the standard is a cocktail of a benzodiazepine and a narcotic to achieve conscious sedation. Such medications often lead to less than ideal outcomes with longer times to ambulation, delayed discharge and in many cases, drug addiction. This practicum study evaluates the effects of IV acetaminophen (Tylenol) on breakthrough pain during invasive heart procedures in order to replace the current standard of moderate sedation. The results indicate that not only is IV Tylenol as effective as midazolam and fentanyl combined, but it causes a significant decrease in pain response as well.Item Creating novel purification and biochemical characterization protocols for C. collagenase from Clostridium histolyticum, developing a new emergency medicine product, and formulating several novel therapies for chronic and acute wound treatment(2017-05-01) Mars, Jason P.; Jerry W. Simecka; Patricia A. Gwirtz; Aleksa JovanovicThe pharmaceutical industry not only includes infinite areas of specialization, but also consists of distinct areas that do not typically overlap. Biotechnology is the branch of medicinal research that bridges the gap between the fields within the pharmaceutical industry by being able to take on the challenges that require knowledge of a vast range of information. This practicum was organized to put the scientific knowledge and the interdisciplinary practices of biotechnology to use in a modern day, pharmaceutical company specializing in wound therapy and skincare: Smith & Nephew Biotherapeutics. Wound therapy has the widest range of application due to being one of the few fields that affects everyone, regardless of medical disposition. The specific goals of this practicum were: to develop novel purification and biochemical characterization protocols for C. collagenase from Clostridium histolyticum to replace current production methods of Santyl®, to develop a working prototype of a venom-based, hemostatic film, and perform reformulation, quality control, troubleshooting, and verification testing on samples of Regranex®, Iodosorb “Max”, and EU-Collagenase. Every goal presented was approached with the end results of saving Smith & Nephew costs, reducing bioburden of production, and creating more efficient protocols to bring Smith & Nephew into the modern age.Item Effects of Cervical Manipulation on Cardiac Autonomic Control(2006-05-01) Giles, Paul David; Michael Smith; Scott Stoll; Walter WitryolGiles, Paul David, Effects of Cervical Manipulation on Cardiac Autonomic Control. Master of Science (Clinical Research and Education – OMM), May 2006, pp, 1 table, 8 figures, references. Objective: Osteopathic Manipulative Medicine treatment (OMT) regimes often focus on treating the Autonomic Nervous System (ANS) in addition to biomechanics. Techniques focused on the upper cervical spine are theorized to affect the function of the vagus nerve and thereby influence the parasympathetic branch of the ANS. This study was conducted to observe the effect of upper cervical spine manipulation on cardiac autonomic control as measured by heart rate variability (HRV). Methods: Nineteen healthy, young adult subjects were randomly assigned an order in which they would undergo three different experimental protocols: OMT, Sham, and a time control. Six minutes of electrocardiographic data was collected before and after each intervention to be analyzed by power spectral analysis. Results: All baseline data for each protocol and all parameters studied were the same. The OMT protocol and all parameters studied were the same. The OMT protocol caused a change in the standard deviation of the normal-to-normal (SDNN) intervals (0.121 +/- 0.0822 sec, p=0.005) and the change in the high frequency HRV was different from the changes caused by other interventions (p=0.038). Conclusions: This preliminary data supports the hypothesis that under cervical spine manipulation affects the parasympathetic nervous system; however, more data on more subjects needs to be collected in order to clarify some points, and to reach statistical significance in certain measures.Item Evaluation of respiratory outcomes in pre-term infants receiving nCPAP versus surfactant and mechanical ventilation during transport.(2012-12-01) Jacobs, Linda M.; Sumihiro SuzukiThe initial respiratory treatment for premature infants is trending toward nCPAP but this therapy sometimes fails requiring surfactant plus mechanical ventilation. This retrospective review of respiratory interventions by referral hospitals and a neonatal transport team evaluated predictors of respiratory support at 72 hours. A unique variable of time between birth and transport arrival was added to the multivariate regression model. This study confirmed that gestational age and high levels of respiratory support in the first hours of life are strong predictors for higher levels of respiratory support at 72 hours. Time to transport arrival was not a factor. The transport team has trended toward utilizing nCPAP more often over the past two years.Item Gender Differences in Hemoglobin Level at the Onset of Symptoms of Cancer-Related Anemia(2003-12-01) Levar, Joshua M.; Victoria RudickLevar, Joshua M., Gender Differences in Hemoglobin Level at the Onset of Symptoms of Cancer-Related Anemia. Masters (Clinical Research Management), December, 2004, 39 pp., 2 tables, 5 illustrations, bibliography, 47 titles. The purpose of this study was to assess whether the previously demonstrated relationship between quality of life and anemia in cancer patients was influenced by gender. Two hundred and fifty one patients of various diagnoses completed the Functional Assessment of Cancer Therapy – Anemia (FACT-An) subscale to measure quality of life. Regression analysis revealed a significant positive correlation between hemoglobin and FACT-An subscale score, as well as a negative correlation between Eastern Cooperative Oncology Group (ECOG) performance status and FACT-An subscale score. Mean comparison demonstrated a significant difference in FACT-An score between patients currently and not currently receiving chemotherapy. An analysis of covariance, controlling for current therapy and ECOG performance status as confounders, found that men score more poorly on the FACT-An within the hemoglobin range of 10.-13.0 g/dL. In conclusion, the normalization of hemoglobin levels improves quality of life; however, gender differences should be taken into account when determining optimal hemoglobin levels.Item Immediate Effects of Osteopathic Manipulative Treatments on Immune Function in a Healthy Population: A Pilot Study(2006-05-01) John, Janice Thomas; Stoll, Scott; Simecka, Jerry; Atkinson, BarbaraJanice Thomas, D.O., M.S. Immediate Effects of Osteopathic Manipulative Treatments on Immune Function in a Healthy Population: A Pilot Study. Master of Science (Clinical Research and Education – OMM), May 2006, 75 pp, 3 tables, 5 figures, 66 references, 24 titles. Objectives: The purpose of this pilot study was to investigate the immediate effects of Osteopathic Manipulative Treatment (OMT) on immune function in a healthy population. Methods: This was a randomized, blinded and controlled clinical trial. 50 healthy individuals, ages 18 to 40, were recruited. Subjects were randomly assigned to one of two groups: OMT or Rest (control). Blood and saliva samples were collected pre and post-intervention (thirty minutes of OMT or Rest). Samples were analyzed for a CBC, salivary IgA, and various lymphocyte populations. Results: This study successfully demonstrated the feasibility of this protocol. No statistically significant differences in outcome measures were identified between the two groups, nor were any apparent trends identified. Conclusion: This study established a framework for future research investigating the effects OMT on acute and chronic infection, chronic pain, and immunocompromised populations in human and/or animal populations.Item Intravenous pyruvate to protect heart and brain during closed-chest resuscitation and recovery from cardiac arrest(2014-08-01) Cherry, Brandon H.; Mallet, Robert T.; Olivencia-Yurvati, Albert H.; Raven, Peter B.Cardiac arrest is a leading cause of death in the United States and Western Europe. Cardiopulmonary resuscitation (CPR) is the only means of sustaining the victim until application of defibrillatory countershocks. Although it has been over 50 years since its advent, CPR remains a work in progress. Many initially resuscitated victims later die from the damage sustained from ischemia-reperfusion, and treatments to combat the extensive ischemia-reperfusion injury sustained during cardiac arrest-resuscitation remain elusive. The major mechanism of injury underlying ischemia-reperfusion is the intense overproduction of reactive oxygen and nitrogen species (RONS) that accumulate during reperfusion and compromise normal cell function. RONS formed during resuscitation trigger lipid peroxidation, disable enzymes vital for cell metabolism and survival and, ultimately, induce cell death within affected organs. In order to prevent extensive damage to the central nervous system culminating in permanent neurocognitive disability and death, prospective treatments must possess robust antioxidant properties, traverse the blood-brain barrier between the cerebral circulation and brain parenchyma, and be non-toxic at effective doses. Pyruvate is a natural intermediary metabolite, energy-yielding substrate and antioxidant. Pyruvate neutralizes RONS, thereby dampening oxidative stress and preventing covalent oxidative modification of enzymes and lipid membranes, and generates ATP to support brain function. Pyruvate readily traverses the blood-brain barrier and is non-toxic over a wide range of doses, including those previously demonstrated to protect the heart during cardiopulmonary bypass and the brain during stroke, thereby supporting oxygen and fuel delivery to the recovering brain. Moreover, pyruvate has been shown to promote cardiac electromechanical and metabolic recovery following cardiac arrest and open-chest CPR. This study tested whether infusion of pyruvate during, CPR and early recovery can decrease the biomarkers of oxidative stress after cardiac arrest. Isoflurane-anesthetized pigs were subjected to 6 min electrically-induced, untreated ventricular fibrillation, followed by 4 min closed-chest CPR, defibrillation and either 1 or 4 h recovery. Beginning at 5.5 min arrest, either sodium pyruvate or NaCl control were infused iv for the duration of CPR and for the first 60 min after recovery of spontaneous circulation (ROSC). Arterial blood was sampled pre-arrest and at 5, 15, 30, 60, 120, 180, and 240 min ROSC for analyses of blood gases and plasma constituents. At either 1 h (i.e. end of treatment infusion) or 4 h ROSC, a craniotomy was performed, the pig was euthanized, the brain was removed, and biopsies from hippocampus and cerebellum were snap-frozen in liquid nitrogen for biochemical analysis. The first phase of this project tested the hypothesis that intravenous administration of sodium pyruvate during precordial compressions and the first 60 min ROSC restores hemodynamic, metabolic, and electrolyte homeostasis in a closed chest porcine model of cardiac arrest. Resuscitation with pyruvate sharply decreased the incidence of lethal pulseless electrical activity (PEA) following defibrillatory countershocks, and lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure. Pyruvate also enhanced glucose clearance, elevated arterial bicarbonate, and raised arterial pH. The second phase of this project tested the hypothesis that pyruvate prevents the decrease in activity of the brain’s antioxidant enzymes following cardiac arrest and hyperoxic (100% O2). Activities of glutathione peroxidase and glutathione reductase were decreased at 60 min ROSC vs. sham in both the hippocampus and cerebellum. Pyruvate partially preserved glutathione peroxidase activity at 1 h ROSC, but by 4 h, after 3 h of pyruvate clearance from the circulation, the enzyme’s activity fell to the same extent as in NaCl-infused pigs. Interestingly, the glutathione peroxidase/reductase activity fell sharply in non-arrested sham pigs between the time points corresponding to 1 and 4 h ROSC, suggesting that hyperoxia resulting from ventilation with 100% produced sufficient oxidative stress to inactivate the enzymes. Similarly, lactate dehydrogenase activity fell between 1 and 4 h ROSC in hippocampus and especially cerebellum. In sham pigs, lactate dehydrogenase activity decreased from the time points corresponding to 1 and 4 h ROSC, and pyruvate had no effect on lactate dehydrogenase in either region of the brain. Thus, cardiac arrest and hyperoxic ventilation disabled a critical antioxidant system in two ischemia-sensitive brain regions. Pyruvate afforded partial protection of these enzymes which waned after pyruvate cleared from the circulation. We conclude that 1) Pyruvate infusion during cardiac arrest, CPR and early recovery promotes conversion from ventricular fibrillation to a productive sinus rhythm instead of lethal PEA; 2) Pyruvate hastened glucose clearance, a prognostic measure used clinically; 3) Pyruvate elevated the arterial bicarbonate concentration and raised arterial pH, which combats the acidemia normally observed following ROSC; 4) Cardiac arrest-resuscitation and hyperoxic ventilation disabled the glutathione peroxidase-reductase system, a critical component of the brain’s antioxidant defenses, in hippocampus and cerebellum; and 5) Pyruvate delayed oxidative inactivation of glutathione peroxidase in the cerebellum, but this effect subsided as pyruvate elevated. These investigations demonstrate the therapeutic effects and limitations of pyruvate as a resuscitative treatment to hasten electrocardiographic and metabolic recovery post cardiac arrest.Item Long Term Compliance and Withdrawal Rates in Gynecologic Oncology Clinical Research Studies(2003-12-01) Kersey, Jen Kelley; Robert Kaman; LaChelle Arredondo; Robin NewmanOncology is an area of study that is greatly affected by time. Patients with cancer need safe and effective treatment immediately. For some, current treatments have not worked to eliminate their disease. Their recurrent condition reinforces the need for safer and more effective treatment. This treatment must not only destroy the cancerous cells, but it must also allow for the continuation of their lives. This life can be measured by time and quality. Ideally, both would be maximized for proper treatment, yet current science has not found this model cure. For some regimens, quality of life could be maximized at the expense of quantity of life and vice versa. Both the patient and the healthcare provider should evaluate the balance of expectations. The potential of each life should be maximized for length and quality. The investigator/physician must do everything in their power to ensure that the patient’s needs are met medically. In treatment involving recurrent cancer patients, time is of the essence. Therapy, in every form, must be given immediately to extend and improve their remaining lives. If QOL assessments can predict the outcome of retention, acknowledgement of the subject’s well-being can allow for greater insight into the physical and emotional effects of the experimental treatment. The use of this information can help future generations of cancer patients by providing data that describes the therapy. The study of “Long Term Subject Compliance and Withdrawal Rates in Gynecologic Oncology Clinical Research Studies” is necessary for the evaluation of QOL assessments to subject retention, patient care practices in research and private practice may be affected through incorporation of the QOL assessment. There are many benefits that may result if significance is found in this study. The main objective of the QOL assessment is to observe the QOL of the subject in relation to the effect of the treatment on the individual. If subject retention can be predicted from the evaluation of QOL assessments, future clinical research studies, in gynecologic oncology and beyond, may modify their protocols to include the assessments. Improved subject retention will ultimately improve the accuracy of data collection. Improved accuracy will help evaluate the outcomes of treatment for cancer patients. If the QOL assessment shows a correlation to subject retention, the survey could be used as a tool, not only to assess the patient’s well being, but also to predict withdrawal. This project could identify “at-risk” factors of the subject population. Subjects who are at-risk for withdrawal should be followed closely. Excluding subjects that meet the clinical protocol would be unethical. To protect the validity of the study and ensure ethical measures are taken, those subjects that have factors associated with drop-out should be enrolled in the study and monitored closely. The survey may give insight to the types of ancillary or palliative care that may be needed during the patient’s fight with cancer. This research hopes to identify that quality of life assessments are an integral dimension of the research practice. The care of the patient rests in the hands of the physicians providing treatment. They are responsible for the needs and best interest of the patients. Wide discrepancies between the rating of specific outcomes of treatment by the patient and physician have been noted in current literature. This study may help show that the patient driven quality of life assessment is an important aspect of patient care, and should be integrated as a common tool for the care of gynecologic oncology patients. The use of this tool outside of the research setting should also be explored. Future studies in quality of life, beyond that of gynecologic oncology, may be investigated. This study hopes to initiate further research for the quality of life assessment because the QOL assessment gives data regarding well-being from the patient’s perspective. Future studies should also research the confounding factors that may influence subject withdrawal. These additional collaborative factors may contribute to compliance or those “at-risk” for drop out. By maximizing subject retention and protecting subject safety, healthcare research can provide results that reflect the true investigational question.Item Molecular Regulation of Cardiac Stem Cell Growth and Differentiation by Extrinsic Factors and Novel Intracellular Signaling Pathways(2008-05-01) Bartosh, T.J.; Rouel S. Roque; Harold Sheedlo; Robert WordingerT. J. Bartosh, Molecular regulation of cardiac stem cell growth and differentiation by extrinsic factors and novel intracellular signaling pathways. Doctors of Philosophy (Biomedical Sciences), May 2008, 293 pp., 4 tables, 59 illustrations, bibliography, 353 titles. Insufficient myocardial regeneration following ischemic injury provokes cardiac dysfunction, adverse tissue remodeling, and ultimately heart failure. Stem cell replacement therapy appears to be a promising strategy for improving cardiac function, however, challenges involving inadequate stem cell differentiation, engraftment, and survival following transplantation currently impede the efficacy of regeneration protocols. These limitations emphasize the significance of identifying extrinsic factors and corresponding molecular mechanisms regulating cardiac cell differentiation. Recent reports recognizing a stem cell component in the adult heart (i.e. cardiac stem cells, CSC) have provided additional targets and/or tools for myocardial repair. This investigation verified the presence of CSC in the adult dog heart, described methods to generate three dimensional (3D) cardiac microtissues (‘cardiospheres’) from CSC, and revealed features of cardiospheres potentially useful for identifying extrinsic procardiogenic factors, evaluating myocardial response to stress, and delivering functional CSC into the damaged heart. Specifically, cardiosphere formation was facilitated by culturing CSC in growth medium on a poly-L-ornithine substratum. Cardiospheres are comprised of interior cells that exhibited characteristics of CSC; differentiating cardiomyocytes at the periphery with organized contractile machinery; and/or vascular cells capable of forming vessel-like networks. Upon co-culture with neonatal cardiomycocytes, spheres developed foci of contracting regions. Furthermore, cardiospheres exhibited increased resistance to oxidative stress and survived subcutaneous injections without undergoing neoplastic transformation further supporting their ability to effectively promote myocardial regeneration. Retionic acid (RA), the active form of vitamin A, augmented expression of myocyte-specific proteins in cardiospheres, thus, RA may improve the success of cardiac regenerative therapies and provide an appropriate stimulus to model underlying mechanisms fundamental for CSC differentiation. The tendency for cultured stem cells to undergo various levels of multi-lineage commitment is, however, cumbersome for deciphering precise cues that direct cell fate decisions. In this study, the molecular pathways important for RA-induced cardiac differentiation were examined using h9c2 cardioblasts as a stable cell model. In h9c2 cells, RA treatment promoted transcriptional enhancement of the muscle-enriched gene regulatory protein MEF2C, morphological alterations indicative of differentiation, and a robust increase in expression of myocyte differentiation genes including cardiac myosin heavy chain (cMHC) and ventricular myosin light chain-2 (vMLC2). These changes were preceded by rapid events involving elevation of intracellular Ca^2+ and phosphorylation of p38 MAPK. The effects of RA were attenuated using CA^2+ buffering agents or chemical inhibitors of L-type Ca^2+ channels (LTCC) and the phosphatase calcinuerin, but not by RA receptor antagonists. Furthermore, overexpression of dominant negative (dn) MEF2C, dnp38 MAPK, or CAIN, a physiological calcineruin inhibitor, abrogated MEF2 activity and RA-induced differentiation. These results imply that RA promotes cardiomyocyte differentiation, independent of RA response element activation, via induction of CA^2+ -regulated signaling pathways by activating the LTCC-calcineurin/38MAPK-MEF2 axis. Taken together, these findings enhance our understanding of extrinsic factors and molecular mechanisms indispensable for myocyte differentiation and subsequently provide novel therapeutic targets and cellular tools for regeneration and repair of damaged myocardium.Item Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model(2002-12-01) Kern, Jami RaDel; Thomas Yorio; Robert W. Gracy; Peter B. RavenKern, Jami RaDel, Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model. Doctor of Philosophy (Biomedical Sciences, Biochemistry and Molecular Biology), December 2002, 156 pp., 1 table, 27 illustrations, references, 112 titles. The focus of these studies was to characterize a novel connective tissue model for use in experiments examining possible contraction initiators in the wound healing process, i.e. endothelin-1 (ET-1). Through these studies, it has been shown that use of a telomerized dermal fibroblast cell line addresses the concerns relating to variations due to heterogeneity of normal human cells cultured in vitro, without creating a cancerous cell line or interfering with normal phenotypic changes. In addition, the incorporation of telomerized cells into our TE, which does not spontaneously contract (US Patent #6471958), provides a unique model to study the contraction and scar formation process. Using the TE populated with hTERT fibroblasts, an innovative technique was developed to identify the initiation of tissue contraction using an optical fiber interferometry system. The process allows observation of contraction within five minutes of stimulus addition and also enables continuous data capture over a period of several hours. The greatest strength of this system is its sensitivity, since optic interferometer allows measurement of displacement (contraction) to the tens of nanometers. Along those lines, the current studies have identified ET-1 as a potential early initiator in wound healing and suggest a novel pathway through which it functions. This proposed mechanism includes both direct effects of ET-1 through the Rho-associated kinase pathway and indirect effects potentiated by TGF-β. Future studies addressing whether TGF-β converges on the Rho-associated kinase pathway or acts independently through other signaling mechanisms should be initiated. The discovery of early initiators of tissue contraction is essential in the identification of potential therapeutic targets in the quest to reduce prolonged and severe tissue contracture and scaring.Item North Texas Health & Science - 2011, Issue 1(University of North Texas Health Science Center at Fort Worth, 2011-01-01)Item North Texas Health & Science - 2011, Issue 2(University of North Texas Health Science Center at Fort Worth, 2011-01-01)Item North Texas Health & Science - 2012, Issue 1(University of North Texas Health Science Center at Fort Worth, 2012-01-01)Item North Texas Health & Science - 2012, Issue 2(University of North Texas Health Science Center at Fort Worth, 2012-06-01)