Tesis sobre el tema "Fibrosis Assessment"

Cystic fibrosis (CF) is an inherited disease leading to disrupted function of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. CF affects many organ systems including the pancreas, liver, intestine, sweat glands, and gallbladder. The leading cause of morbidity and mortality, however, is lung disease. A porcine model of CF was developed, and over time it develops lung disease that recapitulates many of the characteristics observed in humans with CF including airway remodeling, mucus accumulation, infection, and inflammation. At birth, and despite the absence of inflammation and infection, the CF pig airways exhibit a host of abnormalities including tracheal cartilage ring defects, abnormal appearing smooth muscle bundles, reduced trachea diameter, and reduced mainstem bronchi diameter. The primary objectives of this study were to construct an experimental method that allowed for the attainment of airway size information at multiple inflation pressures, to assess the extent of airway narrowing in the newborn CF porcine lung at 20 cmH2O, to determine the tracheal lobe volume for CF and non-CF, and to perform morphologic assessment of the parenchymal airspaces for CF and non-CF newborn pigs. Micro-computed tomography (micro-CT) was selected as the primary analysis tool. The volumetric, high resolution data sets of micro-CT provided a means to virtually track airways through the three dimensional space of the lung, and to image airways as small as 250 microns in diameter. Due to experimental constraints, only one lobe was analyzed: the tracheal lobe; it is the porcine equivalent of the human right upper lobe. Each excised tracheal lobe was cannulated and micro-CT scanned five times. Each lobe was scanned at multiple inflation pressures ranging from 0 to 20 cmH2O. The airways were segmented with a custom designed, substantially-automated computer algorithm. Quantitative analysis of airway size was done with the Pulmonary Workstation 2 software package. At a pressure of 20 cmH2O, the CF airway narrowing was most pronounced in the large airways of the tracheal lobe, and the percent difference in airway cross sectional area between CF and non-CF lessened for airways of smaller size. The volume of the newborn CF pig tracheal lobe was approximately twenty percent smaller than non-CF, but no differences were observed in tracheal lobe airspace histology between the groups. Airway size deviations at birth imply developmental abnormalities in utero that are dependent upon CFTR function. Additionally, the observation that reduced airway caliber exists only in relatively large airways suggests a time-dependent role of CFTR on airway development, as the large airways develop before the small ones in utero. These findings may provide insight to the early pathogenesis of CF lung disease.

Despite affecting many organ systems, the leading cause of morbidity and mortality in the cystic fibrosis (CF) population is lung disease. For the current studies we investigated elements of CF lung disease in a porcine model of CF and in people with CF. Our primary analysis tool was chest computed tomography (CT). To investigate early CF lung disease we examined three week old CF and non-CF pigs. We found three week old CF pigs to have large, irregular tracheal smooth muscle bundles, airways of reduced size, airways of irregular shape, and airways of abnormal distensibility. Three week old CF pig lung parenchyma was more heterogenous in density than three week non-CF pigs, especially in the right cephalad lung. The degree of lung tissue heterogeneity in CF pigs correlated with the degree of lung infection. Three week old CF pigs also had significantly more air trapping upon exhalation, evidence of airflow obstruction, than non-CF pigs. The degree of air trapping correlated with the degree of mucus accumulation in the airways. These data show that CF pigs spontaneously develop hallmark features of CF lung disease within weeks of birth, and that abnormal airway growth and development in CF may contribute to lung disease. This study helped set the foundation for future comparative studies involving CF therapeutics, for example, antibiotics and mucolytics. In adults with CF we performed a before drug, after drug study. The drug was ivacaftor, and it restores the basic underlying defect in a subset of people with CF: impaired function of a particular anion channel. We hypothesized that abnormal airway smooth muscle behavior in people with CF, known as “CF asthma,” is, in part, a primary pathogenic mechanism of CF lung disease. We tested our hypothesis by assaying smooth muscle tone before and after administration of ivacaftor. We limited the time duration to two days. We reasoned two days was long enough for ivacaftor to become effective, but not long enough to reverse long standing lung infection and inflammation which could affect smooth muscle function independently. The implication being, that observed changes would be directly due to restoration of the CF defect. We found evidence suggesting relaxation of airway and vascular smooth muscle tone. And, the change in airway smooth muscle tone correlated with the change in vascular smooth muscle tone. These data suggest that impaired smooth muscle function is a primary element of CF lung disease. Many of the people in our two day ivacaftor study returned for follow up after one year of ivacaftor therapy. We hypothesized that radiographic features of lung disease would improve following one year of ivacaftor therapy. We observed no change in lung volume upon inspiration, but a reduction in expiratory lung volume, approximately half of which occurred within two days. Our airway measurements were confounded by errors in scan reconstruction, however, other published studies report airway wall thinning over long term ivacaftor administration. Taken together, these studies of pigs with CF and people with CF, help us understand this disease.

Schistosomiasis is a water-borne tropical disease plaguing approximately 240 million people worldwide. Approximately 3-70 million disability-adjusted life years are lost due to the disease. In the Philippines, schistosomiasis japonica is currently endemic in 28 provinces, 190 municipalities, and 2230 barangays (villages). Approximately 12 million residents of these endemic areas are vulnerable to infection. Preventive chemotherapy with praziquantel has been the cornerstone for schistosomiasis control in the country. The current national control program comprises annual free mass drug administration (MDA) (40 mg/kg PZQ) in all schistosomiasis-endemic communities with a prevalence of >10%. The Philippine National Schistosomiasis Control Program has recently reported that human prevalence has declined to less than 3% nationally. However, contradictory reports claim the national program is faltering. Poor drug coverage, poor drug compliance, infrequent monitoring and evaluation, and high reinfection rates daunt control efforts. Zoonotic transmission further complicates control efforts. Furthermore, advanced disease cases and schistosomiasis-related deaths are currently being reported in endemic areas throughout the country.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Background: Measurements of lung function are routinely used in patients with cystic fibrosis (CF) to provide information that may be clinically relevant. Spirometry is the conventional lung function measurement used, however young children find spirometry difficult to perform and often cannot achieve the strict acceptability criteria for the test. The forced oscillation technique (FOT) is a lung function measurement that only requires tidal breathing and is easy for young children to perform. However, there is limited information about the utility of this technique in the clinical assessment of young children with CF who are unable to perform spirometry. Aims: The aim of this project was to evaluate the FOT for clinical assessment in 2 to 7 year old children with CF. Specifically this involved: 1. Technical assessment of the FOT in children with CF; 2. Comparisons of lung function using the FOT in children with CF and healthy children; 3. Evaluation of associations with factors known to be associated with lung disease including: i) inflammation ii) infection and iii) structural damage. Methods Lung function was measured in a cohort of 59 children between the ages of 2 and 7 years with CF at the time of quarterly clinic visits. Resistance and reactance at 6, 8 and 10Hz (Rrs6, Rrs8, Rrs10, Xrs6, Xrs8, Xrs10, respectively) were reported and expressed as Z scores. Children were classified as asymptomatic or symptomatic based on a respiratory questionnaire and physical examination at the time of testing. Bronchoalveolar lavage and high resolution computed tomography (HRCT) were performed annually under general anaesthesia annually. BAL fluid was assessed for the presence of micro-organisms and quantification of a range of inflammatory markers and HRCT used to determine the extent of structural abnormalities. Results: The between test repeatability (n=25) for lung function was within limits previously described in healthy children. No systematic bias was observed and repeatability was not affected by the presence of respiratory symptoms. Children with CF (n=57) had significantly increased Rrs6-10 (p<0.0001) and decreased Xrs6-10 (p<0.004) compared to healthy children. Rrs6 and Xrs6-10 were significantly worse in the presence of respiratory symptoms, and Rrs6-10 progressively worsened from an asymptomatic to a symptomatic clinic visit. Children with CF (n=48) had no greater bronchodilator response (BDR) compared to healthy children. BDR was not influenced by the presence of an infection or respiratory symptoms. No relationships between inflammatory markers and lung function (n=39) were identified when the presence of an infection was adjusted for. Children with a current infection (n=20) had increased Rrs6-10 (p<0.01) and decreased Xrs6-10 (p<0.04) compared to children who were uninfected (n=23). These relationships were most marked for children infected with Pseudomonas aeruginosa, with children having a reduced lung function between 0.95 and 1.47 of a Z score. No relationships with the presence or absence of mild structural abnormalities (bronchiectasis, bronchial wall thickening and air trapping) and lung function at the time of HRCT were identified (n=34). Conclusion: The FOT is a repeatable measurement of lung function in children with CF and reliable results can be obtained in children as young as 2 years old. Young children with CF exhibit altered respiratory function which was affected by the presence of factors known to be associated with lung disease. The FOT has the potential to provide useful information about changes in clinical status in young children with CF and may be used to direct management of patient lung disease.

Cystic fibrosis (CF) is the most common fatal inherited single gene defect in Caucasian populations. CF lung disease is characterised by infection, inflammation and progressive lung destruction. Lung inflammation is measurable in CF patients even with early disease. Gene therapy offers a theoretical cure for CF lung disease, with large clinical trials now being planned. There is not only a clear need for the development of new therapies in CF but also the means to measure the success of these therapies. One possible approach is to measure biomarkers of airway inflammation non-invasively (in sputum or serum). In this thesis I have employed a number of techniques to measure potential biomarkers in sputum and blood in both cross sectional and serial samples following treatment. Sputum was collected from patients with CF and a number of control groups including Asthma, Bronchiectasis, COPD and healthy controls. SELDI-TOF mass spectrometry was utilised to identify candidate protein biomarkers in sputum. Candidate biomarkers were then identified and compared to established biomarkers by ELISA in sputum. Emission spectroscopy was used to measure metal ions as non-protein biomarkers in sputum. SELDI TOF, ELISA and optical spectroscopy were used to measure biomarkers in CF sputum before and after exacerbation treatment. Calprotectin was also measured in serum before and after exacerbation. SELDI TOF identified calprotectin as a marker of CF lung disease, which highly discriminated CF from control. This could also be measured by ELISA and compared favourably to other inflammatory markers such as Interleukin-8 (IL-8). Emission spectroscopy identified sputum zinc and iron as discriminatory markers of CF. Sputum calprotectin and zinc levels changed significantly following treatment of CF exacerbation. Serum calprotectin also changed significantly and could predict future outcome in these patients. In this thesis I demonstrate the discover}' and application of novel biomarkers of CF lung inflammation. I describe calprotectin (sputum and serum) as useful in the monitoring of exacerbation therapy, with similar findings being displayed for sputum zinc. Further work is now required to fully validate these findings for translation into clinically useful tools.

Introduction: Non-invasive assessment of steatosis and fibrosis is of growing relevance in non-alcoholic fatty liver disease (NAFLD). 1H-Magnetic resonance spectroscopy (1H-MRS) and the ultrasound-based controlled attenuation parameter (CAP) correlate with biopsy proven steatosis, but have not been correlated with each other so far. We therefore performed a headto- head comparison between both methods. Methods: Fifty patients with biopsy-proven NAFLD and 15 healthy volunteers were evaluated with 1H-MRS and transient elastography (TE) including CAP. Steatosis was defined according to the percentage of affected hepatocytes: S1 5-33%, S2 34–66%, S3 $67%. Results: Steatosis grade in patients with NAFLD was S1 36%, S2 40% and S3 24%. CAP and 1H-MRS significantly correlated with histopathology and showed comparable accuracy for the detection of hepatic steatosis: areas under the receiveroperating characteristics curves were 0.93 vs. 0.88 for steatosis $S1 and 0.94 vs. 0.88 for $S2, respectively. Boot-strapping analysis revealed a CAP cut-off of 300 dB/m for detection of S2-3 steatosis, while retaining the lower cut-off of 215 dB/m for the definition of healthy individuals. Direct comparison between CAP and 1H-MRS revealed only modest correlation (total cohort: r = 0.63 [0.44, 0.76]; NAFLD cases: r = 0.56 [0.32, 0.74]). For detection of F2–4 fibrosis TE had sensitivity and specificity of 100% and 98.1% at a cut-off value of 8.85 kPa. Conclusion: Our data suggest a comparable diagnostic value of CAP and 1H-MRS for hepatic steatosis quantification. Combined with the simultaneous TE fibrosis assessment, CAP represents an efficient method for non-invasive characterization of NAFLD. Limited correlation between CAP and 1H-MRS may be explained by different technical aspects, anthropometry, and presence of advanced liver fibrosis.

The purpose of this thesis was to extend our understanding of the assessment and interpretation of aerobic exercise function of paediatric patients with cystic fibrosis (CF). The first investigation sought to establish (1) the validity of traditional criteria to verify maximal oxygen (V ̇O2max) during a maximal cardiopulmonary exercise test (CPET); and (2) the utility of supramaximal verification (Smax) to confirm V ̇O2max. Traditional criteria significantly underreported V ̇O2max, whilst Smax was shown to provide a valid measurement in this patient group. The reproducibility of this CPET protocol, over the short- (48 h) and medium- (4-6 weeks) term, was then established in study two. V ̇O2max was repeatedly determined with no learning effect over 48 h (typical error (TE): ∆150 mL; ∆9.3%) and 4-6 weeks (TE: ∆160 mL; ∆13.3%). Supplementary maximal and submaximal CPET parameters should be incorporated for a comprehensive evaluation of a patient, however they are characterised by greater variability over time. The influence of mild-to-moderate CF on aerobic exercise function and the matching of muscle O2 delivery-to-O2 utilisation during ramp incremental exercise to exhaustion were then examined in study three. Aerobic function was impaired in CF, indicated by very likely reduced fat-free mass normalised V ̇O2max (mean difference, ±90% CI: -7.9 mL∙kg-1∙min-1, ±6.1), very likely lower V ̇O2 gain (-1.44 mL∙min-1∙W-1, ±1.12) and a likely slower V ̇O2 mean response time (MRT) (11 s, ±13). Arterial oxygen saturation was lower in CF, supporting the notion that centrally mediated O2 delivery may be impaired during ramp incremental exercise. Although a faster rate of fractional O2 extraction would be expected in the face of reduced O2 delivery, this was not observed, suggesting additional impairment in O2 extraction and utilisation at the periphery in CF. The fourth study then demonstrated the clinical utility of CPET to assess the response to 12 weeks treatment with Ivacaftor, using a case-based design. Whilst one patient with relatively mild disease demonstrated no meaningful change in V ̇O2max, the second demonstrated a 30% improvement in V ̇O2max, due to increased O2 delivery and extraction. Furthermore, changes in aerobic function were detected earlier than spirometric indices of pulmonary function. This study demonstrated that CPET represents an important and comprehensive clinical assessment tool and its use as an outcome measure in the functional assessment of patients is encouraged. Study five investigated the V ̇O2 kinetics in this patient group. During moderate intensity cycling, the phase II V ̇O2 time constant (τ) (p = 0.84, effect size (ES) = 0.11) and overall MRT (p = 0.52, ES=0.33) were not slower in CF. However, both were slowed during very heavy intensity cycling (p = 0.02, ES = 1.28 and p = 0.01, ES = 1.40, respectively) in CF. Cardiac output and muscle deoxygenation dynamics were unaltered in CF, however, the arterial-venous O2 content difference (C(a-v ̅)O2) was reduced (p=0.03) during VH and ∆C(a-v ̅)O2 correlated with the phase II τ (r= -0.85; p=0.02) and MRT (r = -0.79; p=0.03) in CF. This study showed that impaired oxidative muscle metabolism in this group is exercise intensity-dependent and mechanistically linked to an intrinsic intramuscular impairment, which limits O2 extraction and utilisation. In conclusion, this thesis has provided guidelines for a valid and reproducible CPET protocol for children and adolescents with mild-to-moderate CF, demonstrated the utility of CPET as clinical outcome measure and furthered our understanding of the factors responsible for impaired aerobic exercise function in this patient group.

Cystic Fibrosis (CF) causes chronic lower respiratory tract infection leading to morbidity and mortality. CF Pulmonary Exacerbations (CFPEs) cause accentuated symptoms and increase mortality. The definition and aetiology of CFPEs has however proved elusive. Recently, culture independent techniques have shown that there is much greater diversity of bacteria than previously detected by culture dependent methods. Building on this, in the work presented here, the bacteria in respiratory samples from adults with CF were studied over a 12 month period. Each subject provided thrice weekly sputum samples for analysis by culture and culture independent microbiological methods. Concurrently an in-depth assessment of their subjective and objective health using Visual Analogue Scales (VAS) and spirometry (FEV1) was undertaken. Inflammation markers were also measured. A total of 2061 samples from fourteen adults (mean age 30.2; mean FEV1% predicted 53.3%; 6 females; 8 ?F508 homozygotes) were collected. Subjective VAS measures correlated with objective spirometric measures. However, previously unsuspected complexity of subjective symptomatology was found. Ribosomal clone sequence analysis identified 90 different species, including 15 not previously reported in CF lung disease. Notably 44% of species detected were obligate anaerobes, and 72% were species previously associated with the human oro-pharynx. During the study period, subjects experienced 42 CFPEs requiring treatment. New species were not seen to enter the bacterial community as aetiological agents for CFPEs. However, whilst treatment for CFPEs caused a large fall in the proportion of anaerobic species, no significant change in the proportion of Pseudomonas aeruginosa was detected. Significant and potentially important differences in bacterial community composition, structure and stability between subjects separated by gender, genotype and lung function were observed. Moreover, the presence of certain species correlated with subjects suffering frequent CFPEs. The results presented here give new insights in to the complexity of symptoms and bacterial diversity in CF pulmonary disease

Introduction. Cystic fibrosis (CF) is the most common fatal genetic disease in the Western world. Fifty years ago, most sufferers died before their first birthday. With improved medical care, survival has now increased to adulthood. Respiratory failure, due to recurrent pulmonary exacerbations, is the main cause of mortality and morbidity. To enable clinical trials of new therapies, biomarkers of pulmonary disease are required. Traditionally, spirometry is used, but this is no longer sensitive enough. The development of chronic hyperglycaemia in CF is associated with accelerated pulmonary decline. Thus this thesis aims to determine if increased airway glucose concentration, 'breath glucose', can be used as a biomarker of pulmonary disease in CF. Methods. Exhaled breath condensate (EBC) was collected and analysed for glucose in 254 adult patients with CF when clinically stable. Breath glucose was estimated from condensate glucose (quantified using high performance liquid chromatography) corrected with a dilution factor (estimated using EBC cation concentration). Intra- and inter-day repeatability for breath glucose was determined. The relationship between breath glucose and clinical variables was established, including: lung function; infection; time to next exacerbation; exacerbation rate. Results. Breath glucose is statistically repeatable, but results are variable and inaccurate for clinical use. The coefficient of variation was 81 ± 38% for within day and 109 ± 39% for between days. Breath glucose was not related to any clinical parameter, including spirometry (p=O.867), sputum microbiology (p=O.251), diabetic status (p=O.706). Breath glucose did not predict time to (p=0.487) or rate of (p=0.463) pulmonary exacerbation within 52 weeks. Conclusion. Breath glucose is not a useful biomarker. Analysis of EBC is technically challenging, so ASL glucose is difficult to quantify accurately. Other studies do suggest an association between hyperglycaemia, airway glucose and pulmonary inflammation. Therefore, this negative result is likely due to methodology.

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

BACKGROUND Aortic stenosis affects not only the valve but also the myocardium. In response to the increased afterload, left ventricular hypertrophy initially occurs as a compensatory response to maintain wall stress and cardiac output but ultimately, decompensation and heart failure ensues. The transition from adaptation to decompensation is driven by myocyte death and myocardial fibrosis. The aims of the thesis are to investigate cardiovascular magnetic resonance assessment of disease severity and myocardial fibrosis, and explore its relationship with other biomarkers of disease activity and clinical outcome in patients with aortic stenosis. METHODS AND RESULTS The conventional assessment of aortic stenosis relies heavily on two-dimensional and Doppler echocardiography but there are inherent limitations in echocardiography that can affect the severity classification. I demonstrated that cardiovascular magnetic resonance offered a more accurate estimation of left ventricular volumes and mass, and excellent myocardial characterization. Indeed, inaccurate stroke volume estimation by Doppler echocardiography and inconsistent thresholds in current guidelines accounted for more than 40% of patients with discordant small-area, lowgradient aortic stenosis. These data may explain the variable prognosis reported in this unique group of patients, and argue for more accurate assessment of borderline cases with cardiovascular magnetic resonance. Late gadolinium enhancement imaging detects focal areas of established myocardial fibrosis. In many conditions, including aortic stenosis, a more diffuse form of fibrosis predominates, which is potentially reversible and not readily identified by late gadolinium enhancement. Recently several myocardial T1 mapping approaches have been developed to quantify diffuse fibrosis. Using a standardized and systematic approach, I compared several commonly used T1 mapping techniques and identified that extracellular volume had the best profile (reproducibility and discriminatory potential) for the identification of diffuse fibrosis in patients with aortic stenosis. Cardiac troponin is a structural protein present in the cardiac myocytes. Recent advances in assay technology have substantially improved sensitivity, allowing quantification of troponin concentrations with a high degree of precision in everyone. In more than 250 patients with aortic stenosis, I demonstrated that cardiac troponin I concentrations were independently associated with markers of left ventricular decompensation (hypertrophy and fibrosis) and predicted clinical outcome in patients with aortic stenosis. This suggests that myocardial fibrosis detected by cardiovascular magnetic resonance is consequent on myocardial injury secondary to left ventricular decompensation. Left ventricular hypertrophy with strain pattern on a 12-lead electrocardiogram is associated with poor outcome in patients with aortic stenosis, but the mechanism of this electrocardiographic pattern has not been described. In more than 300 patients with aortic stenosis, I demonstrated that these characteristic repolarization abnormalities were a highly specific marker of focal mid-wall myocardial fibrosis (specificity of 99% and sensitivity of 54%). Moreover, the prognostic value of this electrocardiographic pattern was again confirmed with markedly worse long-term outcomes in these patients. CONCLUSION I have demonstrated that cardiovascular magnetic resonance can assist in the assessment of disease severity in patients with aortic stenosis and discordant echocardiographic findings. Moreover, I have validated the assessment of diffuse myocardial fibrosis, as well as, demonstrated the close association between myocardial fibrosis and biomarkers of myocardial injury and electrocardiographic strain pattern that predicted an adverse outcome in patients with aortic stenosis.

Clinical research in cystic fibrosis (CF) requires study endpoints that are sensitive to airways disease, repeatable and non-invasive. Despite significant advances in the treatment of CF, lung function assessments continue to rely on the forced expiratory volume in 1 second (FEV1). Although simple to perform, it lacks sensitivity, is difficult for younger subjects, and changes over time. An alternative method of assessing lung physiology is to derive measures of ventilation heterogeneity from inert gas washout tests. In early lung disease, measures of gas mixing appear to be more sensitive than spirometry. In addition, since only tidal breathing is required, they are more physiological and are more straightforward for younger subjects. Widespread use has been impaired by the lack of a robust and cost effective gas analyser technology. The work presented in this thesis concerns the adaptation, validation and then use of a novel gas analyser (Innocor) in a clinical system for the performance of multiple breath washouts. Lung clearance index (LCI), a simple measure of ventilation heterogeneity, has been calculated from washouts in 52 adults with CF and 50 healthy controls. LCI was more sensitive to disease than FEV1 in CF, being elevated in 11 of the 12 CF patients with normal spirometry. In healthy subjects, LCI has been shown to be repeatable and reproducible, with a narrow range of normal that is stable over a wide age range. In a separate study of 19 patients, LCI has also been shown to improve with treatment of an exacerbation in CF. Correlation with changes in other biochemical (serum CRP, peripheral blood white cell count, sputum IL-8, sputum neutrophil) clinical (symptom score) or structural (computed tomography) markers was poor. Short term change in LCI has also been demonstrated in CF patients in response to chest physiotherapy, although there was considerable heterogeneity of response in terms of both LCI and volume of lung ventilated by tidal breathing (as measured by washout functional residual capacity). In addition to LCI, multiple breath phase III slope analysis has been performed on washouts of CF patients and healthy controls, and this has been compared to other measures of lung physiology. Proposed measures of convective and diffusive gas mixing have been shown to be unreliable in CF. These studies have also been the first to demonstrate multi-centre use of washout tests as endpoints. The technology described here offers the possibility of a simple and reliable system for performing multiple breath washouts, though at present it is not available commercially. The studies have added to the understanding of the utility and reliability of washout tests, as well as some of their limitations. It is hoped that in future LCI will be an important clinical endpoint in therapeutic intervention studies in CF, and that it will also offer new ways to follow changes in lung physiology in other diseases.

The present study aims at demonstrating phosphorus metabolite concentration changes and alterations in uptake/excretion of a hepatocyte specific contrast agent in patients with diffuse - or suspected diffuse - liver disease by applying two non-invasive quantitative MR techniques and to compare the results with histo-pathological findings, with focus on liver fibrosis. In the first study phosphorus-31 MR spectroscopy using slice selection (DRESS) was implemented. Patients with histopathologically proven diffuse liver disease (n = 9) and healthy individuals (n = 12) were examined. The patients had significantly lower concentrations of phosphodiesters (PDE) and ATP compared with controls. Constructing an ‘anabolic charge’ (AC) based on absolute concentrations, [PME] / ([PME] + [PDE]), the patients had a significant larger AC than the control subjects. The MRS technique was then, in a second study, applied on two distinct groups of patients, one group with steatosis and none-to-moderate inflammation (n = 13) and one group with severe fibrosis or cirrhosis (n = 16). A control group (n = 13) was also included. Lower concentrations of PDE and a higher AC were found in the cirrhosis group compared to the control group. Also compared to the steatosis group, the cirrhosis group had lower concentrations of PDE and a higher AC.  A significant correlation between fibrosis stage and PDE and fibrosis stage and AC was found. Using an AC cut-off value of 0.27 to discriminate between mild (stage 0-2) and advanced (stage 3-4) fibrosis yielded an AUROC value of 0.78, similar as for discriminating between F0-1 vs. F2-4. Dynamic contrast enhanced MRI (DCE-MRI) was performed prospectively in a third study on 38 patients referred for evaluation of elevated serum alanine aminotransferase (ALT) and/or alkaline phosphatase (ALP) levels. Data were acquired from regions of interest in the liver and spleen by using single-breath-hold symmetrically sampled two-point Dixon 3D images time-series (non-enhanced, arterial and venous portal phase; 3, 10, 20 and 30 min) following a bolus injection of Gd-EOB-DTPA (0.025 mmol/kg). A new quantification procedure for calculation of the ‘hepatocyte specific uptake rate’, KHep, was applied on a two-compartment pharmacokinetic model. Liver-to-spleen contrast ratios (LSC_N) were also calculated. AUROC values of 0.71, 0.80 and 0.78, respectively, were found for KHep, LSC_N10 and LSC_N20 with regard to severe versus mild fibrosis. Significant group differences were found for KHep (borderline), LSC_N10 and LSC_N20. In study four no significant correlation between visual assessments of bile ducts excretion of Gd-EOB-DTPA and histo-pathological grading of fibrosis or the quantified uptake of Gd-EOB-DTPA defined as KHep and LSC_N. In conclusion 31P-MRS and DCE-MRI show promising results for achieving a non-invasive approach in discriminating different levels of fibrosis from each other.

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Liver biopsy has been considered the gold standard method to assess the progression of fibrosis in chronic hepatitis C viral infection. Despite its prevalent use, significant complications along with patient discomfort are often reported. A reliable, non-invasive method to assess rapid fibrosis progression in patients with HCV infection must be determined. This study involved 57 subjects that underwent liver transplantation due to end stage liver disease as a result of chronic HCV infection. Serum samples were collected from all subjects at time of biopsy. Measurement of YKL-40 was performed using ELISA assays. Serum samples collected for all subjects at time of biopsy had a total YKL-40 concentration range from 38-1442 ng/mL with a mean of 228.74 ng/mL and a standard deviation of 271.26 ng/mL. The median YKL-40 concentration was 128 ng/mL. Serum YKL-40 is a reliable marker of rapid fibrosis progression and can be used in combination with other markers to accurately predict rapid fibrosis progression in HCV infected patients post orthotopic liver transplantation.

Development of Serum Markers for the Non-Invasive Assessment of Longitudinal Change and Adjacent Stage Differentiation in Hepatic Fibrosis Fibrogenesis is a dynamic process involving extracellular matrix synthesis and degradation that is dependent upon disease specific liver injury. The accepted standard measurement of hepatic fibrosis is through liver biopsy. Non-invasive markers of fibrosis were mostly developed in cross-sectional chronic hepatitis C (CHC) study cohorts. There is a need to develop valid non-invasive biomarkers of fibrosis that can follow dynamic changes in fibrogenesis. Our hypothesis was that current serum fibrosis marker panels were likely to have poor diagnostic performance for following changes in CHC fibrosis, or for the diagnosis of early stage nonalcoholic steatohepatitis (NASH). For this thesis, we utilized well-characterized biorepository data and samples from tertiary referral centers and phase II-III CHC clinical studies. Sequential algorithms that include FibroTest (FT-AT) reduce the need for biopsy in CHC in most patients for a diagnosis of cirrhosis, but only one-third of patients were correctly classified in a prognostic fibrosis classification. FT-AT and Hepascore are associated with poor directional change on longitudinal fibrosis assessment. In CHC non-responders, FT-AT declined by 5% despite an increase in histologic collagen area of 30% over 12 months. Current serum markers had modest diagnostic accuracy for NASH, and were not able to differentiate bland steatosis from early NASH. Next generation serum fibrosis markers should incorporate specific targeted proteins that have been linked to fibrogenesis at various stages in the disease process. Our multiplex panel study of 37 candidate serum biomarkers in >800 CHC patients noted that varying combinations of markers had poor accuracy for adjacent fibrosis stage. Our quantitative differential proteomic expression profile study in CHC liver tissue identified > 1700 proteins and >260 functional protein classes associated with fibrosis stage, providing feasibility for new approaches to fibrogenesis biomarker discovery. Keywords: Fibrogenesis, biomarkers, liver biopsy, hepatitis C

Orientador: Antonio Fernando Ribeiro
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A Fibrose Cística (FC) é a doença de maior prevalência dentre as doenças hereditárias incidentes nos indivíduos caucasóides. A doença é progressiva e apresenta como manifestação clínica à obstrução respiratória crônica, infertilidade masculina e deficiência de ganho de peso pelo dano ao pâncreas exócrino. A maioria dos pacientes tem níveis elevados de eletrólitos no suor. O objetivo do presente trabalho consistiu na importância do diagnóstico precoce das repercussões nutricionais sobre a evolução e prognóstico da doença, pois avaliou o perfil nutricional dos micronutrientes (vitaminas lipossolúveis e oligoelementos) em pacientes com Fibrose Cística atendidos no ambulatório de Fibrose Cística do Hospital das Clínicas da UNICAMP. Foram estudados 86 pacientes, além de 34 voluntários saudáveis para o grupo controle. Amostras de sangue foram coletadas para quantificação dos parâmetros bioquímicos séricos, por métodos enzimáticos colorimétricos. As vitaminas lipossolúveis foram determinadas após seu isolamento por cromatografia líquida rápida de proteínas (High Protein Liquid Chromatograpy-HPLC).Observamos que para a análise dos níveis séricos das vitaminas lipossolúveis e dos oligoelementos na população estudada mostrou valores adequados, exceto para a vitamina A e E, mesmo com uma dieta insuficiente para elementos
Abstract: The Cystic Fibrosis (FC) is the illness of bigger prevalence amongst the incident hereditary illnesses in the caucasian subjects. The illness is gradual and presents as clinical manifestation to the chronic respiratory blockage, masculine infertility and deficiency of profit of weight for the damage to the exocrine pancreas. The majority of the patients has high electrolyte levels in the sweat The present work consisted of the importance of the precocious diagnosis of the nutrition repercussions on the evolution and prognostic of the illness, therefore it evaluated the nutritional profile of the micronutrients (fat-soluble vitamins and oligoelements) in patients with Cystic Fibrosis taken care of in the clinic of Cystic Fibrosis of the Hospital of the Clinics of the UNICAMP. We studied 86 patients and 34 healthy volunteers in the control group. Blood samples were collected for measurement of serum biochemical parameters for enzymatic colorimetric methods. Soluble vitamins were determined after their isolation by fast protein liquid chromatography (High Protein Liquid Chromatograpy-HPLC).We observe that for the analysis of the serum levels of fat-soluble vitamins and the oligoelements in the studied population it showed adjusted values, except for the vitamin A and the E, exactly with a insufficient diet for elements
Doutorado
Saude da Criança e do Adolescente
Doutora em Saúde da Criança e do Adolescente

Suboptimal growth and nutritional status are problematic for children with cystic fibrosis (CF). Optimal nutrition predicts better lung function and longevity. Daily nutrition therapy for children with CF requires adequate food resources, knowledge of appropriate nutrition and behavior management skills, and confidence in one's ability to correctly apply the necessary skills. The Mountain West Cystic Fibrosis Consortium Questionnaire (MWCFC-Q) was designed as an educational needs assessment for parents of children with CF. The goal was to identify areas of concern that could be targeted for educational intervention to ultimately improve children's growth and nutritional status. Data analyzed from 305 returned surveys included household food security, use of food assistance programs, knowledge of nutrition and general CF therapy, and self-confidence in ability to manage CF care. Questions regarding food security and knowledge of CF nutrition and general therapies were multiple choice. A ten point Likert-type scale was employed for determining confidence around management of CF related issues. Respondents' median accuracy for questions regarding nutrient content of commonly used foods was 71.4% and 57.9% for CF nutrition therapy, respectively. Although overall confidence in CF management was high, scores for confidence in nutrition and behavioral management were significantly lower than for confidence in CF respiratory/medical management and CF Center recommendations. In the second phase of this project, a pilot study using the chronic care model was developed for enhancement of nutrition and behavior management skills of parents of children with CF. Participants attended a series of four classes, each with a short didactic presentation, group activity, and discussion. Important features of this evidence-based educational program included nutrition and behavior management, self-efficacy, problem solving skills, and peer mentoring. A pre-, post-, follow-up, follow-up format was used to evaluate changes in participants' knowledge and self-confidence regarding nutrition and behavioral management. Comparisons were made with responses to the mailed survey using the same questionnaire. Secondary outcomes were changes in the rate of weight gain and growth for participants' children with CF. Participants showed statistically significant improvement in knowledge of nutrition therapy for CF at post-intervention compared with respondents to the mailed survey.

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Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Departamento de Ensino. Programa de Pós-Graduação em Saúde da Criança e da Mulher. Rio de Janeiro, RJ, Brasil.
O estado nutricional (EN) é marcador de prognóstico para pacientes com Fibrose Cística (FC) . O objetivo deste trabalho foi avaliar a associação do EN com o perfil inflamatório, prática de atividade física e ingestão alimentar de crianças e adolescentes com FC. O estudo foi observacional do tipo transversal com indivíduos acompanhados no Instituto Fernandes Figueira, da Fundação Osvald o Cruz. Foram selecionados 46 crianças de oito a 18 anos com diagnóstico con firmado de FC. A avaliação do estado nutricional foi feita pelos indicadores altura/idade, pelo índice de massa corporal/ Idade (IMC/I) e pelo IMC em kg/m2 (OMS 2006). Para compos ição corporal utilizou - se a Equação de Slaughter (1988), Circunferência e área muscular do braço e dobra cutânea tricipital. A ingestão dietética foi avaliada pelo recordatório alimentar de 24horas. As citocinas (IL - 1, IL6 e IL 8) por método Elisa e a prot eína C reativa ( PCR ) por nefelometria Avaliou - se a associação entre estado nutricional e as variáveis estudadas por modelos bivariados e multivariados de regressão linear com significância p<0,05. A média de idade encontrada foi de 11,9±2,83 anos, sendo 6 0,9% do gênero feminino, 57,8% apresentou comprometimento do estado nutricional pelo IMC/I, 37,8% e 52,2% pela CMB e AMB respect ivamente e 15,6% baixa estatura . A DCT apresentou - se r eduzida em 17,8% dos pacientes , 26,1% das crianças estavam com percentual de gordura baixo segundo a equação de Slaughter, . A med ia da razão DCSe/DCT apresentou risco e/ou elevação da distribuição de gordura na região abdominal em 55,6% dos pacientes , 22,9% praticou de atividade física, 76,1% atingiu a recomendação de ingest ão energética, sendo significativamente maior no grupo de pacientes em risco ou desnutridos. A razão de ingestão de n6/n3 foi adequada mas os l ipídeos foram consumidos a baixo do percentual recomendado em 54,3% dos casos e os carboidratos ac ima em 32,6% . Os valores de PCR foram inferiores a 0,5mg/dL (prova inflamatória negativa) em 71,1% dos casos. A maioria , 52,2% e 62,5% dos pacientes desnutridos segundo a AMB e CMB respectivamente, possuía esta medida maior que 0,5mg/dL. O TNF - α e a Il 8 foram as únicas citocinas que se associaram de mod o significativo com o IMC. N a análise de regressão linear múltipla as variáveis estatisticamente significativas foram: IL - 8 , Atividade física e VET. Os desfechos estudados foram, percentual de g ordura, adequação do IMC , CMB e AMB. A análise dos compartimentos corporais foi o método mais sensível de avaliação do estado nutricional, o tecido gorduroso foi preservado e a ingestão calórica adequada não foi capaz de preservar a MCM o que sugere um mec anismo semelhante ao da caquexia. O consumo total de lipídeos ainda foi baixo apesar da sua importância para FC, com adequação da razão n6/n3. A dosagem sérica de citocinas não parecem demonstrar a inflamação sistêmica. A única citocina que se associou p ositivamente com o percentual de gordura corporal foi a IL - 8 e parece mais relacionada ao tecido adiposo visceral do que a inflamação presente na FC. Desta forma na FC também deve ser dada maior atenção aos fatores como a pratica da atividade física e ingestão alimentar principalmente de lipídeo, assim como a medida da adiposidade abdominal na avaliação nutricional para prevenção de doenças cardiovasculares.
Nutritional status is a prognostic marker in patients with Cystic Fibrosis. The aim of this study was to evaluate the association of the inflammatry status, physical activity and food intake of children and adolescents with CF. The stud y was observational, cross - sectional subjects followed by the Fernandes Figueira Institute, Oswaldo Cruz Foundation. We selected 46 patients from 8 to 18 years old with a confirmed diagnosis of CF. The nutritional status was made by anthropometric height/a ge and body mass index (BMI) in kg/m2 (WHO 2006). For body composition the equation of Slaughter (1988) was used, mid - upper arm circumference and upper arm muscle area and triceps skinfold thickness. Dietary intake was assessed by a 24 - hour dietary recall. Cytokines by ELISA and CRP for nefelometria evaluated the association between nutritional status and the variables studied by bivariate models and multivariate linear regression with significance p <0.05. The average age was 11.9 ± 2.83 years, 60.9% femal e, 57.8% had poor nutritional status by BMI/ I, 37.8% and 52.2% for the MUAC and UAM respectively an d 15.6% short stature. The TSF was presented reduced in 17.8% of patients and 26.1% according to the equation of Slaughte r. The average of the ratio SST / TSF showed a distribution of abdominal fat, 22.9% practiced physical activity, 76.1% reached the recommended energy intake, was significantly higher in patients at risk or malnourished. The ratio n6/n3 intake was adequate but lipids were consumed below the rec ommended percentage in 54.3% of cases and 32.6% up on carbohydrates. CRP values in 71.1% o f cases were less than 0.5 mg/ dL. Most malnourish ed patients according to the UMA and this measur e had CRP greater than 0.5 mg/dL. TNF - α and Il 8 were the only cyto kines that were associated significantly with BMI. After multiple linear regression analysis the variables were statistically significant: IL - 8 , Physical activity and VET. The body composition analysis was the most sensitive method for assessing the nutri tional status, the fat tissue was preserved and adequate caloric int ake was not able to preserve FFM suggesting a mechanism similar to cachexy . The total lipid was still low despite its im portance for CF , with adequacy ratio n6/n3. Serum cytokines do not a ppear to systemic inflammation. The only cytokine that was positively associated with body fat percentage was the IL - 8 and seems more related to visceral adipose tissue than inflammation present in CF. CRP may be a marker of malnutrition. Thus CF should also be given greater attention to environmental factors such as the practice of physical activity and food intake mainly lipid ,as the measurement of waist circumference in the nutritional assessment for cardiovascular disease prevention.

Introduction: Chronic hepatitis B (CHB) is a viral infection that can lead to development of fibrosis and hepatocellular carcinoma (HCC). Several factors affecting disease progression have been reported, such as sex and region of origin. Liver stiffness and fibrosis can be evaluated using transient elastography. The degree of fibrosis is an important parameter when deciding if treatment and HCC surveillance is indicated. Aim1) To compare patients with CHB according to sex and region of origin regarding the parameters liver stiffness, presence of significant fibrosis, hepatitis B e antigen (HBeAg) positivity, frequency of elevated alanine aminotransferase (ALT) levels and HCC surveillance.2) To identify factors associated with significant liver fibrosis. Methods: 410 patients with a registered doctor’s visit 2015–2018 at the Department of Infectious Diseases at Örebro University Hospital were included. A systematic review of medical records was performed and groups (women-men, regions of origin) were compared. Multivariate logistic regression was used to identify factors associated with significant fibrosis. Results: Men had significantly higher liver stiffness values, higher presence of significant fibrosis, and were more frequently under HCC surveillance compared to women. No other significant differences were found regarding the studied parameters, neither related to sex, nor to region of origin. Factors associated with significant fibrosis were: male sex, elevated ALT levels and hepatitis D virus (HDV) co-infection. Conclusions: Men had a higher frequency of significant fibrosis compared to women. Factors associated with significant fibrosis were male sex, elevated ALT values and HDV co-infection.

Body composition (BC) is an important prognostic factor in patients with cystic fibrosis (CF). International guidelines recommend monitoring growth and nutritional status in children with CF using simple anthropometry. However, methodological issues with simple techniques are more significant in children due to growth and maturation, and even more problematic in patients, perhaps accounting for inconsistent findings in previous BC research in children with CF. My thesis addressed three aims: 1) comparison of BC in young children with CF and controls using the criterion four-component model (4CM), cross-sectionally and longitudinally using pair-, group-match and reference database comparison ; 2) investigation of relationships between BC and lung function (FEV1); and 3) evaluation of simpler BC techniques for clinical assessment of children with CF. Results 1) Using the 4CM I found sex differences not identified by simpler techniques; girls with CF had abnormal baseline body composition, whilst longitudinal analysis showed deteriorating fat-free mass (FFM) in both sexes. Conclusions differed according to the comparison group used, perhaps accounting for some inconsistencies between previous studies. 2) Contrary to previous research, using the 4CM fat mass was positively associated with FEV1 in girls; this association was not apparent at 2 year follow-up despite declining FEV1. FEV1 was associated with FFM in boys and bone mass in girls, in accord with previous research. 3) Simple BC techniques were not interchangeable, and dual-energy X-ray absorptiometry (DXA) on its own or in combination with bio-electrical impedance (BIA) gave results closest to the criterion method. Conclusion Using the 4CM, abnormal BC and associations between BC and lung function were detected, which were not apparent using simple anthropometry. The findings emphasise the importance of using appropriate techniques to measure BC in children with CF, and suggest that DXA with or without BIA may be most appropriate in clinical practice.

The focus of this work is to understand the current state of permeability measurement and prediction methods for fibrous, porous media and to suggest improvements. For this purpose the most widely used and accepted measurement technique, the channel flow method, is used to experimentally investigate the effects of fiber sizing and fluid viscosity on the permeability of glass and carbon fibers. Experiments have shown that the variation in permeability occurs due primarily to the fluid viscosity and not the nature of fluid, which other researchers have proposed. Studies were also carried out on both sized and unsized fibers to show that significant permeability variation occurs when fluids of different viscosity are used. Further, experimental studies on the effect of secondary flow have revealed that, for fiber products representative of the aerospace industry, secondary flow has little effect, which challenges models proposed by other researchers. Previous studies had shown a dual scale flow for fiber products with a significantly lower fiber volume fraction. A novel acoustical method based on standardized impedance tube measurements has been developed to predict physical properties—both permeability and characteristic length—of the porous medium. The predicted permeability values from the acoustical method for the range of porosity studied in this work compare well enough with existing permeability models’ predictions to warrant further study. The acoustical method is quick and repeatable, and when compared with the existing flow methods may provide a convenient alternative. It also provides a measure of fiber arrangement (via the “viscous characteristic length”) that should be studied further to explain variations in permeability measurements due to alternative fiber product architecture.
Thesis (Ph.D.)--Wichita State University, College of Engineering, Dept. of Mechanical Engineering

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Introduction: Instruments to objectively assess the perception of symptoms in patients with BCQ are scarce. Patients with COPD and bronchiectasis (BCQ) share similar symptoms. Therefore, COPD Assesment Test (CAT), originally developed for COPD may be suitable for use in these patients. Objective: To validate and test the reproducibility of the CAT for non-cystic fibrosis (non–CF) bronchiectasis patients. Methods: Hundred patients (FEV1 52 ± 25%; FVC 67 ± 22%; 48 ± 14 years; 42 males) underwent spirometry, cardiopulmonary exercise testing (peak workload and VO2), and shuttle walk test (ST). They answered the Saint George Respiratory Questionnaire (SGRQ) and the Medical Research Council (MRC) scale, and the CAT in two different days (CAT-1 and CAT-2). A pedometer recorded the No. of steps/day (NSD). Results: CAT showed a positive correlation with SGRQ domains (r = 0.74, p< 0.001) and MRC (r = 0.49, p< 0.001). A negative correlation was observed among CAT and lung function (FVC%: r= -0.33, p= 0.001; FEV1%: -0.28, p = 0.004), peak workload (r = -0.31, p = 0.001), VO2 (r = -0.44 p< 0.001), distance walked on ST (%, r= -0.46, p< 0.001), and NSD (r = -0.74, p< 0.001). There was an excellent reliability in the scores from CAT-1 and CAT-2 (21 13.25 – 26.75 and (19 13 – 26.75, respectively) with high intraclass correlation coefficient (ICC: 0.92 [95% CI: 0.83 – 0.96], p< 0.001). Conclusion: CAT is a valid instrument for measuring the impact of bronchiectasis in well-being and daily life of patients with BCT, and is a reproducible questionnaire.
Introdução: Instrumentos que avaliam objetivamente a percepção dos sintomas em pacientes com BCQ são escassos. Pacientes com DPOC e bronquiectasia (BCQ) compartilham sintomas semelhantes. Portanto, o COPD Assesment Test (CAT), originalmente desenvolvido para DPOC, pode ser adequado para uso nesses pacientes. Objetivo: Validar e testar a reprodutibilidade do CAT para BCQ. Métodos: 100 pacientes (42 homens, 48 ± 14 anos, VEF1 52 ± 25% prev, CVF 67 ± 22% prev) foram submetidos à espirometria, shuttle walk test incremental (SWTI) e teste de exercício cardiopulmonar exercício (TECP, carga e VO2 pico). Eles responderam ao Saint George’s Respiratory Questionnaire (SGRQ), à escala de dispneia Medical Research Council (MRC) e ao CAT (este em dois dias diferentes CAT-1 e CAT-2). O número de passos por dia (NP) foi registrado por pedômetro. Resultados: Os escores do CAT correlacionaram-se positivamente com os escores dos domínios SGRQ (r de 0,74, a p< 0,001) e MRC (r= 0,49, p= 0,001). Foi observada correlação negativa entre o CAT e a função pulmonar (CVF%: r= -0,33, p= 0,001; VEF1%: -0,28, p= 0,004), carga (r= -0,31, p= 0,001), VO2 (r= -0,44 p< 0,001), distância no SWTI (r= -0,46, p< 0,001), e NP (r= -0,42, p< 0,001). Houve excelente reprodutibilidade entre os escores de CAT-1 e CAT-2 (21 13,25 – 26 e (19 13 – 26,75, respectivamente), com elevados coeficiente de correlação intraclasse (ICC: 0,91 [95% CI: 0,87-0,94], p<0,001). Conclusão: CAT é um instrumento válido para medir o impacto da BCQ no bem-estar na vida diária dos pacientes com BCQ, e é um questionário reprodutível.

CHAPTER 2 Five experiments were conducted to investigate the dietary factors within the rumen environment which could contribute to the rate of degradation of straw. Three sources of digestible cellulose and/or hemicellulose (unmolassed sugar beet pulp, citrus pulp and dried grass) and two sources of natural proteins (fish meal and soya bean meal) were investigated for their ability to increase degradation of straw In. sacco in sheep, as supplements to untreated straw. The untreated straw used in these experiments was adequately supplemented with rumen degradable nitrogen, sulphur, vitamins and minerals. Unmolassed sugar beet pulp and dried grass when given at a level of 150 g.kg DM-1, increased both the rate and extent of DM degradation of straw In sacco. Citrus pulp and soya bean meal had no effect on straw degradation while fish meal increased the extent of straw DM degradation. The rumen NH3 levels and pH in both the control and supplemented animals were above the range that would be expected to cause an inhibition in fibre digestion. It was concluded that digestible cellulose/hemicellulose and fish meal improved the conditions in the rumen for fibre degradation in animals given straw diets. CHAPTER 3 The supplementary effects of unmolassed sugar beet pulp and fish meal on intake, digestibility and growth performances of sheep, given either untreated or ammonia treated straw, were investigated using twenty four sheep. The experimental design was a 2 x 2 x 2 factorial layout. Unmolassed sugar beet pulp and fish meal increased untreated straw digestibility by 10% and intake by 16-22%. The supplements had no effect on the digestibility or intake of basal ammonia treated straw. Ammonia treatment alone however increased the intake and digestibility of untreated straw by 76% and 16%, respectively. The growth achieved in this experiment was highly correlated to the intake of total digestible DM. CHAPTER 4 Two experiments were conducted to study the supplementary effects of unmolassed sugar beet pulp and fish meal on intake, digestibility and growth performance of cattle given either untreated or ammonia treated straw. Twenty four Hereford x Friesian steers and thirty two Friesian cross steers and bulls were used for the two experiments respectively. The first experiment was conducted for ten weeks, while the second experiment was continued for twenty weeks. Both the experiments were of 2 x 2 x 2 factorial design. Unmolassed sugar beet pulp and fish meal when given in a combination increased the intake and digestibility of untreated straw. Similar to the results of the sheep experiment (Chapter 3) these supplements did not change the intake or digestibility of ammonia treated straw. Ammonia treatment however increased the intake by 22 and 2 and digestibility by 26 and 14% in these two experiments. The growth observed in the first experiment was higher than expected, but in both experiments growth was related to total digestible DM intake. CHAPTER 5. Methods to predict the rate and extent of roughage degradation in different rumen environments were investigated. The activities of two particle-bound microbial enzymes were measured, glutmate dehydrogenase (GDH) and carboxymethyl cellulase (CMCase). A method was developed and standardized to measure particle-bound microbial enzymes after incubating straw contained in nylon bags in the rumens of sheep given different diets. Using this method particle-bound enzyme activities were correlated with dry matter degradation in the rumen. Particle-bound NAD-linked GDH activity showed no relationship to dry matter degradation while particle-bound CMCase activity showed a very high correlation with the rate and extent of straw degradation. It was concluded that measuring particle-bound CMCase activity at 8 or 16 hr incubation periods could be useful in predicting the rate and extent of DM degradation of straw. CHAPTER 6 The results were discussed in relation to the view that the 'rate limiting: steps' that control digestion and intake of low quality roughages such as rate of fibre degradation, rate of particle size reduction, and the rate of passage of undigested material from the rumen depend upon: (a) proportion of the fibre-degrading organisms in the total microbial population (b) factors related to the roughage such as 'fragility' (c) animal species (i.e. sheep or cattle). Practical implications of the findings for animal production were evaluated on a nutritional and economical basis and it was concluded that the judicious supplementation of untreated straw with a source of digestible cellulosic/hemicellulosic material and a slowly degrading natural protein could replace ammonia treatment of straw, but has to be reassessed in different parts of the world depending on the availability and cost of the supplements.

In the manufacturing process of mineral wool insulation plates, defects arise, such as unmelted base minerals and uncured binder which gets embedded within the plates. To be able to sort out these defective plates from a manufacturing line, a reliable quality assessment is needed. The aim is to find an ultrasonic non-destructive testing method that can identify the embedded defects. This was achieved through experiments on defective insulation plates using three different ultrasonic non-destructive testing methods that were of interest. These methods were higher harmonics, pulse-echo and through transmission. Of these three, the through transmission method showed the most promising results in finding the defects that were sought after. The through transmission method utilizes two aligned transducers, one acting as a transmitter and one as a receiver. When the defective area passes through the sound beam between the transducers the intensity of the beam drops, indicating that a defect is present. The weakened intensity is due to the signal attenuation, mainly caused by the higher density of the defects compared to the base material in the surrounding insulation plate. The method is well suited for being implemented in a production line since it’s a fast method and, therefore, suited for moving objects. More measurements are needed to establish a reliable reference value to consistently distinguish the defects from the surrounding plate. The method was only evaluated in a small scale experimental environment so further experiments on a larger scale are needed to mimic and evaluate the reliability in the real case scenario of the production lines.
I tillverkningsprocessen av isolerskivor i stenull uppstår inneboende defekter i isolerskivorna, dessa defekter består av osmälta basmineraler och ohärdat bindemedel. För att kunna sortera bort dessa skadade skivor från tillverkningslinjen behövs en pålitlig metod för kvalitetsbedömning. Avsikten med det här arbetet är att hitta en oförstörande provningsmetod baserad på ultraljud som kan identifiera de inneboende defekterna. Detta genomfördes genom experiment på defekta isolerskivor med tre olika oförstörande provningsmetoder baserade på ultraljud. Dessa metoderna var, higher harmonics, pitch-echo och through transmission. Through transmission visade lovande resultat i att identifiera de båda typerna av skador. Metoden är baserad på att en sändare sänder ut ultraljud till en mottagare placerad i linje med sändaren. När ett defekt område passerar ultraljudsvågen mellan sändaren och mottagaren försvagas intensiteten av signalen. Försvagningen av signalen beror mestadels på att densiteten är högre hos defekterna än hos basmaterialet i isolerskivan. Denna försvagning indikerar att en defekt befinner sig i mätområdet. Metoden är väl implementerbar i en tillverkningslinje, då det är en snabb metod vilket den behöver vara då objektet är i rörelse. Mer mätningar behövs för att fastställa ett pålitligt referensvärde för att konsekvent kunna sortera ut de defekta isolerskivorna. Metoden är endast utvärderad i en småskalig laborationsmiljö och det behövs fler tester i en större skala undersöka pålitligheten i det verkliga scenariot med tillverkningslinjen.

碩士
臺灣大學
應用力學研究所
98
Congenital muscular torticollis ( CMT ) caused by the fibrotic change of the sternocleidomastoid muscle ( SCM ) can be detected by ultrasonography. The incidence of CMT varies from 0.3% to 2% in literature and is the third skeletal muscular diseases in infants. This study aimed to combine the advantages of ultrasound and the accuracy of histopathology to construct objective diagnostic reference by animal model. We proposed a K value based on the ultrasound image’s difference of mean echo intensity in injured and normal side, and K value has positive correlation with the fibrosis ratio of histopathology, Pearson correlation r=0.75 (p&lt;0.01). In five follow-up cases of infants ( 3.1 1.8 months ), K value decrease by time because the degeneration of the fibrotic tissue.

碩士
國立臺灣大學
應用力學研究所
99
This study is dedicated to the application of liver fibrosis assessment using ultrasound imaging, focusing on the envelope signal converted from RF signal to investigate the differences of the statistical distributions between healthy and disease livers. Liver cirrhosis is considered as an irreversible process that there is almost no effective course of treatment to recover the fibrosis liver. Fibrosis is marked by the gradual replacement of hepatocytes by extracellular collagen, which is a chronic and long-term degeneration of the liver function. Invasive assessment like liver biopsy often accompanied with sequela and is prone to sampling error when small biopsy samples are analyzed. Therefore, there is a clinical need to establish an effective and reliable method for the noninvasive assessment, espesically the prediction of early stage liver fibrosis. Ultrasound speckle in B-mode images, which is the result of a wave interference phenomenon of backscattering signal, has been used for clinical liver fibrosis and steatosis diagnosis. The characteristic of speckle is associated with the density of scatterers in tissue, indicating different patterns among various tissue properties. To analyze the RF signal derived from liver ultrasound, three methods were implemented: Nakagami-m parameter method, Yamaguchi method and ACRA (adaptive criteria-referenced assessment) method. Nakagami-m parameter method uses m-value to describe the distribution within the ROI (region of interest), proved to be a valid approach characterizing breast tumors and cataract lens. Yamaguchi method determines whether the ROI is a fiber area or not based on the assumptive parameters derived from the phantom experiment. ACRA is a method, which was originally developed by our lab and could be adapted to various system conditions; it reflects the degree of fibrosis liver via the unassessed tissues in comparison with a criteria-reference constructed by healthy tissues. Results showed that Nakagami-m and ACRA method were able to discriminate the degree of fibrosis from livers in vitro rat experiment. Area under receiver operating characteristic curve (AUC) was used to judge the performence. In the in vivo linear array experiment, three methods performed well in the early stage F≥1 fibrosis (Nakagami-m: 0.86, Yamaguchi: 0.98, ACRA: 0.95). In the in vivo convex array experiment, three methods performed well in the stage F≥1 and F≥2 fibrosis (Nakagami-m: 0.96, 0.82, Yamaguchi: 0.93, 0.92, ACRA: 0.99, 0.93). In the in vitro operation linear array experiment, only ACRA successfully identified stage F≥2 and F=4. It is concluded that the concept of ACRA could exend to other domains, observing the degree of the difference between tissues to indicate the abnormity. This method possesses clinical potential and application value.

碩士
國立成功大學
資訊工程學系
105
In general, the liver biopsy is the most accurate method to assess liver fibrosis. The liver biopsy is a procedure to remove a small piece from liver tissue and it is invasive that can cause many complications. Besides, rats are widely used in liver fibrosis experiments and a lot of rats are sacrificed to verify, assess the liver fibrosis level and reduced the sampling errors. Therefore, ultrasound and statistical parameters were used to assess the liver fibrosis as a non-invasive method in this study. Sprague Dawley rats were induced liver fibrosis by CCl4. A 7.5MHz single element transducer was used in ultrasound system. Nakagami parameter and IB were used to quantitatively assess the liver fibrosis as a non-invasive method. B-mode images of mild and severe groups were significantly brighter than control group, but the difference of brightness between mild and severe groups were not significant. IB of control group was –131.9±3.3dB, mild group was –117.3±0.8dB, and severe group was –114.0±2.5dB. The p-value of IB between control group and mild group was small than 0.01 when the p-value of IB between mild group and severe group was small than 0.05. IB was the strength of backscattering signals, which was easily affected by ultrasound system and the angle of incident signals. Nakagami statistical parameter could indicate the scatterer properties by ultrasonic backscattering signals. Nakagami parameter of control group was 0.58±0.03, mild group was 0.78±0.02, and severe group was 0.83±0.02. The scatterer distribution of control group, mild group, and severe group were pre-Rayleigh distribution. The p-value of Nakagami parameter between control group and mild group was small than 0.01 when the p-value of Nakagami parameter was small than 0.05. Nakagami images could show the local scatterer properties which B-mode images could not. In the end, B-mode images are still widely used in medical field, so the Nakagami images can provide the local information to B-mode images and make the more accurate assessment of liver fibrosis stages.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease, which is characterized by a multi-organ involvement and increased mortality, mainly due to cardiovascular complications. Myocardial fibrosis (MF) is common in SLE, affecting up to 30% of these patients. Cardiac magnetic resonance imaging allows an accurate assessment of myocardial tissue in SLE patients, but it is costly, time consuming, and unfit for patients with coexisting chronic kidney disease. Recent advanced echocardiographic techniques allow an accurate assessment of MF. In particular, speckle tracking echocardiography (STE) is a reproducible technique that provides information about MF by detecting abnormalities in myocardial active deformation. Scar imaging echocardiography with ultrasound multi-pulse scheme (eSCAR) is another novel technique that has been validated for detecting ischemic myocardial scars in patients with prior acute myocardial infarction. Aim: To examine whether STE and eSCAR may detect the presence of subclinical myocardial involvement in patients with SLE. Methods: We consecutively recruited 29 patients (M/F=3/26; age 45±11 years) with established SLE, who had a disease duration of 15±10 years. Their median SLE Disease Activity Index (SLEDAI) score was 2 (0-6). Patients with current cardiac symptoms or prior history of any heart disease were excluded from the study. We also recruited a sample of 32 control individuals, who were comparable for age, sex and traditional cardiovascular risk factors to the cases. All participants underwent a complete echocardiography examination, using both STE and eSCAR. Results: Global longitudinal strain (GLS) was significantly impaired in most myocardial segments in SLE patients than in control subjects, except for the myocardial apical region that was comparable between the two groups. Higher SLEDAI was associated with an impaired GLS-4 chamber (r=0.470, p=0.01) and GLS infero-septal wall (r=0.464, p=0.01). A higher daily dosage of prednisone was also associated with an impaired GLS in the infero-septal myocardial segment (r=0.414, p=0.02). Myocardial scar by eSCAR was observed in 5 (17%) out of 29 SLE patients, mainly in the infero-septal myocardial segment. A significant association was found between the infero-septal GLS and the presence of scar by eSCAR technique (r=0.569, p<0.001). Conclusions: Advanced echocardiography techniques detected the presence of subclinical myocardial dysfunction in SLE patients with no history of cardiac disease compared to controls. An ‘apical sparing’ GLS pattern was also observed in SLE patients, with possible important diagnostic implications. In about one fifth of SLE patients a myocardial scar by eSCAR technique was identified, mainly in the infero-septal segments. Larger prospective studies are certainly needed to confirm these findings and to better elucidate the diagnostic and prognostic significance of advanced echocardiography techniques (including GLS and eSCAR) in patients with SLE.

博士
國立陽明大學
生物醫學影像暨放射科學系
107
Abstract Chronic liver disease usually accompanies parenchymal fibrosis and hepatic dysfunction. The consequences also affect prognosis and treatment outcome. Tissue proof is considered as gold standard to monitor the disease progression; however, repeated procedure is not practicable due to its invasiveness and possible complications. Non-invasive methods for assessing liver function and parenchymal fibrosis have been developed. Gd-EOB-DTPA is a liver-specific MR contrast agent, which can provide anatomical and functional information for imaging diagnosis of liver disease. The clearance of intravenous indocyanine green (ICG) is regarded as liver function and widely used reference before hepatectomy. Liver parenchymal cell volume (PCV) is a way of estimation of residual hepatocytes from surgical specimen. We used Gd-EOB-DTPA-enhanced MRI to assess liver function and validate with ICG tests and PCV. Nineteen patients scheduled for operation were enrolled. Functional liver volume (FLV) was computed from 20 min post-contrast MR images. FLV showed significantly correlated with ICG-R15 (ICG retention measured in serum 15 min after injection) (r = −0.47, p = .042) and PCV (r = 0.814, p <.001). This MR-derived index was proven to be clinically feasible for quantifying liver function in patients with chronic liver disease. Acoustic radiation force impulse (ARFI) is an ultrasound technique for measurement of liver parenchymal stiffness. ARFI has been reported to be a reliable method to assess fibrosis severity in a variety of liver parenchymal diseases. We aimed to compare the diagnostic abilities of Gd-EOB-DTPA-enhanced MRI and ARFI with the METAVIR system for staging parenchymal fibrosis in patients with chronic liver disease. Twenty-three consecutive patients with histopathologically proven hepatic fibrosis were prospectively recruited. Shear wave velocity (SWV) measurements were used to determine liver stiffness by ARFI. During the MR study, T1-weighted images were acquired before and 20 min after injection of Gd-EOB-DTPA and the liver-enhancement ratio (LER) was calculated. The diagnostic abilities of ARFI and MRI were compared using receiver operating characteristic analysis. Among the patients, 43.5%, 17.4%, 21.7%, and 17.4% had METAVIR F4, F3, F2, and F1, respectively. The area under ROC curve for F2-F3-F4 vs. F1, F3-F4 vs. F1-F2, and F4 vs. F1-F2-F3 was 0.842, 0.794, and 0.792 for SWV, and 0.855, 0.778, and 0.669 for LER, respectively. The correlation coefficient was 0.573 (p = 0.004) between the SWV and METAVIR score, -0.451 (p = 0.031) between LER and the METAVIR score, and -0.474 (p = 0.022) between the SWV and LER. Liver parenchymal enhancement of Gd-EOB-DTPA-enhanced MRI could be another noninvasive method for assessing the stiffness and fibrosis of liver parenchyma via comparison with the ARFI and METAVIR scoring systems. In the last part of the thesis, various advanced MR techniques to evaluate liver parenchyma are discussed, including MR elastography, diffusion-weighted MRI, multi-parametric MRI, and artificial intelligence. In conclusion, Gd-EOB-DTPA-enhanced MRI is clinically feasible for quantifying liver function and reflecting the severity of liver parenchymal fibrosis.

碩士
長庚大學
醫學生物技術暨檢驗學系
100
Idiopathic pulmonary fibrosis (IPF) is characterized by an intricate cytokine network and abnormal deposition of mesenchymal cells. Recent studies indicated that Oncostatin M (OSM), a member of IL-6 family cytokine, was up-regulated in the bronchoalveolar lavage fluid (BALF) of patients with interstitial lung disease. Furthermore, exogenous administration of OSM into the lungs of mice resulted in a significant recruitment of inflammatory cells, as well as a dose-dependent increase of collagen deposition in the lungs. These results suggested that OSM is a potent mediator of lung inflammation and lung fibrosis. To date, there are no effective treatments for IPF. Recent studies indicated that mesenchymal stem cells (MSCs) significantly reduced damage in the alveoli and were engrafted at injury sites. Interestingly, OSM induced proliferation, differentiation of MSCs, and also induced Oncostatin M receptor (OSMR) expression in the cells. Moreover, OSM also activated HGF expression in fibroblast and might participate in repair after lung injury. Taken together, to improve the protective effect of the transplanted MSCs, we proposed to enrich OSMR overexpressed cell population of MSCs by OSM pretreatment, upon transplantation, OSM binding to the OSM receptor lead to activate signaling pathway from MSC and further induced cytoprotective genes for pulmonary fibrosis treatment. Our data showed that OSM-pretreatment induced OSMR, gp130 and HGF mRNA and protein expression in MSCs. When cocultured with OSM-pretreated MSCs and TGF-β1 treated MRC-5s, the fibrotic marker, the fibronectin mRNA expression was significantly down-regulated. Furthermore, intratracheal instillation of OSM-pretreated MSCs into BPF mouse model at day 3, eighteen days after treatment, the pulmonary respiratory function have shown significant improvement, the mRNA expression of inflammatory factors IL-1β, IL-6 and fibrotic factor collagen III, MMP-9 were significantly decreased in the lung tissues when compared to MSCs group. Our data indicated that anti-inflammation and anti-fibrotic ability were better than MSCs. In conclusion, we demonstrated that OSM-pretreated MSCs significant improves the therapeutic effect of the pulmonary fibrosis.

Classification of chronic lung disease (CLD) in children has traditionally distinguished cystic fibrosis (CF), the most common lethal inherited genetic disorder worldwide, from less common non-CF disorders, a heterogeneous group of conditions with different etiologies, but overlapping clinical features. While in CF the multi-organ involvement has traditionally required a systemic approach to patients’ care, not limited to respiratory issues, in non-CF CLD this aspect has often been neglected. However, an increasing awareness is emerging regarding the need to adequately address issues such as growth, nutrition and psychological aspects, sometimes underestimated by pediatric respiratory physicians. In particular, nutritional status and growth in pediatric non-CF CLD are strongly related to lung impairment and these two elements may deeply influence each other. Furthermore, in non-CF CLD poor attention has been dedicated to the impact of the disease on patients’ life, whereas quality of life represents a crucial parameter to assess disease severity, efficacy of treatments and to highlight areas of possible improvement in patients’ care. Given these premises the present phD thesis aimed at (1) evaluating currently used and emerging tools in the diagnosis and monitoring of pediatric non-CF CLD and at (2) assessing the role of nutritional status assessment in the management of non-CF CLD children

碩士
中山醫學院
營養科學研究所
88
Abstract Oral submucous fibrosis (OSF), an obscure oral pathosis, is characterized by an abnormal accumulation of oral submucous collagen fibers, epithelium atrophy, and the resultant limitation of mouth opening. Studies indicated that OSF was one of precarcinogenic lesions and mortality of oral cancer is the fifth cause of cancer-related death among men. The reasons for the occurrence of OSF and oral cancer were still unknown. Some studies showed that it is closely related to nutritional deficiency, but it is limited to the related evidence. The purpose of this study is to understand the life style, diet behavior, nutritional assessment and immune function of patients with OSF and oral cancer, and the data colleted could be served as a reference on clinical therapy. Subjects were recruited from Taichung and Changhua. To understand the life style, diet behavior and daily intake of patients with OSF and oral cancer. The subjects got to complete a life a style questionnaire, 24-h diet recall, and the blood samples were collected to examine the nutritional status and immune function. The results show that 96.4% of OSF and oral cancer patients had the habit of chewing betel quid. And, 92.8% of them favored smoking and 65% of them favored drinking. The intake of energy, vitamin E, B2, B6, C, niacin, calcium, and iron for all patients were all lower than daily recommended nutrient allowance (RDNA). The nutritional status showed that the content of vitamin E, B1, selenium and zinc were lower in all patients and vitamin B6 was decreased with the severity of disease. The evaluation of the immune showed that, the relative percentage of T-lymphocyte and the ratio of CD4 and CD8 were also gradually decreased with the severity of disease. In conclusion, patients with OSF and oral cancer had poor diet intake, malnutrition and depressed immune-response sensitivity, etc. Correct diet advice and nutrition supplement could be greatly helpful to prevention and clinical therapy of OSF and oral cancer. Key words: OSF, oral cancer, nutrition, immune, diet behavior

Background and Aims: There is an ongoing effort to identify non-invasive surrogates for staging liver disease and detecting clinically significant portal hypertension (CSPH), defined as the presence of gastro-oesophageal varices on endoscopy. Transient elastography performed by Fibroscan (F-TE) has a well-established role in the non-invasive assessment of liver fibrosis and is widely used in clinical practice. Point Share Wave Elastography (pSWE) is a new software-based technique that measures liver stiffness during B-mode ultrasonography. Data about its performance in primary cholestatic liver disease are limited. Aim of this study was to evaluate the performance of pSWE in primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) and its correlations with F-TE and the current scores of liver fibrosis and disease severity. The ability of liver stiffness measurement (LSM) and spleen stiffness measurement (SSM) performed by pSWE to detect CSPH in PSC and PBC was also assessed. Methods: Demographics, biochemistry, clinical and ultrasonographic data were prospectively collected from 152 PSC and 154 PBC patients. F-TE (Fibroscan®, Echosens, median kPa, IQR ＜30, SR ＞60%), pSWE (ElastoPQ, Philips Affiniti70G) and the most common clinical scores of fibrosis were obtained on the visit date. Correlations of TE and pSWE with scores and clinical variables were investigated. Since a recent liver biopsy was not available for most patients, F-TE was used as surrogate of histological fibrosis, adopting the fibrosis stage cut-offs validated in PSC and PBC. ROC curves were obtained in order to define optimal cut-offs for pSWE. Predictors of CSPH were evaluated in patients who had an upper-GI endoscopy within 12 months of the elastographic assessment. ROC curves were constructed to establish the performance of elastographic techniques and fibrosis scores in predicting CSPH. Results: pSWE LSM correlated significantly with F-TE, all clinical scores of fibrosis and the currently validated prognostic scores in PSC and PBC (P＞0.001). AUROCs (95%CI) for LSM pSWE were ≥0.93 for all fibrosis stages, with optimal cut-offs for fibrosis stage ≥1, ≥2, ≥3, =4 of 7.4, 8.5, 10.5 and 12.1 kPa in PSC and 7.7, 8.9, 9.6 and 13.3 kPa in PBC, respectively. 46 PSC and 53 PBC patients had an upper-GI endoscopy within 12 months of the elastographic assessment. CSPH was detected in 18 and 12 cases, respectively. SSM showed the best performance in predicting CSPH, with best cut-off 40.2 kPa for PSC and 50.1 kPa for PBC. In this cohort, the diagnostic performance of ElastPQ SSM in detecting CSPH was superior to the recently validated Baveno VI and Expanded Baveno VI criteria. Conclusions: This study provides evidence of the suitability of liver and spleen ElastPQ pSWE as valuable diagnostic and prognostic markers in PSC and PBC. In particular, ElastPQ SSM can be used as a reliable tool for the detection of CSPH in primary cholestatic liver disease.

Tese de Doutoramento em Ciências da Saúde, Medicina, apresentada à Faculdade de Medicina da Universidade de Coimbra.
Hypertension (HT) is the most common modifiable cardiovascular risk factor and is responsible for significant mortality and morbidity. It is estimated that 30-45% of the adult population has HT, with its prevalence increasing with advancing age. The employment of lifestyle changes and drug therapy reduces the risk of major cardiovascular events such as acute myocardial infarction, stroke and heart failure and increases life-expectancy. However, despite the availability of safe and efficacious anti-hypertensive drugs, the prevalence of uncontrolled HT continues to rise, with non-adherence to therapy being the most important contributor of poor blood pressure control. HT is considered true resistant when a documented failure to achieve blood pressure guideline-recommended targets is observed and, secondary HT, non-adherence and pseudo-hypertension have been excluded. The pathophysiology of resistant HT is complex and not clear. The vasoconstrictor effects of the sympathetic nervous system and its implication on the pathogenesis of refractory hypertension have long been known, turning it into an attractive therapeutic target. Renal denervation (RDN) is a minimally invasive percutaneous technique which consists on the delivery of radiofrequency energy on the renal artery wall in order to achieve a selective disruption of sympathetic nerve endings. First generation randomized trials were rather controversial, specifically HTN-3, given the results were not the expected and renal denervation did not achieve a higher blood pressure reduction than the control arm. Several questions have been raised by this ‘negative’ study and prompted investigation around the technique. Up to the current date, RDN has been employed to treat patients with resistant HT and second generation trials have included a wider cohort of patients, in order to assess safety and efficacy of the procedure, based on the development of more advanced devices and the recent knowledge regarding renal nerve distribution. In the current study, we used RDN to treat patients with true resistant HT, in order to assess i) the safety of the technique, through the performance of intra-arterial optical coherence tomography (OCT) in a porcine model and, its efficacy through the histologic evaluation of the treated renal artery; ii) the impact of the technique on the sympathetic cardiac innervation, in a subgroup of patients; iii) the impact of the technique in the immune system and, possibly, identify a biomarker to predict response; iv) the efficacy of the technique through a clinical evaluation and ambulatory blood pressure monitoring v) the impact of renal denervation on several parameters such as left ventricular ejection fraction, diastolic function, arrhythmic profile, renal function, glycemic and lipid profiles, NT pro-BNP and vitamin D. In the first part of our research, we performed unilateral pre-bifurcation RDN, with a multi-electrode (second generation) catheter, in twelve domestic pigs. The contralateral artery was untreated (control). OCT was performed pre and immediately post procedure and follow-up with renal angiogram and OCT was performed after one month. We demonstrated that RDN caused acute vessel wall changes (intimal disruption (edema/spasm) and intraluminal thrombus formation) visible by OCT but, after one month, the vessel was completely healed, with no evidence of renal stenosis in any of the treated subjects. The histological analysis revealed nearly absent tyrosine hydroxylase immunostaining and a statistically significant increase in the amount of collagen fibers in the denervated artery, compatible with a decrease in nerve terminals and an increase in fibrosis, compared to the control, suggesting an efficacious delivery of the radiofrequency energy to the vessel wall. No differences were found in the norepinephrine or epinephrine renal tissue levels between the treated and contralateral kidney. Subsequently, we performed 123I-labelled meta-iodobenzylguanidine (MIBG) cardiac scintigraphy, in a subgroup of patients, before and six months after RDN, to evaluate the impact of the procedure on cardiac sympathetic activity. Early heart to mediastinum ratio (HMR) was significantly lower in responders at baseline but similar after six months follow-up. Late HMR was statistically similar in both groups pre- and post RDN but reduced in comparison to values reported in healthy subjects. Accordingly, there were no statistically significant differences regarding the washout rate but above normal baseline values were detected, a difference more evident in the non-responders (sympathetic overdrive? increased risk of cardiovascular events?), with both group values converging after the RDN. None of the evaluated rates altered significantly at follow-up, translating an absence of deleterious sympathetic nerve disruption. In conclusion, RDN is a safe procedure, in terms of cardiac nerve integrity, but none of the evaluated MIBG parameters were useful to predict response. In the third part our research, we investigated the cellular immune profile in our cohort of 23 patients with resistant HT and treated with RDN. A preliminary assay, which included an extended analysis of T, B and natural killer cells, monocytes and dendritic cells, guided the forthcoming study in order to select T cells (CD4 and CD8, memory and activated subsets) as possible biomarkers of response to RDN. Blood samples were obtained in six timings, pre and post procedure. Response to RDN was evaluated at six-months and one year and was observed in 69.6 and 82.6% of patients, respectively. Absolute values of HLA-DR+ double negative T cells were significantly lower in the group of responders at one year. Additionally, interaction between the timings were found in three T cell subsets (T CD4, T CD8 and naïve T CD8 cells), with the responders tending to present with lower absolute values and little inter-timing variation. Afterwards, our research focused on investigating the behavior of cytokines in 21 RDN patients. We analyzed 45 protein targets which included cytokines, chemokines and growth factors. Response to RDN was evaluated as described previously. Venous blood samples were obtained at four timings, pre and post procedure. 66.7% of the patients were responders at six months and 85.7% were late-responders. Levels of regulated on activation, normal T cell expressed and secreted (RANTES) were significantly lower in responders, both at baseline and at 30 days (p=0.037). As there is evidence that Angiotensin II inhibits RANTES expression and, the renin-angiotensin-aldosterone system is directly linked to the sympathetic nervous system, we could hypothesize that low levels of RANTES are associated with higher levels of angiotensin II and therefore to an over activated sympathetic nervous system, turning these patients more prone to a RDN response. Finally, we assessed serum vitamin D concentration as a predictor of blood pressure response to RDN. Additionally, we evaluated overall long-term safety and efficacy, as well as echocardiographic parameters, in the cohort of 24 patients submitted to RDN. Response at six-months (early-responders) and one year was evaluated. We observed that responders at six-months had significantly higher baseline and six-month follow-up values of vitamin D than non-responders. Responders at one year (including late-responders) continued to present with higher vitamin D levels than non-responders, although it didn’t reach statistical significance, probably due to the low number of the later at this stage. In the long-term follow-up (mean 52 months), 70.8% of the patients maintained a clinical response. Regarding echocardiographic parameters, there was an improvement in diastolic function in non-responders, finding that could reflect benefit from the RDN, even though no effect on blood pressure was observed. No other relevant echocardiographic differences were found. In conclusion, RDN effectively lowered the blood pressure in the majority of the studied patients, with an optimal safety pattern. Our study contributed to better comprehend the clinical, immunological and hemodynamic profiles of patients submitted to this procedure and, to identify potential biomarkers of denervation success. Our findings may allow for a better identification of patients who could really derive benefit from RDN and prompt further investigation around this area.
A hipertensão (HT) é o factor de risco cardiovascular modificável mais comum e é responsável por morbilidade e mortalidade significativas. É estimado que 30-45% da população adulta tenha HT sendo que, a sua prevalência aumenta com o avanço da idade. A implementação de alterações do estido de vida e a terapêutica com fármacos reduz o risco de eventos cardiovasculares major com o enfarte agudo do miocárdio, acidente vascular cerebral e insuficiência cardíaca e, aumenta a expectativa de vida. No entanto, apesar da disponibilidade de medicamentos seguros e eficazes para controlar a HT, a prevalência de HT não controlada continua a aumentar sendo que, o mais importante contribuidor para um mau controle tensional é a não aderência à terapêutica. A HT é considerada verdadeiramente resistente quando não se conseguem atingir os valores de pressão arterial recomendados pelas guidelines e, quando são excluídos HT secundária, não-aderência e pseuso-hipertensão. A patofisiologia da HT resistente é complexa e pouco clara. Os efeitos vasoconstrictores do sistema nervoso simpático e a sua implicação na patogénese da HT resistente é conhecida, tornando-o num alvo terapêutico atrativo. A desnervação renal (RDN) é uma técnica percutânea minimamente invasiva que consiste na aplicação de energia de radiofrequência na parede da artéria renal, com o objectivo de provocar lesão nos terminais nervosos simpáticos. Os ensaios randomizados de primeira geração foram controversos, especificamente o HTN-3, uma vez que os resultados não foram os esperados e a RDN não se associou a uma maior queda na pressão arterial, em relação ao grupo de controlo. Várias questões foram colocadas por este estudo ‘negativo’ e motivou uma maior investigação da técnica. Até ao presente, a RDN tem sido usada para tratar doentes com HT resistente e os ensaios de segunda geração já incluiram uma população de doentes mais ampla, para avaliação da segurança e eficácia do procedimento, baseando-se no desenvolvimento de um catéter mais avançado, multi-eléctrodo, e no conhecimento recente relativo à distribuição dos nervos renais. Na presente tese, utilizámos a RDN para tratar doentes com HT resistente verdadeira, com o objectivo de avaliar i) a segurança da técnica, através da realização de tomografia de coerência óptica (OCT) intra-arterial num modelo porcino e, a sua eficácia através da avaliação histológica da artéria renal tratada; ii) o impacto da técnica na inervação simpática cardíaca, num subgrupo de doentes; iii) o impacto da técnica no sistema imunitário e, potencialmente, identificar um biomarcador predictor de resposta; iv) a eficácia da técnica através de uma avaliação clínica e de monitorização ambulatória da pressão arterial; v) o impacto da RDN em vários parâmetros como a fração de ejeção do ventrículo esquerdo, função diastólica, perfil disrítmico, função renal, perfis glucídico e lipídico, NT pro-BNP e vitamina D. Na primeira parte da nossa investigação, realizámos, em doze porcos domésticos, RDN unilateral, pré-bifurcação, com um catéter multi-eléctrodo (segunda geração). A artéria contra-lateral não foi tratada (controlo). Foi realizado OCT pré e imediatamente após o procedimento e, após um mês, follow-up com angiografia renal e OCT. Demonstrámos que a RDN causou alterações agudas na parede do vaso (alterações da intíma (edema/espasmo) e formação de trombo intra-luminal) visíveis por OCT mas, após um mês, o vaso estava totalmente recuperado, não se observando estenose da artéria renal em nenhum caso. A análise histológica revelou uma quase ausência de coloração para tirosina hidroxilase e um aumento estatisticamente significativo na quantidade de fibras de colagénio na artéria tratada, alterações compatíveis com uma redução dos terminais nervosos e um aumento da fibrose, comparativamente com o controlo, sugerindo uma entrega eficaz da energia de radiofrequência na parede arterial. Não se encontraram diferenças estatisticamente significativas em relação aos níveis tecidulares renais de norepinefrina e adrenalina, entre o rim tratado e o contra-lateral. Subsequentemente, realizámos cintigrafia cardíaca com 123I meta-iodobenzylguanidina (MIBG), num subgrupo de doentes, antes e seis meses depois da RDN, para avaliação do impacto do procedimento na actividade simpática cardíaca. O ratio coração/mediastino (HMR) precoce foi significativamente mais baixo nos respondedores pré-procedimento, mas semelhante entre os grupos após seis meses de follow-up. O HRM tardio foi estatisticamente semelhante entre os grupos, pré- e pós-RDN, mas apresentando valores mais baixos do que os reportados em indivíduos saudáveis. Por conseguinte, não se verificaram diferenças estatisticamente significativas na taxa de washout, mas valores basais acima do normal foram detectados, sendo esta diferença mais evidente nos não-respondedores (hiperactivação simpática? risco aumentado de eventos cardiovasculares?), verificando-se uma aproximação entre os 2 grupos após a RDN. Nenhum dos parâmetros avaliados se alterou significativamente no follow-up, o que poderá traduzir uma ausência de lesão dos nervos simpáticos. Em conclusão, a RDN é um procedimento seguro, em termos de integridade nervosa cardíaca, mas nenhum dos parâmetros de MIBG avaliados foram úteis para predizer resposta. Na terceira parte da nossa investigação, avaliámos o perfil imunológico celular na nossa população de 23 doentes com HT resistente e tratada com RDN. Um estudo preliminar, que incluiu uma análise exaustiva de células T, B e natural killer, monócitos e células dendríticas, orientou o o estudo posterior e permitiu identificar as células T (CD4 e CD8, memória e activadas) como possíveis biomarcadores de resposta na RDN. Foram obtidas amostras de sangue em seis instantes de tempo, pré e pós-procedimento. A resposta à RDN foi avaliada aos seis meses e um ano e observou-se em 69.6 e 82.6% dos doentes, respectivamente. Os valores absolutos das células T duplas-negativas activadas foram significativamente inferiores no grupo de respondedores ao ano. Verificou-se adicionalmente uma interação entre os diferentes tempos em 3 subgrupos de células T (T CD4, T CD8 e T CD8 naive), com tendência para os respondedores apresentarem valores absolutos inferiores e pouca variação entre os tempos. Seguidamente, a nossa investigação focou-se em avaliar o comportamento das citocinas em 21 doentes tratados com RDN. Foram analizados 45 alvos proteicos que incluiram citocinas, quimiocinas e factores de crescimento. A resposta à RDN foi avaliada de acordo com o descrito previamente. Amostras de sangue venoso foram obtidas em quatro instantes de tempo, pré e pós-procedimento. 66.7% dos doentes foram respondedores aos 6 meses e 85.7% foram respondedores-tardios. Os níveis de RANTES (regulada sob activação, expressada e secretada por células T normais) foram significativamente mais baixos nos respondedores, tanto basalmente como aos 30 dias (p=0.037). Como há evidência que a Angiotensina II inibe a expressão da RANTES e, o sistema renina-angiotensina-aldosterona está directamente conectado ao sistema nervoso simpático, podemos colocar a hipótese de que baixos níveis de RANTES estão associados a valores elevados de angiotensina II e consequentemente, a um sistema nervoso simpático sobreactivado, tornando estes doentes mais susceptíveis de responderem à RDN. Por último, avaliámos a concentração da vitamina D sérica, colocando a hipótese de potencial preditora de resposta à RDN. Adicionalmente, avaliámos a segurança e eficácia a longo-prazo, assim como os parâmetros ecocardiográficos, na população de 24 doentes submetida a RDN. Foi avaliada a resposta aos 6 meses (respondedores precoces) e ao ano. Observámos que os respondedores apresentaram valores basais e aos seis meses significativamente mais elevados de vitamina D que os não-respondedores. Os respondedores ao ano (que inclui os respondedores tardios) continuaram a apresentar valores mais elevados de vitamina D que os não-respondedores, apesar de ausência de significado estatístico, provavelmente pelo baixo número de não-respondedores nesta altura. No follow-up a longo-prazo (média de 52 meses), 70.8% dos doentes mantiveram resposta clínica. Em relação aos parâmetros ecocardiográficos, verificou-se uma melhoria da função diastólica nos não-respondedores, achado que poderá reflectir benefício da RDN, apesar de não se ter observado efeito na pressão arterial. Não foram encontradas outras diferenças de relevo nos parâmetros ecocardiográficos. Em conclusão, a RDN efectivamente reduziu a pressão arterial na maioria dos doentes estudados, apresentando um óptimo padrão de segurança. O nosso estudo contribuiu para uma melhor compreensão do perfil clínico, imagiológico, imunológico e hemodinâmico dos doentes submetidos a este procedimento e, para a identificação de potenciais biomarcadores de sucesso. Os nossos resultados poderão permitir uma melhor seleção de doentes, para que possam realmente beneficiar da RDN, e potenciar investigação nesta área.

Fibre reinforced polymer (FRP) composite is one of the most promising material system of 21st century due to high strength to weight ratio, non-corrosive nature, flexibility and easy fabrication. FRP has enormous demand for application in diversified fields like aerospace, submarines, underwater pipeline, automobiles, architectural components, satellites etc. Major dilemma is faced by researchers, when FRP composites are exposed to harsh and hostile environments. FRP composites are prone to degradation when exposed to humid environments, cryogenic environments,high temperature, different radiation environments such as UV and microwave, thermal spikes and shocks etc. Due to this, environmental degradation of FRP composite is a progressing area of research. The current experiment deals with ‘Durability assessment of carbon nano tubes (CNT) modified advanced fibrous polymeric composite in different marine environment’. The material system used were GFRP, GFRP+0.1wt.%CNT, GFRP+0.3wt.%CNT and GFRP+0.5wt.% CNT, which were exposed to different environmental conditions , 35°C water, 5°C water and hygrothermal conditioning. All the samples showed decrease in properties when exposed to moisture due to plasticization, but the degradation shown by GFRP+0.1%CNT was the least due to minimum absorption of water in the first two environmental conditioning and showed the best mechanical properties due to uniform dispersion and higher surface area. Hence, nano filler modification should be done judiciously to derive the best properties from FRP composites. If more wt % of CNT is used then it would result in agglomeration, formation of voids and provide sites for micro crack nucleation which will ultimately result in failure of composite.

This thesis describes the electrical and physiological characterization of cardiac tissue with myocardial infarction (MI) responsible for abnormal cardiac rhythms such as ventricular tachycardia (VT), using a newly-developed magnetic resonance imaging (MRI) electrophysiology system. In electrophysiology (EP), radiofrequency (RF) catheter ablation combined with cardioverter-defibrillator implantation is a first-line action to manage ventricular VT. Unfortunately, this therapy is known to have sub-optimal success rates in a large number of patients because of difficulties to accurately identifying the arrhythmic target regions. Currently, characterization of post-MI scars is performed by using catheters to measure electrical signals of the endocardial tissue (electroanatomical mapping), under x-ray fluoroscopy guidance. Prolonged radiation exposure to both the cardiologist and the patient have made the use of MRI extremely attractive; further, unlike x-ray imaging, MRI provides post-MI scars with direct visualization, characterization in three dimensions and the ability to visualize ablation lesions. Although recent research has focused on registration between pre-acquired MR images and electroanatomical maps, a potentially more useful approach is to use real-time MRI to directly locate and characterize potential arrhythmogenic regions during the EP procedure. A real-time MR-guided EP system was developed and validated to perform EP diagnostic procedures, such as mapping and pacing. In a series of animal studies, the system demonstrated the ability to use active catheter tracking and intra-procedural MR imaging to navigate to specific regions in the left ventricle and record intracardiac electrical signals. A study correlating myocardial fibrotic scar detected by multicontrast late enhancement (MCLE) MRI and electroanatomical voltage mapping demonstrated that MRI information (transmurality, tissue classification, and relaxation rate) can accurately predict areas of myocardial fibrosis identified with bipolar voltage mapping. Finally, MCLE-derived gray zone was shown to have a high correspondence to regions with a high proportion of abnormal intracardiac signals. The methods described in this thesis help advance the understanding of infarcted tissue responsible for ventricular tachycardia. Further studies are proposed to perform RF ablation lesions and correlate pre- and post-ablation tissue electrophysiological properties with MRI.

Advanced fibrous polymeric composites are one of the most successful composite material systems due to its wide range of advantages such as high specific strength and stiffness, fatigue properties and corrosion resistance. Composite structures undergo different loading conditions i.e. from static to dynamic during their service life. During a cruise cycle an aircraft structure undergo different temperatures starting from ambient temperature on ground to during flight at 30,000 ft (-50˚C) and +50˚C during stays at the tropic and arid places. The polymer matrix is more susceptible to these changes than the fiber and thus dominates the mechanical behavior of FRP composites. Polymers are characterized as visco-elastic materials that their mechanical properties are strain rate dependent or they are called as sensitive to the rate at which loaded. The present experimental investigation uses flexural test to assess the effects of thermal conditioning at above- (50˚C) and below- (-50˚C) ambient temperature for multilayered laminates of 60 weight percentages of silane treated E-Glass fiber/epoxy composites and also with PAN based high strength epoxy compatible carbon fiber/epoxy composites. The state of interaction between the fiber and matrix was reflected in the ILSS values measured by 3 point-bend test with an Instron tensile testing machine with five increasing crosshead speed ranging from 1, 10,100,200 and 500 mm/min. Thermal conditioning at +50˚C is to induct further polymerization process in terms of epoxy embrittlement and along with the development of penetrating and/or semi penetrating network at the fiber/matrix interface. Whereas at −50˚C, the polymer chains get frozen due to which the deformation process is reduced results in less polymer relaxation i.e. it gets hardened. At higher crosshead speed due to shorter load assisted relaxation time, there is reduction in ILSS. The polymer gets more time for relaxation at lower crosshead speeds; as a result there is enhancement of ILSS values. The failure mechanisms are changing with changing in loading rate from static to dynamic. Fracture processes at the crack tip are controlled by thermal relaxation time and mechanical relaxation. At higher strain rates the heat generation was much faster than heat removal due to quasi-adiabatic heating which increases the fracture strain. In both the systems the locus of failure will shift from fiber polymer interface to the matrix itself that means instead of adhesion failure the predominating failure may be cohesive failure and that too shear cusp formation. FTIR analysis depicts that the band at 2609 cm-1 in the spectrum of carbon/epoxy composite can’t be seen properly in the spectra of the glass/epoxy systems. Carbon fiber may react with the OH groups which supports that the ILSS values are higher for CFRP than GFRP. DSC analysis shows an increase in glass transition temperature (Tg) after thermal conditioning for glass/epoxy composites. But for carbon /epoxy systems due to strong adhesion between the fiber and matrix Tg value is more as compared to glass/epoxy systems. But with increase in thermal conditioning time the Tg decreases due to the breakage of secondary bonds. AFM surface topography reveals that fiber/matrix height difference gradually increased with the increase of thermal treatment time at 50˚C for 5 hours suggested that residual stresses are developed due to this shrinkage. Implication of thermal conditioning most often lead an improved adhesion of the interface (at above ambient) and/ or increased crack density (at below ambient) temperature. These changes might lead further complications in accessing the loading rate sensitivity which itself as contradictory as on today.