No description available.

Background. Myocardial hypertrophy is a risk factor for cardiovascular morbidity and mortality. Independent of underlying disease, the cardiac muscle strives in different ways to compensate for an increased workload. This remodelling of the heart includes changes in the extracellular matrix which will affect systolic and diastolic cardiac function. Furthermore, signal transduction, molecular diffusion and microcirculation will be affected in the hypertrophic process. One important extracellular component is the glycosaminoglycan hyaluronan. It has been shown to play a major role in other conditions that feature cellular growth and proliferation, such as wound healing and malignancies. The aim of this thesis was to investigate hyaluronan and its role in both an experimental rat model of cardiac hypertrophy as well as in cultured mouse cardiomyocytes and fibroblasts. Methods. Cardiac hypertrophy was induced in rats by aortic ligation. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were quantified after 1, 6 and 42 days after operation, in cardiac tissue from the left ventricular wall. Localization of hyaluronan and its receptor CD44 was studied histochemically. Hyaluronan synthesis was correlated to gene transcription using microarray gene expression analysis. Cultures of cardiomyocytes and fibroblasts were stimulated with growth factors. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were detected. Hyaluronan size was measured and crosstalk between cardiomyocytes and fibroblasts was investigated. Results. Increased concentration of hyaluronan in hypertrophied cardiac tissue was observed together with an up-regulation of two hyaluronan synthase genes. Hyaluronan was detected in the myocardium and in the adventitia of cardiac arteries whereas CD44 staining was mainly found in and around the adventitia. Hyaluronan synthesis correlated to the expression of genes, regulated by transcription factors known to initiate cardiac hypertrophy. Stimulation of cardiomyocytes by PDGF-BB induced synthesis of hyaluronan. Cardiomyocytes also secreted a factor into culture media that after transfer to fibroblasts initiated an increased synthesis of hyaluronan. When stimulated with hyaluronan of different sizes, a change in cardiomyocyte gene expression was observed. Different growth factors induced production of different sizes of hyaluronan in fibroblasts. The main synthase detected was hyaluronan synthase-2. Cardiomyocytes were also shown to secrete microvesicles containing both DNA and RNA. Isolated microvesicles incubated with fibroblasts were observed by confocal microscopy to be internalized into fibroblasts. Altered gene expression was observed in microvesicle stimulated fibroblasts. Conclusion. This study shows that increased hyaluronan synthesis in cardiac tissue during hypertrophic development is a part of the extracellular matrix remodelling. Cell cultures revealed the ability of cardiomyocytes to both synthesize hyaluronan and to convey signals to fibroblasts, causing them to increase hyaluronan synthesis. Cardiomyocytes are likely to express receptors for hyaluronan, which mediate intracellular signalling causing the observed altered gene expression in cardiomyocytes stimulated with hyaluronan. This demonstrates the extensive involvement of hyaluronan in cardiac hypertrophy.

This thesis deals with the aggregation behaviour of palmitoylhyaluronan in two degrees of substitution, namely 10 % and 16 %. Using a fluorescence spectroscopy method with pyrene as a fluorescence probe, we found a critical micellar concentration. The results were confirmed by measuring the dynamic light scattering, which also showed an increase in the size of aggre-gates with an increasing concentration. System stability is reduced by increased ionic strength as evidenced by zeta potential measurement. Experiments found that 16 % substitution sample can form a gel. The gel is very stiff and has excellent properties, which was confirmed by rhe-ology. We also managed to incorporate pyrene in the gel, which was demonstrated by the presence of highly solvated domains that could be polymer micelles. Based on these results, 16% substitution sample was subjected the MTT assay to cytotoxicity. The results confirmed that the examined sample was not toxic.

The effect of hydrophobically modified hyaluronan on surfactants aggregation has been studied in this master’s thesis. The value of critical micelle concentration of anionic surfactant SDBS (sodium dodecylbenzensulfonate), cationic surfactant CTAB (cetyltrimethylamonnium bromide) and nonionic surfactant Triton X-100 (octylphenol ethoxylate) was determined by fluorescence spectroscopy using pyrene probe. Aggregation behavior of surfactants was performed with addition of hydrophobically modified hyaluronan of two molecular weights (17 kDa, 206 kDa) in aqueous solution. The greatest influence of hydrophobized hyaluronan on aggregation behavior was observed in system with cationic surfactant CTAB. Stability of system containing cationic surfactant and hydrophobically modified hyaluronan was established through zeta potential. Last part of thesis deals with size determination using dynamic light scattering.

No description available.

This disertation thesis is focused on the study of physico-chemical interactions of hyaluronan (with molecular weights from 10 to 1750 kDa) with cationic surfactants measured using uncommon technique named high resolution ultrasonic spectroscopy. Densitometer was also used for the study of these interactions, in measuring of density and ultrasonic velocity of hyaluronan with different molecular weight in dependence on elevated temperature (25 50 °C). The aim is the determination of critical micelle concentration (CMC) and critical aggregation concentration (CAC) of the suractants in the absence and in the presence of hyaluronan with various molecular weights. Interactions in this system are important for the design of the systems for the targeted delivery, especially for the drugs. The experiments were made in water and sodium chloride solution. The significant breakpoint in the ultrasonic velocity showed changes in the system hyaluronan-surfactant.

<p>The kidney regulates the excretion of water and electrolytes, which maintains homeostasis and enables control of arterial blood pressure. Hyaluronan, a large negatively charged interstitial glucosaminoglycan, is heterogeneously distributed within the kidney, primarily found in the medulla.</p><p>Medullary hyaluronan content changes depending on the state of body hydration and plays a part in fluid regulation through its water binding and viscoelastic properties. </p><p>The aim of this thesis was to provide new insight into the regulation of intrarenal hyaluronan during different states of body hydration, during completion of kidney development, and during diabetes mellitus.</p><p>Dehydration reduces medullary interstitial hyaluronan in parallel with reduced hyaluronan synthase 2 gene expression and increased urinary hyaluronidase activity. Acute hydration results in an increase in medullary hyaluronan, an increase that requires nitric oxide and prostaglandins. Urinary hyaluronidase activity decreases during hydration. The elevation of hyaluronan is important for reducing water permeability of the interstitium i.e. favoring diuresis.</p><p>Changes in hyaluronan concentration constitute a morphoregulatory pathway that plays a key role in nephrogenesis. The reduction in neonatal hyaluronan depended on an angiotensin II mediated process that does not appear dependent on lymph vessel formation. If angiotensin II is blocked with an ACE inhibitor, hyaluronan accumulates, which results in structural and functional abnormalities in the kidney. </p><p>Renomedullary hyaluronan is elevated during uncontrolled diabetes, which coincides with induction of hyaluronan synthase 2 mRNA, hyperglycemia, glucosuria, proteinuria and overt diuresis. The levels of hyaluronan are probably at a <i>terminus ad quem</i> as no further response was seen during hydration. The higher interstitial expression of hyaluronan during diabetes may be involved in the progression of diabetic nephropathy.</p><p>This thesis in physiology provides new mechanistic insights into the regulation of renal hyaluronan during various aspects of fluid handling.</p>

The kidney regulates the excretion of water and electrolytes, which maintains homeostasis and enables control of arterial blood pressure. Hyaluronan, a large negatively charged interstitial glucosaminoglycan, is heterogeneously distributed within the kidney, primarily found in the medulla. Medullary hyaluronan content changes depending on the state of body hydration and plays a part in fluid regulation through its water binding and viscoelastic properties. The aim of this thesis was to provide new insight into the regulation of intrarenal hyaluronan during different states of body hydration, during completion of kidney development, and during diabetes mellitus. Dehydration reduces medullary interstitial hyaluronan in parallel with reduced hyaluronan synthase 2 gene expression and increased urinary hyaluronidase activity. Acute hydration results in an increase in medullary hyaluronan, an increase that requires nitric oxide and prostaglandins. Urinary hyaluronidase activity decreases during hydration. The elevation of hyaluronan is important for reducing water permeability of the interstitium i.e. favoring diuresis. Changes in hyaluronan concentration constitute a morphoregulatory pathway that plays a key role in nephrogenesis. The reduction in neonatal hyaluronan depended on an angiotensin II mediated process that does not appear dependent on lymph vessel formation. If angiotensin II is blocked with an ACE inhibitor, hyaluronan accumulates, which results in structural and functional abnormalities in the kidney. Renomedullary hyaluronan is elevated during uncontrolled diabetes, which coincides with induction of hyaluronan synthase 2 mRNA, hyperglycemia, glucosuria, proteinuria and overt diuresis. The levels of hyaluronan are probably at a terminus ad quem as no further response was seen during hydration. The higher interstitial expression of hyaluronan during diabetes may be involved in the progression of diabetic nephropathy. This thesis in physiology provides new mechanistic insights into the regulation of renal hyaluronan during various aspects of fluid handling.

Ο καρκίνος του παχέος εντέρου αποτελεί αναμφισβήτητα ένα παγκόσμιο πρόβλημα, όντας η δεύτερη αιτία θανάτου από καρκίνο. Τα γεγονότα που οδηγούν σε καρκίνο του παχέος εντέρου ακολουθούν στις περισσότερες περιπτώσεις συγκεκριμένη αλληλουχία. Έτσι, ξεκινώντας από υπερπλαστικό επιθήλιο, εξελίσσεται σε αδένωμα/δυσπλασία, σε ενδοεπιθηλιακή νεοπλασία και καταλήγει σε καρκίνο. Κατά την εξέλιξη σε καρκίνο σημαντικές αλλαγές λαμβάνουν χώρα στη σύσταση και οργάνωση του εξωκυττάριου χώρου του εντερικού τοιχώματος. Για το λόγο αυτό απαιτείται μελέτη του μεταβολισμού των εξωκυττάριων μακρομοριακών συστατικών, με στόχο την κατανόηση και διασαφήνιση της διηθητικής και μεταστατικής συμπεριφοράς των κακοήθων νεοπλασιών του παχέος εντέρου. Στην παρούσα εργασία κρίθηκε χρήσιμη η μελέτη των ενζύμων μεταβολισμού του υαλουρονικού οξέος (ΗΑ), συστατικό του εξωκυττάριου χώρου που φαίνεται να εμπλέκεται στο μηχανισμό της ανάπτυξης του όγκου, της διείσδυσης των καρκινικών κυττάρων και στη διάδοση των μεταστάσεων. Για τον παραπάνω λόγο μελετήθηκε η δραστικότητα των υαλουρονιδασών (Hyals) με ζυμογράφημα-ΗΑ, σε εκχυλίσματα ιστών και σε ορούς, και των συνθασών του ΗΑ (HAS-1, HAS-2) καθώς και του υποδοχέα του ΗΑ, CD44, με RT-PCR ανάλυση. Τα αποτελέσματα που προκύπτουν από το ζυμογράφημα-ΗΑ σε εκχυλίσματα ιστών συναινούν σε αυξημένη δραστικότητα των Hyals σε καρκινικά σε σύγκριση με τα μακροσκοπικώς φυσιολογικά δείγματα στην πλειονότητα των σταδίων, ενώ και οι δύο δραστικότητες είναι σημαντικά αυξημένες σε σχέση με τα δείγματα υγιών ιστών. Όσον αφορά τα δείγματα ορών, τα επίπεδα των Hyals φαίνεται να μειώνονται αισθητά 7 ημέρες μετεγχειρητικά σε σχέση με την ημέρα πριν την εγχείρηση, ενώ 1, 3 και 6 μήνες μετά εμφανίζεται και πάλι σταδιακή αύξηση των επιπέδων τους. Σε γονιδιακό επίπεδο, τα επίπεδα της HAS-1, HAS-2 και του CD44 εμφανίζονται αυξημένα στα καρκινικά δείγματα έναντι των μακροσκοπικώς φυσιολογικών. / Colon cancer is indisputably a great problem, being the second cause of death by cancer. The facts that lead to colon cancer have certain concatenation. Thereby, starting most of the times as hyperplastic epithelium, it develops to endoepithelium adenoma, and it finally leads to cancer. During this development important changes happen at the constitution and organization of the extracellular matrix of the intestinal tract. For this purpose, study of the metabolism of the extracellular matrix constituents is required, so as to understand the metastatic behaviour of the malignant tumors of colon cancer. The present study focuses on the metabolism enzymes of hyaluronic acid (HA), a constituent of the extracellular matrix that seems to be involved in the tumor growth mechanism, the infiltration of the cancerous cells and metastasis. Particularly, hyaluronidases (Hyals) activity (HA-zymography) and hyaluronan synthases (HAS-1, HAS-2) as well as hyaluronan receptors (CD44) expression (RT-PCR analysis) are studied at this study. The results of the study show increased Hyals levels in cancerous samples compared to the macroscopically normal ones, in all anatomic sites of colon examined, while both activities remain significantly increased compared to healthy samples.. as far as it concerns Hyals levels in sera, they seem to decrease perceptibly 7 days postoperatively, while 1, 3 and 6 months afterwards gradually increased to reach the amount preoperatively. In gene level, HAS-1, HAS-2 and CD44 expression levels were increased in cancerous samples compared to the macroscopically normal ones.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.