Strasburg, Gale Marvin.
Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 126-135).
McConnell, Mark, McConnell, Mark
The progression of genetically inherited cardiomyopathies from an altered protein structure to the clinical presentation of the disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information of multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational methods to characterize the baseline function and structure of the Tm overlap, as well as the effects that mutations causing divergent patterns of ventricular remodeling have on both structure and function.
Cardiac troponins are released into the circulation following myocardial injury with measurement of serum troponin concentration integral to the diagnosis of myocardial infarction. The next generation of high-sensitivity cardiac troponin assays have enhanced precision at very low concentrations and for the first time permit troponin concentrations to be reliably quantified outwith acute coronary syndromes. My aim was to evaluate the impact of more sensitive troponin assays on the diagnosis of myocardial infarction and to determine the potential for novel applications in stable cardiovascular diseases. In 2,929 consecutive hospitalised patients with myocardial injury I assessed the impact of implementing a contemporary sensitive troponin assay and a lower diagnostic threshold, on the incidence, management and outcome of patients with primary (type 1) and secondary (type 2) causes of myocardial infarction. Lowering the threshold improved outcomes in patients with type 1 myocardial infarction, but disproportionately increased the diagnosis of type 2 myocardial infarction, and identified a group of patients who remained at high-risk of death over the next 12 months. In 1,126 consecutive patients with suspected acute coronary syndrome I evaluated the impact of a high-sensitivity cardiac troponin I assay and sex-specific diagnostic thresholds on the diagnosis of myocardial infarction. Whilst having little effect in men, the use of sex-specific diagnostic thresholds would double the diagnosis of myocardial infarction in women, identifying those at increased risk of recurrent events. In a second cohort of 4,870 patients, I evaluated the optimal threshold to rule out myocardial infarction. The negative predictive value for myocardial infarction or cardiac death at 30 days was evaluated for a range of troponin concentrations. Troponin concentrations < 5 ng/L on presentation identified half of all patients with suspected acute myocardial infarction as low risk. Implementation of this assay and approach would markedly reduce hospital admissions and improve the diagnosis of myocardial infarction, particularly in women. High-sensitivity cardiac troponin assays may help us to manage patients with chronic cardiovascular diseases, such as aortic stenosis or stable angina pectoris. In patients with aortic stenosis, cardiac troponin was associated with an advanced hypertrophic response on electrocardiography and the presence of myocardial fibrosis on cardiac magnetic resonance imaging. Furthermore, cardiac troponin concentrations were a strong independent predictor of cardiovascular death or aortic valve replacement. In patients with stable angina, cardiac troponin was associated with transient ST-segment depression on ambulatory monitoring, suggesting that troponin concentrations may be modified by silent myocardial ischaemia. My findings suggest that high-sensitivity troponin assays have major potential to improve the assessment, treatment and outcome of patients with suspected acute coronary syndrome. The potential for cardiac troponins to improve risk stratification and monitoring of disease progression in patients with chronic cardiovascular disease now needs to be evaluated in prospective cohort studies.
Armour, Kathryn L.
i) Human cardiac cDNA libraries were constructed and clones encoding human cardiac troponin I (cTnI) isolated. ii) Both the entire <i>cTNI</i> cDNA and a 5'-portion, were expressed in <i>Escherichia coli</i> as fusion products with β-galactosidase. The full-length cDNA was also expressed unfused. iii) The murine monoclonal antibody 29Mu is specific for human and baboon cTnI whereas the 31Mu antibody reacts with cTnI from a range of species. These antibodies might be useful in the imaging of necrotic cardiac tissue. In this study, 31Mu was found to bind to all prepared forms of cTnI antigen, in enzyme-linked immunosorbant assays (ELISAs) and, where tested, in Western blots. Thus, its epitope is localised towards the N-terminus of cTnI. 29Mu bound to the bacterially-produced unfused cTnI but not to the fusion polypeptides or crude bovine cTn. Its ability to bind to human cardiac extracts was related to the method of their preparation, indicating that the epitope of 29Mu shows greater conformational dependency than that of 31Mu. iv) cDNAs encoding the variable domains of 29Mu and 31Mu were cloned and chimaeric antibodies, comprising murine variable and human constant regions produced. Humanised antibodies, in which only the antigen-binding sites were of murine origin, were also produced. Such recombinant antibodies would be expected to exhibit reduced immunogenicity in man. v) Neither the chimaeric nor humanised antibody versions of 29Mu bound cTnI. Chimaerised 31Mu reacted with all forms of cTnI but did not show complete equivalence to 31Mu. An antibody containing the humanised 31 kappa chain and the chimaeric heavy chain was reactive to all forms cTnI in ELISAs but its efficiency of binding, relative to that of the chimaeric antibody, was dependent upon the antigen source. Humanised heavy chains were produced utilising two different human frameworks and the framework, showing closer homology to the 31Mu variable domain, supported antigen binding with fewer murine residue substitutions. However, both successful humanised 31 antibodies showed some cross-reactivity.
The Physiological Consequences of Hypertrophic Cardiomyopathy (HCM) and Restrictive Cardiomyopathy (RCM) Related Mutations in Human Cardiac Troponin IWen, Yuhui 10 July 2008 (has links)
An arginine (R) to a glycine (G) mutation at position 145 in the highly reserved inhibitory domain of cardiac troponin I (cTnI) is associated with hypertrophic cardiomyopathy (HCM), an autosomal dominant disease characterized by left ventricular hypertrophy. An arginine (R) to tryptophan (W) mutation at the same position in cTnI is associated with restrictive cardiomyopathy (RCM), a disease characterized by diastolic dysfunction with normal left ventricular size and normal systolic function. In this study we addressed the functional consequences of the human cardiac troponin I (hcTnI) HCM R145G mutation and hcTnI RCM R145W mutation in transgenic mice. Simultaneous measurements of the ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca2+ activated force without changes in the maximal ATPase activity and an increase in the Ca2+ sensitivity by both ATPase activity and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state) showing that changes in Ca2+ sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared to Tg-WT fibers. The addition of 3mM BDM at pCa 9.0 showed that there was approximately 2~4 percent of force generating cross-bridges attached in Tg-R145G fibers compared to less than 1.0 percent in Tg-WT fibers, suggesting the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca2+] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared to Tg-WT. These results suggest that the phenotype of HCM is most likely caused by the compensatory mechanisms in the cardiovascular system which are activated by: 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge; 2) incomplete relaxation (diastolic dysfunction) caused by prolonged [Ca2+] and force transients; and 3) an inability of the cardiac TnI to completely inhibit activation at low levels of diastolic Ca2+ in Tg-R145G. Simultaneous measurements of the ATPase activity and force in transgenic skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) versus Tg-WT showed that there was a ~13 to ~16 percent increase in the maximal Ca2+ activated force and ATPase activity, respectively. The rate of dissociation of force generating cross-bridges (g) and energy cost (ATPase/force) was the same in all groups of fibers. These results suggest that the increase in force and ATPase activity is associated with an increase in the number of force generating cross-bridges attached at all activation levels. Additionally, there was a large increase in the Ca2+ sensitivity of force development and ATPase activity. In intact fibers, the mutation caused prolonged force and intracellular [Ca2+] transients, as expected due to the increased Ca2+ sensitivity (slower dissociation rate of Ca2+ from cTnC). The above cited results suggest that: 1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca2+ transients, especially at high heart rates; 2) there would be a decrease in ventricular filling (diastolic dysfunction); and 3) an increase in contractility during systole that would off-set the negative effect of a decrease in diastolic filling on ventricle stroke volume thus allowing the heart to maintain normal stroke volume despite the compromise in RCM (Tg-R145W) heart.
Robertson, Ian Michael
No description available.
Structure and dynamics of biomolecules: probing muscle regulation, prion protein unfolding, and drug insertion into DNA by nuclear magnetic resonance spectroscopyJulien, Olivier Unknown Date
No description available.
Structure and dynamics of biomolecules: probing muscle regulation, prion protein unfolding, and drug insertion into DNA by nuclear magnetic resonance spectroscopyJulien, Olivier 06 1900 (has links)
Nuclear magnetic resonance (NMR) spectroscopy is a powerful approach to study the structure and dynamics of macromolecules in a close-to-native solution environment. In the present thesis I present my investigation of protein and nucleic acid structure and dynamics in a wide variety of biological systems using NMR spectroscopy. The center of attention of the Sykes laboratory for the last 35 years has been the role of the Troponin complex in the regulation of muscle contraction. Accordingly, the main focus of this thesis is the study of this important nano-machine, and how its structure and dynamics regulate its biological function. In Chapter II, the perturbation of Troponin C’s structure and dynamics by the attachment of two different bifunctional rhodamine probes is investigated. In Chapter III, the dynamics and position of the bifunctional rhodamine probe when attached on the C helix of Troponin C is studied. In Chapter IV, the structure and dynamics of tryptophan mutants of Troponin C is reported. In Chapter V, the effect of the co-solvent trifluoroethanol on the tryptophan side chain position of mutant F77W of the N-domain of Troponin C is examined. In the following chapter, Chapter VI, the structure and dynamics of a Troponin C – Troponin I chimera is studied using NMR spectroscopy and molecular dynamics simulations to assess the presence or absence of an intrinsically disordered region in Troponin I, and to assess the validity of the flycasting mechanism proposed to regulate muscle contraction. In Chapter VII and VIII, a different topic is introduced. The structural changes occurring during the denaturation process of the bovine prion protein are monitored using NMR spectroscopy to gain insights into the protein misfolding process that causes diseases. In Chapter IX, the structural impact of inserting nucleoside phosphonates into DNA are examined by reporting the NMR structure of a DNA dodecamer duplex containing the modified nucleoside Cidofovir at position 7.
Moderní markery orgánového poškození v klinické praxi / Modern Markers of Organ Damage in Clinical PracticeBrož, Pavel January 2019 (has links)
Physical activity is a useful tool in the prevention of many diseases. Hospitalization after strenuous exercise from cardiac or noncardiac causes, even in young athletes without previous symptoms, can occur. These situations are not uncommon and e.g. after completing a half- marathon clinical symptoms suspicious from cardiac etiology can be present. Limitations of biomarkers used in daily clinical practice can lead to misinterpretation with additional consequences to the patient's outcome. Our goal was to describe changes of markers used in daily clinical practice after extreme physical activity and after exercise under laboratory conditions. We performed two studies in cooperation with Department of cardiology and Department of sports medicine. The goal of our first study was to examine high sensitivity troponin I (hsTnI), galectin-3, cystatin C, NGAL and ultrasensitive CRP (uCRP) after extremely long run during the competition in long distance running. The goal of our second study was to examine high- sensitivity troponin T (hsTnT) and hsTnI, creatinine and cystatin C, and urine albumin and NGAL after a standardized two-hour treadmill run under laboratory conditions and to find possible connection with echocardiographic, laboratory and other assessed parameters. The second goal of study under laboratory...
To compare four methods of CKMB measurement and the qualitative Troponin-T assay as diagnostic discriminants of acute myocardial infarction.January 1996 (has links)
Chui Wai Leung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 120-126). / List of tables and figures --- p.1 / Declaration --- p.6 / Acknowledgments --- p.7 / Summary --- p.8 / Chapter Chapter 1: --- Introduction --- p.10 / Chapter 1.1 --- Acute Myocardial Infarction (AMI) / Chapter 1.2 --- Diagnosis of AMI / Chapter 1.2.1 --- Clinical Signs / Chapter 1.2.2 --- Electrocardiogram (ECG) / Chapter 1.2.3 --- Cardiac enzymes / Chapter 1.3 --- "CKMB,a marker of choice" / Chapter 1.4 --- "Troponin-T, another candidate marker" / Chapter 1.5 --- Objectives / Chapter Chapter 2: --- Analytical evaluation of CKMB measurement by the four methods --- p.20 / Chapter 2.1 --- Analytical methods / Chapter 2.1.1 --- Assay for total creatine kinase / Chapter 2.1.2 --- Assay for CKMB / Chapter 184.108.40.206 --- CKMB mass concentration assay / Chapter 220.127.116.11 --- CKMB EEC & immunoinhibition activity assay / Chapter 18.104.22.168 --- CKMB activity concentration assay1 / Chapter 22.214.171.124 --- CKMB activity concentration assay2 / Chapter 2.2 --- Precision / Chapter 2.3 --- Accuracy / Chapter 2.4 --- Linearity / Chapter 2.5 --- Recovery / Chapter 2.6 --- Interference / Chapter 2.6.1 --- Effect of haemolysis / Chapter 2.6.2 --- Effect of turbidity / Chapter 2.6.3 --- Effect of bilirubin / Chapter 2.7 --- Stability / Chapter Chapter 3 : --- Correlation among the four methods of CKMB measurement --- p.61 / Chapter Chapter 4 : --- Establishment of reference ranges for the four methods of CKMB measurement --- p.71 / Chapter Chapter 5: --- Information on the Qualitative Troponin-T Rapid Assay® --- p.80 / Chapter Chapter 6 : --- Clinical Evaluation of CKMB and Troponin-T in detection of AMI --- p.82 / Chapter 6.1 --- Material and Methods / Chapter 6.1.1 --- Subjects / Chapter 6.1.2 --- Specimens / Chapter 6.1.3 --- Criteria for diagnosis / Chapter 6.1.4 --- Analytical methods / Chapter 6.1.5 --- Statistical methods / Chapter 6.2 --- Results / Chapter 6.3 --- Discussion / Chapter Chapter 7 : --- General Discussion --- p.105 / Appendix 1: study protocol sheet --- p.113 / Appendix 2: diagnostic criteria for a definite AMI --- p.115 / Appendix 3: criteria for exclusion of AMI --- p.117 / Appendix 4: enzyme criteria --- p.118 / References --- p.120
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