Proefschrift Universiteit Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

A classical radioimmunoassay for hypoxanthine was developed and validated. Hypoxanthine, being a low molecular weight compound (M.W. 136.11) is not immunogenic unless first attached to a macromolecule such as a protein. Derivatives of hypoxanthine were synthesised for this purpose. In addition to the standard preparation of 6-trichloromethyl purine, a derivative of this compound with a 4-carbon spacer arm was prepared, namely purine-6-carboxypropanamide. Hypoxanthine itself showed an unusual degree of stability, having no reactivity towards reagents when considered in either the keto or the enol form. The use of 6-chloropurine, a far more reactive analogue of hypoxanthine, resulted in the synthesis of a novel carboxymethoxylamine derivative, purine-6-carboxymethyl oxime. Four conjugates were prepared using ovalbumin as carrier protein. Hypoxanthine derivatives with a free carboxyl group were conjugated using the mixed anhydride method. 6-trichloromethyl purine was reactive enough to be coupled directly. Hypoxanthine-9-B-D-arabinofuranoside was coupled using the periodate oxidation method for sugar derivatives with vicinal hydroxyl groups. As the hapten was linked via the E-amino groups of lysine residues in each case, the molar derivatisation of each conjugate was calculated by measuring the number of free amino groups in the protein before and after conjugation using 2, 4, 6-trinitrobenzene sulphonic acid. With one of the conjugates ultraviolet spectrophotometric analysis, and calculation of the amount of purine removed during dialysis were also used for comparison and confirmation of the value obtained. The antisera were affinity purified and twelve reagents were compared for their ability to elute anti-hypoxanthine antibodies, whilst retaining immunoreactivity of the eluted fractions. For the determination of hypoxanthine by radioimmunoassay two phase separation systems were investigated, namely chemical precipitation of antigen-antibody complexes using ammonium sulphate, and adsorption of free hypoxanthine using activated charcoal. The antisera were shown to be highly specific for hypoxanthine, with cross-reactivities to six analagous compounds being < 0.1%, and crossreactivity to two further compounds, adenine and allopurinol being 1.9 and 3.2% respectively. Recovery of hypoxanthine added to samples was of the order of 97.3%, and the limit of detection was > 150 nmole/g. Inter-assay coefficient of variation for the data points for the hypoxanthine standard curve was < 10% for hypoxanthine concentrations below 125 nmole/ml. Inter-assay coefficient of variation for samples of fish extract containing hypoxanthine was approximately 12%. Hypoxanthine levels increased with time in samples of trout and whitebait, so that its concentration was indicative of quality, but hypoxanthine levels in liver decreased with length of storage time. Hypoxanthine concentrations in fish samples, as determined by radioimmunoassay, were compared with the values obtained using a well established spectrophotometric method. The correlation coefficient for the two methods was 0.84507 (n = 45) using hypoxanthine solutions extracted from whitebait and 0.93298 (n = 33) for samples of trout muscle, so establishing the radioimmunoassay as a technique for measuring the quality of such foods.

This is the first systematic large-scale study of lesbian health that has been conducted in the U.K. Its purpose is to provide data about lesbians' breast and cervical screening behaviour and experiences of health care. Comparable studies in the U.S.A. suggest that lesbians do not attend for routine screening tests and are less likely, than heterosexual women, to practise breast self examination. A questionnaire (the Lesbians and Health Care Survey) was distributed to 1066 lesbians in the UK. Four follow-up focus groups (n = 30) were used to explore some of the issues arising from the survey. The major quantitative survey findings include: 12 per cent of lesbians have never attended for a cervical smear; 20 per cent have never practised BSE, and only 11 per cent attend for a mammogram every three years. The qualitative survey data were content analysed in order to identify the reasons given by lesbians for their healthcare behaviour. In the follow-up focus groups, breast health is taken as a case study. This thesis contributes to defining a lesbian feminist health agenda by its valuing of lesbians' own perspectives; by providing alternative conceptions of lesbians' health that do not rely on biomedical, disease models; and it locates lesbians' health experiences within a socio-political framework. By providing a range of data about-lesbians' health, the findings may help to inform the understanding of health providers about lesbians' health needs, improve the practice of health care delivery for lesbians and be of value to lesbians in making decisions about their health care behaviour.

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Existing screening tools are not suitable for the detection of HIV-associated neurocognitive disorders (HAND) in South Africa. Study 1 of the current thesis aimed to establish construct validity of a new screening tool, the Cognitive Assessment Tool-Rapid(CAT-Rapid), in a sample of cognitively healthy South African undergraduates (n = 122).Study 2 investigated the tool¡¯s diagnostic validity in a sample of HIV-positive adult South Africans (n = 89). In Study 1 and Study 2, correlational analyses characterizing the associations between CAT-Rapid subtests and analogous standardized neuropsychological tests sought to establish construct validity. In Study 2, ROC curves, and estimates of sensitivity and specificity values, characterized the CAT-Rapid¡¯s diagnostic validity. Results from Study 1 demonstrated adequate construct validity for the CAT-Rapid in the cognitively healthy sample. Results from Study 2 did not demonstrate construct validity of the tool in the clinical sample. Regarding diagnostic properties, at the recommended cut off score ¡Ü 10, the CAT-Rapid did not demonstrate optimal sensitivity and specificity in the detection of HAND. Future research should investigate how the CAT-Rapid compares to paper-and-pencil screening tests that have demonstrated promising results in studies emerging from the global north.

Background: Undiagnosed chronic obstructive pulmonary disease (COPD) and asthma remain prevalent health issues. The current global and Canadian prevalence reported for obstructive lung disease do not reflect the true prevalence since undiagnosed cases remain missed and uncounted. Spirometry testing is viewed as the current gold standard for diagnosing obstructive lung disease. However, barriers associated with inaccessibility and underuse have contributed to undiagnosed lung disease. While guidelines advise against spirometry for asymptomatic persons, active case-finding for persons at-risk and those presenting with symptoms has been recommended. Given early treatment and management has the potential to improve health-related quality of life and reduce the progression of lung decline, identifying undiagnosed lung disease is critical to preventing adverse health outcomes. To date, this marks the first study to incorporate both obstructive lung diseases into a single-case finding instrument. Objective: To develop and validate a case-finding questionnaire to identify undiagnosed COPD and asthma in community-dwelling adults, and to prospectively evaluate reliability and predictive performance. Methods: This study uses data obtained from the Undiagnosed Chronic Obstructive Pulmonary Disease and Asthma Population (UCAP) study from June 2017 to March 2020. Eligible participants were >18 years, had a history of chronic respiratory symptoms, and had no previous physician diagnosis of obstructive lung disease. Presence of obstructive lung disease was confirmed with spirometry. Multinomial logistic regression and recursive partitioning were used to develop a case-finding questionnaire. Predictors available from six questionnaires completed during spirometry visit. Diagnostic accuracy of the models was used to evaluate performance. Risk score externally validated in a cohort of participants recruited between October 2020 and January 2021 at study sites open during the COVID-19 pandemic. Results: Derivation cohort included 1615 participants, with 136 ultimately diagnosed with asthma and 195 diagnosed with COPD. A 13-item questionnaire was developed using logistic regression: age, pack-years of cigarette smoking, wheeze, cough, sleep, chest tightness, level of tiredness, physical activity limitation, occupational exposure, primary or second-hand smoke exposure, frequency of chest attacks, and salbutamol medication. Internal validation showed an area under the curve (AUC) of 0.79 (0.70-0.90) for COPD and 0.64 (0.45-0.80) for asthma. At a predicted probability of greater than or equal to 6%, specificity was 17% for no OLD, sensitivity was 91% for asthma, and sensitivity was 96% for COPD. External cohort included 74 subjects, with 8 diagnosed with COPD and 6 diagnosed with asthma. The AUC for COPD was 0.89 (95% CI: 0.62-0.90) and AUC was 0.65 (95% CI: 0.63-0.72) for asthma. Sensitivity was 100% for both asthma and COPD, specificity was 13%, and positive predictive value was 23%. Conclusion: The 13-item case-finding questionnaire was shown to be reliable and with modest predictive ability in identifying COPD and asthma. Prospective evaluation with the UCAP study is still ongoing to recruit a larger sample to re-evaluate predictive performance.

Exploration of peroxisome proliferator-activated receptor-gamma (PPAR") as a drug target holds applications for treating a wide variety of chronic inflammation-related diseases. Type 2 diabetes (T2D), which is a metabolic disease influenced by chronic inflammation, is quickly reaching epidemic proportions. Although some treatments are available to control T2D, more efficacious compounds with fewer side effects are in great demand. Drugs targeting PPAR" typically are compounds that function as agonists toward this receptor, which means they bind to and activate the protein. Identifying compounds that bind to PPAR" (i.e. binders) using computational docking methods has proven difficult given the large binding cavity of the protein, which yields a large target area and variations in ligand positions within the binding site. We applied a combined computational and experimental concept for characterizing PPAR" and identifying binders. The goal was to establish a time- and cost-effective way to screen a large, diverse compound database potentially containing natural and synthetic compounds for PPAR" agonists that are more efficacious and safer than currently available T2D treatments. The computational molecular modeling methods used include molecular docking, molecular dynamics, steered molecular dynamics, and structure- and ligand-based pharmacophore modeling. Potential binders identified in the computational component funnel into wet-lab experiments to confirm binding, assess activation, and test preclinical efficacy in a mouse model for T2D and other chronic inflammation diseases. The initial process used provided "-eleostearic acid as a compound that ameliorates inflammatory bowel disease in a pre-clinical trial. Incorporating pharmacophore analyses and binding interaction information improved the method for use with a diverse ligand database of thousands of compounds. The adjusted methods showed enrichment for full agonist binder identification. Identifying lead compounds using our method would be an efficient means of addressing the need for alternative T2D treatments. / Ph. D.

CQ und HCQ werden häufig zur Behandlung von Erkrankungen aus dem rheumatischen Formenkreis wie z.B. SLE oder RA eingesetzt. Die lange Anwendung birgt das Risiko der Entwicklung einer CQ/HCQ-assoziierten Makulopathie. Diese ist charakterisiert durch den irreversiblen Verlust von Photorezeptoren und RPE und im Verlauf progredienten Visusverlust. Die QAF-Bildgebung ist eine nicht-invasive, innovative Methode zur Diagnostik krankhafter Netzhautveränderungen. Durch entsprechende technische Modifikationen eines cSLO sind inzwischen quantitative Aussagen bei Verlaufskontrollen der FAF derselben Patienten und Patientinnen sowie interpersonelle Vergleiche möglich. In der vorliegenden Studie wurden 32 CQ/HCQ Patienten und Patientinnen über den Zeitraum von einem Jahr mittels multimodaler Bildgebung (IR-, FAF bei 488 nm und 787 nm, QAF bei 488 nm, rotfreie Aufnahmen sowie SD-OCT Bilder) auf BEM-typische Veränderungen am Augenhintergrund gescreent bzw. Verlaufskontrollen bei bekannter BEM durchgeführt. Die QAF Entwicklung innerhalb eines Jahres wurde quantitativ und räumlich analysiert. Hierbei zeigte sich eine den erwarteten Alterseffekt übersteigende Erhöhung der QAF. Dies könnte durch eine erhöhte Lipofuzingenese oder metabolische Aktivität der Netzhaut erklärt werden. Die vorgestellten Methoden könnten zukünftig eine nützliche Erweiterung zu den bereits bestehenden Diagnostik-Tools für Screening auf BEM sein. Bei CQ/HCQ Patienten und Patientinnen zeigt sich eine grundsätzlich erhöhte QAF gegenüber der Kontrollgruppe ohne das Medikament. Im Ein-Jahres-Verlauf gab es einige Patienten und Patientinnen, die einen überdurchschnittlich starken Anstieg der QAF zeigen. Es bleibt zu klären, ob diese Ausreißer Hinweise auf die spätere Entwicklung einer BEM liefern. So könnte die QAF im klinischen Alltag Anwendung finden und vor allem bei Verlaufskontrollen zusätzliche Informationen bieten. / Systemic CQ/HCQ intake can cause severe ocular side effects including bull's eye maculopathy. This disease is characterized by irreversible loss of photoreceptors, RPE and progressive vision loss. QAF imaging is a non-invasive method for diagnosis of pathological retinal changes. QAF enables the comparison of fundus autofluorescence between different individuals as well as between sessions of the same individual. Thirty two patients currently or previously treated with CQ/HCQ underwent multimodal retinal imaging (infrared, red free, fundus autofluorescence, QAF [488 nm], and spectral-domain optical coherence tomography) and were followed-up after one year. A quantitative and topographic analysis of QAF development was performed. Our study confirms our previous finding of increased QAF in patients taking CQ/HCQ with a further significant QAF increase from baseline to follow-up. Whether pronounced QAF increase might predispose for rapid progression toward structural changes and BEM development is currently investigated in ongoing studies. In addition to standard screening tools during systemic CQ/HCQ treatment, QAF imaging might be useful in CQ/HCQ monitoring and could serve as a screening tool in the future.