Restrictive cardiomyopathy (RCM) is a cardiac muscle disorder characterized by increased ventricular stiffness and diastolic dysfunction. Patients with RCM often present severe cardiac problems which usually lead to heart failure and sudden death. No effective treatment is available for RCM which makes the finding of novel efficient therapies an urgent necessity. Great progress in molecular biology techniques and advances in transgenic animal development provide great opportunities for the study of RCM and other cardiovascular diseases encountered in clinical patients.... Our laboratory is among the first to generate transgenic mouse models of RCM based on cardiac troponin I (cTnI) missense mutations. In this study, transgenic mice that suffer from RCM have been generated to understand the factors behind the diastolic dysfunction associated with that myocardial disease.... The information obtained from this study allows a better understanding of the role of troponin in RCM and the factors behind the physiopathology of the disease. It will also offer a therapeutic strategy taking into account the physiological characteristic of RCM. / by Pierre-Ives Jean-Charles. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Restrictive cardiomyopathy (RCM) is represented in part by left ventricular stiffness and diastolic dysfunction. Missense mutations of the cardiac troponin I (cTnI) gene cause idiopathic RCM. These mutations are located in the C-terminus of cTnI and affect cardiac relaxation. Transgenic mouse models presenting the pathology observed in clinical patients with RCM have been generated previously and express the mutant cTnI in their hearts. RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity and promote diastolic dysfunction in the heart. Previous studies using double transgenic mice (cTnI/R193H/ND) showed that ventricular relaxation is enhanced in the cTnI/R193H transgenic mice. In this study, another double transgenic mouse model, (cTnI/R193H/ND/KO), provides an avenue to investigate its rescuing effects on RCMlinked mutations in the cTnI /R193H/KO mouse. Use of molecular biological techniques, transgenic animal developments and murine echocardiography in this study has culminated into a greater understanding of RCM and diastolic dysfunction. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

INTRODUÇÃO: A tuberculose é um problema grave de saúde pública, acometendo indivíduos em todas as faixas etárias e em todos os estratos socioeconômicos. Apesar de estarem sob grande risco de adoecimento, as crianças carecem de meios diagnósticos sensíveis e específicos. Neste estudo avaliou-se em crianças a acurácia de um teste baseado na dosagem de interferon-gama liberado por linfócitos após estímulo com antígenos específicos do Mycobacterium tuberculosis (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Austrália] ).MÉTODO: Foram incluídas no estudo 184 crianças não infectadas e 11 crianças com infecção pelo Mycobacterium tuberculosis. Todas as crianças receberam previamente o BCG. Foram excluídas crianças com comprometimento do sistema imunológico. Obteve-se amostra de sangue de cada criança, e o material foi processado conforme as instruções do laboratório fabricante. O desempenho do teste foi avaliado pela construção de uma curva de características operacionais (ROC). RESULTADOS: Do total de 184 crianças sem infecção pela micobatéria, 74 (40,2%) eram do sexo feminino, e 130 (70,6%) tinham menos de quatro anos de idade. A idade média neste grupo foi de 35 meses. Seis (3,2%) crianças apresentaram resultado indeterminado do teste, uma criança (0,5%) apresentou um resultado positivo e 177 (96,2%) apresentaram resultado negativo. No grupo de 11 crianças infectadas, sete (63,0%) eram meninas, e a idade média era de 58,5 meses. Duas (18,0%) crianças neste grupo apresentaram resultado negativo do teste. A curva ROC obtida evidenciou uma área sob a curva de 0,876 (I. C 95% - 0,82 a 0,92; p<0,001), refletindo o desempenho preditivo elevado do teste. A sensibilidade do teste foi de 81,8% (IC 95% - 48,2% a 97,2%) e a especificidade de 98,8% (IC 95% - 96,0 a 99,8%), o valor preditivo positivo foi de 81,8% (IC 95%: 46,3% a 97,4%) e o valor preditivo negativo foi de 98,9% (IC 95%: 96,0% a 99,8%). CONCLUSÕES: Neste estudo o teste mostrou ter uma boa acurácia no diagnóstico da infecção pelo Mycobaterium tuberculosis em crianças previamente vacinadas com o BCG, e sua utilização rotineira pode contribuir para a melhor avaliação de crianças expostas a um doente bacilífero e na tomada de decisões sobre a introdução de quimioprofilaxia ou tratamento. / BACKGROUND: Tuberculosis is a major public health problem, affecting people from all ages and diverse socioeconomic incomes. Despite the high risk that children have to develop the disease, accurate methods for diagnosis are not yet available. In this study the accuracy of an interferon-gamma release assay (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Australia]) was evaluated for the diagnosis of Mycobacterium tuberculosis infection in children. METHODS: 195 children were evaluated, 184 children without mycobacterial infection, and 11 children infected by the Mycobacterium tuberculosis. All the children had been previously vaccinated with BCG. Immunocompromised children were excluded from the study. A blood sample was obtained from each child, and it was processed according the manufacturer´s instructions. The performance of the assay was evaluated by a receiver operating characteristic (ROC) curve. RESULTS: In the group of 184 noninfected children, 74 (40.2%) were female and 130 (70.6%) were younger than four years old. The mean age in this group was 35 months. Six children (3.2%) had indeterminate test result, one child (0.5%) had a positive test result, and 177 (96.2%) children had negative test results. In the group of 11 infected children, seven (63.0%) were female, and the mean age in this group was 58.5 months. Two children (18.0%) in this group had a negative test result. The ROC curve determined an area under the curve of 0.876 (95% CI 0.82 to 0.92; p< 0.001), disclosing a high positive predictive value for the test. The assay sensibility was 81.8% (95% CI, 48.2% to 97.2%) and the assay specificity was 98.8% (95% CI, 96.0% to 99.8%), the positive predictive value was 81.8% (95% CI: 46.3% to 97.4%) and the negative predictive value was 98.9% (95% CI: 96.0% to 99.8%). CONCLUSIONS: In this study, the accuracy of the assay was high for the diagnosis of Mycobacterium tuberculosis infection in children previously vaccinated with BCG. The use of this assay for the routine evaluation of children exposed to the disease may help physicians to decide on whether to start chemoprophylaxis or tuberculosis treatment.

Shen, Jiayun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 166-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

INTRODUÇÃO: A tuberculose é um problema grave de saúde pública, acometendo indivíduos em todas as faixas etárias e em todos os estratos socioeconômicos. Apesar de estarem sob grande risco de adoecimento, as crianças carecem de meios diagnósticos sensíveis e específicos. Neste estudo avaliou-se em crianças a acurácia de um teste baseado na dosagem de interferon-gama liberado por linfócitos após estímulo com antígenos específicos do Mycobacterium tuberculosis (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Austrália] ).MÉTODO: Foram incluídas no estudo 184 crianças não infectadas e 11 crianças com infecção pelo Mycobacterium tuberculosis. Todas as crianças receberam previamente o BCG. Foram excluídas crianças com comprometimento do sistema imunológico. Obteve-se amostra de sangue de cada criança, e o material foi processado conforme as instruções do laboratório fabricante. O desempenho do teste foi avaliado pela construção de uma curva de características operacionais (ROC). RESULTADOS: Do total de 184 crianças sem infecção pela micobatéria, 74 (40,2%) eram do sexo feminino, e 130 (70,6%) tinham menos de quatro anos de idade. A idade média neste grupo foi de 35 meses. Seis (3,2%) crianças apresentaram resultado indeterminado do teste, uma criança (0,5%) apresentou um resultado positivo e 177 (96,2%) apresentaram resultado negativo. No grupo de 11 crianças infectadas, sete (63,0%) eram meninas, e a idade média era de 58,5 meses. Duas (18,0%) crianças neste grupo apresentaram resultado negativo do teste. A curva ROC obtida evidenciou uma área sob a curva de 0,876 (I. C 95% - 0,82 a 0,92; p<0,001), refletindo o desempenho preditivo elevado do teste. A sensibilidade do teste foi de 81,8% (IC 95% - 48,2% a 97,2%) e a especificidade de 98,8% (IC 95% - 96,0 a 99,8%), o valor preditivo positivo foi de 81,8% (IC 95%: 46,3% a 97,4%) e o valor preditivo negativo foi de 98,9% (IC 95%: 96,0% a 99,8%). CONCLUSÕES: Neste estudo o teste mostrou ter uma boa acurácia no diagnóstico da infecção pelo Mycobaterium tuberculosis em crianças previamente vacinadas com o BCG, e sua utilização rotineira pode contribuir para a melhor avaliação de crianças expostas a um doente bacilífero e na tomada de decisões sobre a introdução de quimioprofilaxia ou tratamento. / BACKGROUND: Tuberculosis is a major public health problem, affecting people from all ages and diverse socioeconomic incomes. Despite the high risk that children have to develop the disease, accurate methods for diagnosis are not yet available. In this study the accuracy of an interferon-gamma release assay (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Australia]) was evaluated for the diagnosis of Mycobacterium tuberculosis infection in children. METHODS: 195 children were evaluated, 184 children without mycobacterial infection, and 11 children infected by the Mycobacterium tuberculosis. All the children had been previously vaccinated with BCG. Immunocompromised children were excluded from the study. A blood sample was obtained from each child, and it was processed according the manufacturer´s instructions. The performance of the assay was evaluated by a receiver operating characteristic (ROC) curve. RESULTS: In the group of 184 noninfected children, 74 (40.2%) were female and 130 (70.6%) were younger than four years old. The mean age in this group was 35 months. Six children (3.2%) had indeterminate test result, one child (0.5%) had a positive test result, and 177 (96.2%) children had negative test results. In the group of 11 infected children, seven (63.0%) were female, and the mean age in this group was 58.5 months. Two children (18.0%) in this group had a negative test result. The ROC curve determined an area under the curve of 0.876 (95% CI 0.82 to 0.92; p< 0.001), disclosing a high positive predictive value for the test. The assay sensibility was 81.8% (95% CI, 48.2% to 97.2%) and the assay specificity was 98.8% (95% CI, 96.0% to 99.8%), the positive predictive value was 81.8% (95% CI: 46.3% to 97.4%) and the negative predictive value was 98.9% (95% CI: 96.0% to 99.8%). CONCLUSIONS: In this study, the accuracy of the assay was high for the diagnosis of Mycobacterium tuberculosis infection in children previously vaccinated with BCG. The use of this assay for the routine evaluation of children exposed to the disease may help physicians to decide on whether to start chemoprophylaxis or tuberculosis treatment.

Chan, Ka Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 133-150). / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.iv / CONTRIBUTORS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xiii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY 21 --- p.2 / Chapter 1.1 --- Down syndrome --- p.2 / Chapter 1.2 --- Current methods of prenatal diagnosis of fetal trisomy 21 --- p.3 / Chapter 1.2.1 --- Non-invasive procedures --- p.3 / Chapter 1.2.2 --- Invasive procedures --- p.5 / Chapter 1.3 --- Alternative methods for the prenatal diagnosis of fetal trisomy 21 --- p.7 / Chapter CHAPTER 2: --- CELL-FREE FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.13 / Chapter 2.1 --- Circulating fetal cells --- p.15 / Chapter 2.2 --- Circulating cell-free fetal nucleic acids --- p.15 / Chapter 2.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.17 / Chapter 2.4 --- Digital relative chromosome dosage approach --- p.20 / Chapter 2.5 --- Validation of digital RCD approach on artificial DNA mixtures --- p.22 / Chapter SECTION II --- : MATERIALS AND METHODS --- p.25 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.26 / Chapter 3.1 --- Subject recruitment and sample collection --- p.26 / Chapter 3.2 --- Sample processing --- p.26 / Chapter 3.3 --- Nucleic acid extraction --- p.27 / Chapter 3.3.1 --- Extraction of DNA from placental tissues --- p.27 / Chapter 3.3.2 --- Extraction of DNA from maternal blood cells --- p.27 / Chapter 3.4 --- Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) --- p.28 / Chapter 3.5 --- Paralogous sequence assays optimisation workflow --- p.31 / Chapter 3.5.1 --- Monoplex paralogous sequence assays --- p.31 / Chapter 3.5.2 --- Multiplex paralogous sequence assay --- p.38 / Chapter 3.6 --- Digital PCR --- p.42 / Chapter 3.6.1 --- Principle --- p.42 / Chapter 3.6.2 --- Digital multiplex paralogous sequence assay --- p.42 / Chapter 3.7 --- Statistical analysis --- p.46 / Chapter 3.7.1 --- Disease classification of samples --- p.46 / Chapter 3.7.2 --- Poisson distribution --- p.46 / Chapter 3.7.3 --- Data analysis --- p.48 / Chapter 3.7.4 --- Sequential probability ratio test (SPRT) analysis --- p.49 / Chapter SECTION III: --- ASSAY DEVELOPMENT --- p.53 / Chapter CHAPTER 4: --- TESTING OF ASSAY SPECIFICITY WITH CORIELL CELL LINES --- p.54 / Chapter 4.1 --- Coriell cell lines --- p.54 / Chapter 4.2 --- Specificity of initial PCR primers --- p.56 / Chapter 4.2.1 --- Principle --- p.56 / Chapter 4.2.2 --- Materials and methods --- p.56 / Chapter 4.2.3 --- Results --- p.60 / Chapter 4.2.4 --- Conclusion --- p.63 / Chapter 4.3 --- Specificity of the iPLEX® Gold extension primers --- p.63 / Chapter 4.3.1 --- Principle --- p.63 / Chapter 4.3.2 --- Materials and methods --- p.64 / Chapter 4.3.3 --- Results --- p.65 / Chapter 4.4 --- Further analysis on the specificity of PV2107a initial PCR primers --- p.67 / Chapter 4.5 --- Conclusion --- p.71 / Chapter CHAPTER 5: --- ASSAY OPTIMISATION --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Optimisation of initial PCRs with AmpliTaq Gold® DNA polymerase followed by homogeneous MassEXTEN´DёØ (hME) assays (Sequenom) --- p.72 / Chapter 5.2.1 --- Optimisation of initial PCR reactions --- p.72 / Chapter 5.2.2 --- Principle of homogeneous MassEXTEN´DёØ assays (Sequenom)… --- p.75 / Chapter 5.2.3 --- Homogeneous MassEXTEN´DёØ assays (Sequenom) on euploid and T21 samples --- p.76 / Chapter 5.3 --- Assay selection by iPLEX® Gold single base primer extension reactions (Sequenom) --- p.82 / Chapter 5.4 --- Optimisation of multiplex PCR with AmpliTaq Gold® DNA polymerase --- p.88 / Chapter 5.5 --- Optimisation of multiplex iPLEX® Gold single base primer extension reaction --- p.93 / Chapter 5.6 --- Single molecule detection test for the multiplex paralogous sequence assays … --- p.103 / Chapter SECTION IV: --- ANALYSIS OF CLINICAL SAMPLES --- p.107 / Chapter CHAPTER 6: --- DISEASE CLASSIFICATION OF EUPLOID AND TRISOMY SAMPLES WITH MULTIPLEX PARALOGOUS SEQUENCE ASSAY --- p.108 / Chapter 6.1 --- Introduction --- p.108 / Chapter 6.2 --- Materials and methods --- p.109 / Chapter 6.2.1 --- Sample collection --- p.109 / Chapter 6.2.2 --- Experimental design --- p.110 / Chapter 6.3 --- Results --- p.111 / Chapter 6.4 --- Discussion --- p.114 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.122 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.123 / Chapter 7.1 --- Conclusion --- p.123 / Chapter 7.2 --- Future perspectives --- p.124 / Appendix 1 --- p.126 / Appendix II --- p.127 / REFERENCE --- p.133

Over the last 50 years, flow cytometry has evolved from a modest cell counter into an invaluable analytical tool that measures an ever-expanding variety of phenotypes. Flow cytometers interrogate passing samples with laser light and measure the elastically scattered photons to ascertain information about sample size, granularity, and basic morphology. Obtaining molecular information, however, requires the addition of exogenous fluorescent labels. These labels, although a power tool, have numerous challenges and limitations such as large emission spectra and cellular toxicity. To move beyond fluorescent labels in microscopy, a variety of techniques that probe the intrinsic Raman vibrations within a sample have been developed, such as coherent anti-Stokes Raman scattering (CARS) and Raman microspectroscopy. In this dissertation, I present the first development of a label-free flow cytometer that measures the elastically scattered photons and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, a coherent Raman technique that probes a large region of the Raman spectrum simultaneously, provides rich molecularly-sensitive information. Furthermore, I present its application to sorting polymer microparticles and its use in two example biological applications: monitoring lipid bodies within cultures of Saccharomyces cerevisiae, a model yeast with numerous human homologs, and monitoring the affect of nitrogen starvation on Phaeodactylum tricornutum, a diatom, which is being genetically engineered to efficiently produce biofuels.

Brain and central nervous cancer presents a significant clinical burden, accounting for 2.4% of all cancer deaths. High grade glioma is particularly deadly, with 5 year survival times of 35% or less. Traditional treatment includes tumor resection followed by radiation therapy or chemotherapy. Aggressive resection is essential in order to prolong patient life. In fact, several studies have shown that life expectancy increases with increased extent of resection. Extent of resection is burdened by the fact that surgeons must be careful not to remove functional brain tissue. Resection is incomplete more often than not due to lack of visual cues for the surgeon. He must rely on tactile sensation to distinguish tumor from healthy tissue. Methods such as intraoperative MRI and CT exist, but these require expensive equipment and special training that is not available in all surgical environments. Some laboratories have proposed small molecule dyes to solve this problem, but these are insufficient when used in an invasive tumor model. It was the goal of this research to provide an objective cue in the form of a nanoencapsulated visible dye without the need for additional equipment of changes to the surgery process itself other than injection of the dye. We hypothesized that the nanocarrier would allow staining of the tumor through passive targeting by taking advantage of the enhanced permeability and retention effect. Once the nanocarriers have reached the desired target, they would not diffuse out into healthy tissue due to their large size compared to small molecule dyes, which readily diffuse out and stain healthy tissue. To test this hypothesis, we prepared and characterized a liposomal nanocarrier encapsulating Evans blue dye. The nanocarrier was tested for safety in vitro and in vivo, then used to delineate tumor margins in an invasive rat glioma model in vivo. Microscopic analysis was then conducted to ensure only tumor tissue was stained by the nanocarrier. This thesis presents a successful method of tumor border delineation to provide surgeons with positive visual cues without the need for changes in surgical environment or techniques.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences