Motivation Soluble urokinase plasminogen activator receptor (suPAR) is a known inflammatory marker, which is found in various body fluids. SuPAR reflects the immune and pro–inflammatory status of patients caused by HIV and tuberculosis, amongst others. However, recent studies have shown that suPAR is related to cardiovascular function. The cardiovascular health of the black South African population is a major health concern as this group suffers mostly from hypertension and stroke, leading to an alarming increase in cardiovascular morbidity and mortality. SuPAR may be able to contribute to early detection and prevention of cardiovascular diseases. No studies regarding the associations of suPAR with cardiovascular function have been investigated on black South Africans. Objectives To investigate suPAR as a possible marker of cardiovascular function in African and Caucasian men and women, by determining possible gender and ethnic–specific associations of suPAR with cardiovascular function. Methodology There were 207 African and 314 Caucasian men and women (aged 20–79 yrs.) included in this study. High–sensitivity C–reactive protein, glucose, lipids and creatinine were determined in fasting serum and suPAR was analyzed in plasma samples. Blood pressure was measured using the OMRON apparatus (HEM–747), with a 5–min rest interval between measurements. The Finometer device was used to determine the Windkessel compliance and the carotid dorsalis–pedis pulse wave velocity (PWV) was measured with the Complior (SP acquisition system) on the left side of each subject in the supine position. The means, adjusted means and proportions were compared between the groups by using independent t–tests, analysis of co–variance and the chi–square test, respectively. Associations were investigated between cardiovascular variables and suPAR using single and multiple regression analyses with either pulse wave velocity, systolic blood pressure, diastolic blood pressure or Windkessel compliance as dependent variable. Covariates included were age, body mass index, smoking, alcohol use, physical activity, glucose and high–density lipoprotein cholesterol. Results and conclusion SuPAR levels were significantly higher in Africans (P<0.001) compared to Caucasians. After adjusting for body mass index, suPAR increased significantly with age in all groups, except for African women. Moreover, the suPAR levels of African men and women were significantly higher than the Caucasians within each age quartile. While adjusting for age and body mass index, the cardiovascular profiles of the African and Caucasian men were less favourable compared to women, but suPAR levels were significantly higher in Caucasian women compared to men. In single regression, various measures of cardiovascular function correlated with suPAR in African men and Caucasian men and women. After adjusting for confounders the associations disappeared in Caucasian women, and remained nonsignificant in the African women. However, the association between PWV and suPAR remained significant in African men (B=0.19; P=0.030), while the association of systolic blood pressure (B=0.20; P=0.017), diastolic blood pressure (B=0.17; P=0.020) and Windkessel compliance (B=–0.14; P=0.004) with suPAR remained significant in Caucasian men. In conclusion, Africans presented higher suPAR levels compared to Caucasians, even when stratified by age. Gender specific associations indicated that suPAR was associated with arterial stiffness in African and Caucasian men only, therefore, indicating that suPAR could be a possible biomarker for predicting cardiovascular dysfunction. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Motivation Soluble urokinase plasminogen activator receptor (suPAR) is a known inflammatory marker, which is found in various body fluids. SuPAR reflects the immune and pro–inflammatory status of patients caused by HIV and tuberculosis, amongst others. However, recent studies have shown that suPAR is related to cardiovascular function. The cardiovascular health of the black South African population is a major health concern as this group suffers mostly from hypertension and stroke, leading to an alarming increase in cardiovascular morbidity and mortality. SuPAR may be able to contribute to early detection and prevention of cardiovascular diseases. No studies regarding the associations of suPAR with cardiovascular function have been investigated on black South Africans. Objectives To investigate suPAR as a possible marker of cardiovascular function in African and Caucasian men and women, by determining possible gender and ethnic–specific associations of suPAR with cardiovascular function. Methodology There were 207 African and 314 Caucasian men and women (aged 20–79 yrs.) included in this study. High–sensitivity C–reactive protein, glucose, lipids and creatinine were determined in fasting serum and suPAR was analyzed in plasma samples. Blood pressure was measured using the OMRON apparatus (HEM–747), with a 5–min rest interval between measurements. The Finometer device was used to determine the Windkessel compliance and the carotid dorsalis–pedis pulse wave velocity (PWV) was measured with the Complior (SP acquisition system) on the left side of each subject in the supine position. The means, adjusted means and proportions were compared between the groups by using independent t–tests, analysis of co–variance and the chi–square test, respectively. Associations were investigated between cardiovascular variables and suPAR using single and multiple regression analyses with either pulse wave velocity, systolic blood pressure, diastolic blood pressure or Windkessel compliance as dependent variable. Covariates included were age, body mass index, smoking, alcohol use, physical activity, glucose and high–density lipoprotein cholesterol. Results and conclusion SuPAR levels were significantly higher in Africans (P<0.001) compared to Caucasians. After adjusting for body mass index, suPAR increased significantly with age in all groups, except for African women. Moreover, the suPAR levels of African men and women were significantly higher than the Caucasians within each age quartile. While adjusting for age and body mass index, the cardiovascular profiles of the African and Caucasian men were less favourable compared to women, but suPAR levels were significantly higher in Caucasian women compared to men. In single regression, various measures of cardiovascular function correlated with suPAR in African men and Caucasian men and women. After adjusting for confounders the associations disappeared in Caucasian women, and remained nonsignificant in the African women. However, the association between PWV and suPAR remained significant in African men (B=0.19; P=0.030), while the association of systolic blood pressure (B=0.20; P=0.017), diastolic blood pressure (B=0.17; P=0.020) and Windkessel compliance (B=–0.14; P=0.004) with suPAR remained significant in Caucasian men. In conclusion, Africans presented higher suPAR levels compared to Caucasians, even when stratified by age. Gender specific associations indicated that suPAR was associated with arterial stiffness in African and Caucasian men only, therefore, indicating that suPAR could be a possible biomarker for predicting cardiovascular dysfunction. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Syfte: Att utreda sambandet mellan den lösliga formen av urokinas-receptorn (suPAR) i saliv hos patienter med artrit i käkleden (A-TMJ) och friska kontroller, för att skapa en grund för vidare forskning av suPAR som prediktor för inflammationsgraden i käkleden hos patienter med A-TMJ.Material och metod: En fall-kontrollstudie utfördes med 6 kontroller (medelåldern 31±11år) och 5 patienter med A-TMJ (medelåldern 24±5år). Undersökningen bestod av salivprov, registrering av blödning vid sondering (BoP), blodprovstagning, och undersökning av tuggsystemet där antalet smärtsamma käkledsrörelser (PM) mättes. Sist samlades käkledvätska in. Halten suPAR analyserades i saliv, plasma och käkledsvätska. Resultat: En signifikant skillnad mellan suPAR i saliv kunde ej påvisas (A-TMJ 4,4±3,91ng/ml, kontroller 4,96±4,80ng/ml), emellertid hade patienter en signifikant högre halt av suPAR i plasma (A-TMJ 2,71±0,62ng/ml, kontroller: 1,86±0,35ng/ml, P=0,017). Halten av suPAR i käkledsvätska mättes till 1,57±1,50ng/ml hos patienter men kunde inte detekteras hos kontroller. BoP mättes till 16±9% hos patienter och 14±7% hos kontroller, och median(IQR) för PM var 3(1) i höger käkled och 0(3) i vänster käkled hos patienter. Slutsatser: (i) Ingen slutsats kan dras gällande sambandet mellan suPAR i saliv och A-TMJ, men (ii) patienter med A-TMJ har till viss mån en högre smärta i käkleden vid käkledsrörelse medan deras koncentration av suPAR i plasma är högre jämfört med friska kontroller. Det verkar som (iii) BoP skulle kunna vara kopplat till suPAR i saliv. Resultat från denna studie bör tolkas med försiktighet på grund av litet stickprov, fortsatt forskning behövs för att klargöra sambandet mellan suPAR i saliv och A-TMJ. / Aims: To investigate the levels of soluble urokinase plasminogen activator receptor (suPAR) in saliva between patients with arthritis in the temporomandibular joint (A-TMJ) and healthy controls to create a foundation for further research of the potential predictive value of suPAR in patients with A-TMJ.Materials and method: A case- control study was conducted, 6 controls (mean age 31±11years) and 5 patients with A-TMJ (mean age 24±5years) enrolled in the study. Saliva, blood, synovial fluid (SF) were sampled, and the masticatory system was examined according to DC/TMD, and bleeding on probing (BoP) was assessed, as was painful mandibular movement (PM). The level of suPAR was analyzed in saliva, plasma and SF.Results: Level of salivary suPAR did not differ significantly between A-TMJ patients and healthy controls (P > 0.05). Patients had a significantly higher level of suPAR in plasma than controls (A-TMJ 2.71±0.62ng/mL, controls: 1.86±0.35ng/mL, P=0.017). suPAR level in SF was measured to 1.57±1.50ng/mL in A-TMJ patients and not detected in controls. BoP was 16±9% in patients and 14±7% in controls, and median(IQR) of PM was 3(1) in the right TMJ and 0(3) in the left in patients.Conclusions: (i) No conclusion can be drawn regarding suPAR in saliva and A-TMJ, but (ii) to some degree A-TMJ patients have higher PM meanwhile their plasma concentration of suPAR is higher than controls. A trend that (iii) higher BoP is connected with higher suPAR in saliva could be distinguished. Results must be interpreted with caution due to small study sample, more research is required to further elucidate the association between suPAR in saliva and A-TMJ.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.