Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Spoilage, be it due to microbial actions, chemical reactions or both, poses a serious threat to the food and beverage industries. Not only can spoilage lead to great economic losses, but it can also cause industries to lose their competitive edge in the economic and consumer market. Considering all the modern technologies and the range of preservation techniques that are available, it is surprising that spoilage is still an economic problem. Wine spoilage due to unpalatable bitterness, and the role of lactic acid bacteria (LAB) in causing this bitterness, have received much attention over the years, but no definite understanding has yet emerged. The first objective of this study was to isolate, enumerate and identify the LAB from three red grape varieties, viz. Pinotage, Merlot and Cabernet Sauvignon. The LAB populations on the grapes of all three varieties ranged from 102 to 104 cfu/ml during the 2001 and 2002 harvest seasons. The Cabernet Sauvignon grapes had slightly higher numbers than the Pinotage and Merlot. The LAB population in the Cabernet Sauvignon, Pinotage and Merlot wines after completion of the alcoholic fermentation ranged from 102 to 105 cfu/ml, while during 2002 the numbers in wine undergoing malolactic fermentation (MLF) ranged from 104 to 108 cfu/ml. The isolated LAB were divided into the three metabolic groups, with 59% belonging to the facultatively heterofermentative group, 26% to the obligately heterofermentative group and 15% to the obligately homofermentative group. The isolates were identified by means of species-specific primers as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lb. hilgardii (15), Lb. plantarum (98), Lb. pentosus (12), Lb. paraplantarum (3), Lb. paracasei (28), Pediococcus acidilactici (2) and Pediococcus spp. (35). The most predominant species isolated was Lb. plantarum, followed by Pediococcus spp. The results suggest that Pinotage carries a more diverse LAB population in comparison to Merlot and Cabernet Sauvignon. The second objective of this study was to determine the presence of the glycerol dehydratase gene in the LAB strains by using the G01 and G02 primers. Twenty-six strains tested positive, namely Lb. plantarum (15), Lb. pentosus (1), Lb. hilgardii (5), Lb. paracasei (2), Lb. brevis (2) and a Pediococcus spp. (1). Interestingly, 62% of these strains were isolated from Pinotage. The strains all had the ability to degrade glycerol by more than 90%, and no significant differences were observed between the species. The GO-possessing strains exhibited varying degrees of inhibition towards Gram-positive and Gram-negative bacteria, and the results suggest that this inhibition activity may be similar to that of reuterin, which is produced by Lb. reuteri. This study can form the foundation for unravelling the causes of bitterness in red wines. Combining the results of this study with analytical, sensory and molecular data may very well provide the industry with valuable tools with which to combat the occurrence of bitterness. / AFRIKAANSE OPSOMMING: Bederf as gevolg van mikrobiese aksies, chemiese reaksies of beide, hou 'n groot bedreiging vir die voedsel- en drankbedrywe in. Nie net kan bederf lei tot groot ekonomiese verliese nie, maar dit kan ook veroorsaak dat bedrywe hul kompeterende voordeel in die ekonomiese en verbruikersmarkte verloor. As die moderne tegnologie en die reeks preserveringstegnieke wat beskikbaar is, in ag geneem word, is dit verbasend dat bederf steeds 'n ekonomiese probleem is. Wynbederf as gevolg van oormatige bitterheid en die rol van melksuurbakterieë (MSB) in die ontwikkeling van hierdie bitterheid het oor die jare heen baie aandag geniet, maar geen definitiewe verklaring is nog daarvoor gevind nie. Die eerste doelwit van hierdie studie was om MSB vanaf drie rooidruifvariëteite, nl. Pinotage, Merlot en Cabernet Sauvignon, te isoleer, te kwantifiseer en te identifiseer. Die MSB-populasies op die druiwe van al drie variëteite het gedurende die 2001- en 2002-parsseisoene tussen 102 en 104 kvu/ml gevarieer. Die Cabernet Sauvignon-druiwe het effens hoër getalle as die Pinotage- en Merlot-druiwe gehad. Die MSB-populasies in die Cabernet Sauvignon-, Pinotage- en Merlot-wyne aan die einde van die alkoholiese fermentasie het tussen 102 en 1055 kvu/ml gevarieer. Gedurende 2002 het die MSB-getalle in die wyne waarin appelmelksuurgisting (AMG) aan die gang was tussen 104 en 108 kvu/ml gevarieer. Die geïsoleerde MSB was onderverdeel in die drie metaboliese groepe, met 59% wat behoort aan die fakultatiewe, heterofermentatiewe groep, 26% aan die obligate, heterofermentatiewe groep en 15% aan die obligate, homofermentatiewe groep. Die isolate is geïdentifiseer as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lactobacillus hi/gardii (15, Lactobacillus p/antarum (98), Lactobacillus pentosus (12), Lactobacillus parap/antarum (3), Lactobacillus paracasei (28), Pediococcus acidi/actici (2) en Pediococcus spp. (35) deur middel van spes iespesifieke inleiers. Die mees algemeen geïsoleerde spesies was Lb. p/antarum, gevolg deur Pediococcus spp. Die resultate impliseer dat Pinotage 'n meer uiteenlopende MSB-populasie in vergelyking met Merlot en Cabernet Sauvignon dra. Die tweede doelwit van hierdie studie was om die teenwoordigheid van die gliseroldehidratase-geen in die MSB-isolate deur middel van die GD1- en GD2- inleiers te bepaal. Ses-en-twintig isolate was positief, nl. Lb. p/antarum (15), Lb. pentosus (1), Lb. hi/gard;; (5), Lb. paracasei (2), Lb. brevis (2) en 'n Pediococcus spp. (1). 'n Interessante resultaat was dat 62% van hierdie isolate vanaf Pinotage geïsoleer is. Die isolate was almal in staat om meer as 90% van die gliserol te gebruik en geen noemenswaardige verskille is tussen die isolate waargeneem nie. Die GD-bevattende isolate het verskillende grade van inhibisie teenoor Grampositiewe en Gram-negatiewe bakterieë getoon, en die resultate impliseer dat hierdie inhiberende aktiwiteit dieselfde is as dié van reuterin wat deur Lb. reuteri geproduseer word. Hierdie studie kan dus die basis vorm vir die ontrafeling van die oorsake van bitterheid in rooiwyne. Deur die resultate van hierdie studie met analitiese, sensoriese en molekulêre data te kombineer, kan die wynbedryf voorsien word van waardevolle metodes om die voorkoms van bitterheid mee te bekamp.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Proteins from various sources, including grape berry cells, yeast, bacteria and fining agents e.g. albumin and casein, have previously been identified in wine. These proteins play various critical roles in the functioning and survival of the organisms that produced them but also exhibit oenological properties, once secreted in the juice/wine. Some of them can indeed be beneficial to winemaking, by releasing aroma compounds from grape-derived precursors, or detrimental to wine quality, by causing protein haze. Yeasts contribute significantly to the protein pool during and after alcoholic fermentation. However, while the extracellular proteins of Saccharomyces cerevisiae, the main wine yeast species, have been characterised, those of non-Saccharomyces yeasts remain largely unknown, especially under winemaking conditions. Although specific extracellular enzymes released by non-Saccharomyces yeasts have been the focus of many studies in recent years, the targeted approaches used have restricted our knowledge to these specific enzymes and excluded the other secreted proteins. A more comprehensive insight into entire secretomes could improve our understanding of how yeasts survive in wine and interact with other species in mixed culture fermentations. This study aims to characterise the exo-proteome of Saccharomyces and selected non-Saccharomyces yeasts in pure and mixed cultures in a wine-like medium. Fermentation kinetics were monitored and the extracellular proteins isolated at the end of fermentation. M. pulcherrima hardly fermented whereas L. thermotolerans fermented slowly but steadily. As expected S. cerevisiae completed the fermentation rapidly. In sequential fermentations, the kinetics resembled those of the non-Saccharomyces yeasts for a period before switching to that of S. cerevisiae. This period varied from 4 to 15 days for M. pulcherrima and L. thermotolerans respectively. Visual observations of the protein content of the medium at the end of fermentation using 1D and 2D SDS-PAGE gels as well as identification of these proteins using mass fingerprinting revealed the large variety of proteins secreted and the influence of yeast interactions on each other’s secretome. The fermentation kinetics observed could partially be explained by the extent of the contribution of the different yeast to the protein content. Proteins secreted by non-Saccharomyces yeasts lowered the potential of wine to form protein haze, with both M. pulcherrima and L. thermotolerans in pure and mixed culture fermentations showing lower haze formation than S. cerevisiae. As far as we know, this is the first report on the secretome of non-Saccharomyces under winemaking condition and the influence non-Saccharomyces proteins have on the protein haze potential of wine, providing the basis for future investigations. / AFRIKAANSE OPSOMMING: Proteïene vanaf verskeie bronne (insluitend druiwe korrels, gis, bakterieë en verhelderings agente bv. albumien en kaseïen) is reeds in wyn identifiseer. Hierdie proteïene speel verskeie rolle in die funksionering en oorlewing van die organismes wat dit produseer, maar beskik ook oor wynkundige eienskappe sodra dit in die sap of wyn uitgeskei word. Hoewel sommige proteïene in wyn wel voordelig mag wees as gevolg van die vrystelling van aroma komponente vanuit druif‐voorlopers, kan dit ook nadelig wees vir wyn kwaliteit deur die troebelheid wat dit kan veroorsaak Gis dra aansienlik by tot die totale proteïen inhoud van wyn, beide gedurende asook na alkoholiese fermentasie. Alhoewel die ekstrasellulêre proteïene van Saccharomyces cerevisiae (die mees algemeen gebruikte gis vir wynmaak) reeds goed gekarakteriseer is, is die proteïene van nie-Saccharomyces giste grootliks onbekend, veral die wat tydens wynmaak vrygestel word. Gedurende die laaste paar jaar het verskeie studies gefokus op spesifieke ekstrasellulêre ensieme wat deur nie-Saccharomyces giste produseer word, maar geteikende benaderings het ons kennis beperk tot net hierdie spesifieke ensieme, en enige ander afgeskeide proteïene uitgesluit. ʼn Meer omvattende insig oor die algehele afgeskeide proteoom kan ons begrip van hoe gis in wyn oorleef en interaksies tussen gis spesies in gemengde kultuur fermentasies verbeter Hierdie studie streef om die sekretoom van Saccharomyces en geselekteerde nie-Saccharomyces giste in suiwer en gemengde kultuur fermentasies van sintetiese wyn medium te karakteriseer. Fermentasie kinetika is gemonitor en die ekstrasellulêre proteïene is teen die einde van fermentasie geïsoleer. Metschnikowia pulcherrima het swak fermenteer terwyl Lachancea thermotolerans stadig tog reëlmatig fermenteer het. Soos verwag, het S. cerevisiae vinnig tot droog fermenteer. In agtereenvolgend geïnokuleerde fermentasies is die kinetika vir ʼn tydperk soortgelyk aan die van die nie-Saccharomyces giste voordat dit oorskakel na die van S. cerevisiae. Hierdie tydperk wissel respektiewelik vanaf 4 tot 15 dae vir M. pulcherrima en L. thermotolerans. Visuele waarnemings van die proteïen-inhoud van die medium aan die einde van die gisting met behulp van 1D en 2D SDS-PAGE gels asook identifisering van hierdie proteïene met behulp van massa vingerafdrukke onthul die groot verskeidenheid proteïene wat afgeskei word, asook die invloed van die giste se interaksies op mekaar se sekretoom. Die fermentasie kinetika waargeneem kan gedeeltelik verklaar word deur die omvang van die bydrae van die verskillende gis tot die proteïen-inhoud. Proteïene wat afgeskei word deur nie-Saccharomyces giste verlaag die potensiaal van wyn om proteïen troebelheid te vorm, met beide M. pulcherrima en L. thermotolerans (in suiwer en gemengde kultuur fermentasies) wat minder troebelheid vorm as fermentasies met S. cerevisiae. Sover ons kennis strek, is hierdie die eerste verslag oor die sekretoom van nie- Saccharomyces onder wynmaak toestande en ook oor die invloed wat nie-Saccharomyces proteïene op die proteïen troebelheid van wyn het, en vorm die basis vir toekomstige navorsing. / Winetech and THRIP

Στην παρούσα εργασία, μελετήσαμε την συνεισφορά των μεταβολικών μονοπατιών της HDL και των χυλομικρών/VLDL στην εμφάνιση της παχυσαρκίας, στις διαταραχές του μεταβολισμού της γλυκόζης, στην εναπόθεση των τριγλυκεριδίων στο ήπαρ και στην ανάπτυξη της διατροφικά επαγόμενης μη αλκοολικής λιπώδους νόσου του ήπατος (NAFLD). Έτσι, επιλέξαμε να εστιάσουμε στην μελέτη των απολιποπρωτεϊνών Α-Ι (apoA-I) και Ε (apoE) και του ενζύμου λεκιθινο-χοληστερολική ακυλοτρανσφεράση (LCAT). Η apoA-I αποτελεί το κύριο συστατικό των υψηλής πυκνότητας λιποπρωτεϊνών (HDL) και είναι υπεύθυνη για την σύνθεση τους, η LCAT εστεροποιεί την ελεύθερη χοληστερόλη των λιποπρωτεϊνών του πλάσματος και ευθύνεται για το σχηματισμό των ώριμων σωματιδίων HDL και η apoE συμμετέχει στον καταβολισμό των υπολειμμάτων των χυλομικρών, των πολύ χαμηλής πυκνότητας λιποπρωτεϊνών (VLDL) και των χαμηλής πυκνότητας λιποπρωτεϊνών (LDL) από την κυκλοφορία καθώς και στην de novo βιογένεση της HDL. Προκειμένου να μελετηθεί ο ρόλος αυτών των μορίων στις παραπάνω μεταβολικές διαταραχές, μελετήσαμε πειραματικά μοντέλα ποντικών με έλλειψη στα γονίδια αυτά. Συγκεκριμένα, ομάδες ποντικών με έλλειψη στο γονίδιο που κωδικοποιεί την apoA-I (apoA-I-/-), την LCAT (LCAT-/-), την apoE (apoE-/-) αλλά και μια ομάδα ποντικών που εκφράζουν το πλήρες γονιδίωμα (C57BL/6) τέθηκαν σε δίαιτα πλούσια σε λιπαρά (δίαιτα δυτικού τύπου) για 24 εβδομάδες και πραγματοποιήθηκαν ιστολογικές, βιοχημικές και κινητικές αναλύσεις. Στα apoA-I-/- ποντίκια παρατηρήθηκε αύξηση του σωματικού βάρους, έντονη συσσώρευση τριγλυκεριδίων στο ήπαρ, διαταραγμένη ιστολογική εικόνα του ήπατος και ανάπτυξη διατροφικά επαγόμενης NAFLD όπως, επίσης, παρουσίασαν ανοχή στη γλυκόζη και αντίσταση στην ινσουλίνη. Επιπλέον, η ποσοτικοποίηση του mRNA των γονιδίων FASN, DGAT-1 και PPAR-γ απέκλεισε την de novo σύνθεση των λιπαρών οξέων και των τριγλυκεριδίων σαν πιθανή αιτία της εμφάνισης της νόσου στα apoA-I-/- ποντίκια. Παρόμοια το μεταβολικό προφίλ δεν ανέδειξε σημαντικές διαφορές στην ενεργειακή δαπάνη μεταξύ των apoA-I-/- και των C57BL/6 ποντικών. Επίσης, παρατηρήθηκε ενισχυμένη εντερική απορρόφηση, ταχύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων από την κυκλοφορία και μειωμένη ταχύτητα ηπατικής έκκρισης των πολύ χαμηλής πυκνότητας λιποπρωτεϊνών (VLDL) σε σχέση με την ομάδα ελέγχου. Γονιδιακή μεταφορά της apoA-IMilano μέσω αδενοϊού σε apoA-I-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 12 εβδομάδες, είχε ως αποτέλεσμα την μείωση της συγκέντρωσης των ηπατικών τριγλυκεριδίων και την βελτίωση της ιστολογικής εικόνας και αρχιτεκτονικής του ήπατος. Τα ποντίκια αυτά λόγω της έλλειψης της apoA-I δεν συνθέτουν HDL, επομένως η απουσία της HDL σε συνδυασμό με δίαιτα πλούσια σε λιπαρά οδηγεί στην εμφάνιση παχυσαρκίας, διαταραχών στο μεταβολισμό της γλυκόζης και NAFLD. Για να αξιολογήσουμε τη συνεισφορά της ποιότητας της HDL στην εμφάνιση των παραπάνω διαταραχών, μελετήσαμε LCAT-/- ποντίκια που διαθέτουν ¨ανώριμη¨ δισκοειδή HDL. Όπως και στα ποντίκια που δεν εκφράζουν την apoA-I, έτσι και σε αυτή την ομάδα παρατηρήθηκε σημαντική διατροφικά επαγόμενη εναπόθεση τριγλυκεριδίων στο ήπαρ και διαταραγμένη ιστολογική εικόνα και αρχιτεκτονική του ήπατος. Αντιθέτως στα ποντίκια αυτά παρατηρήθηκε σημαντική αύξηση του σωματικού βάρους σε σχέση με την ομάδα ελέγχου. Επιπλέον, τα LCAT-/- ποντίκια δεν παρουσίασαν διαταραχές στο μεταβολισμό της γλυκόζης ενώ οι κινητικές αναλύσεις έδειξαν ότι η απουσία της LCAT σχετίζεται με αυξημένη εντερική απορρόφηση των διατροφικών λιπιδίων, ταχύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων και μειωμένη ταχύτητα ηπατικής έκκρισης των VLDL σε σχέση με τα C57BL/6 ποντίκια. Γονιδιακή μεταφορά της LCAT μέσω αδενοϊού σε LCAT-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 12 εβδομάδες, είχε ως αποτέλεσμα την σημαντική μείωση της συγκέντρωσης των ηπατικών τριγλυκεριδίων και την βελτίωση της ιστολογικής εικόνας και αρχιτεκτονικής του ήπατος. Τα μέχρι τώρα δεδομένα μας λοιπόν υποδεικνύουν πως το μεταβολικό μονοπάτι της HDL είναι κεντρικός ρυθμιστής διαδικασιών σχετιζόμενων με την εναπόθεση διατροφικών τριγλυκεριδίων στο ήπαρ και την εμφάνιση NAFLD. Επιπλέον, τα αποτελέσματα μας υποστηρίζουν πως η συνύπαρξη μειωμένης και πιθανόν δυσλειτουργικής HDL μαζί με NAFLD σε ασθενείς με μεταβολικό σύνδρομο δεν είναι μια απλή σύμπτωση αλλά υποδηλώνει μία ισχυρή μηχανιστική συσχέτιση ανάμεσα στις δύο αυτές καταστάσεις. Προκειμένου να μελετηθεί ο ρόλος του μεταβολικού μονοπατιού των χυλομικρών, μελετήσαμε ποντίκια με έλλειψη στην apoE τα οποία καταβολίζουν βραδέως τα διατροφικά λιπίδια. Τα apoE-/- ποντίκια αντιστάθηκαν στην παχυσαρκία και στην εμφάνιση της διατροφικά επαγόμενης NAFLD σε σχέση με τα C57BL/6 ποντίκια. Επίσης, δεν παρουσίασαν διαταραχές στο μεταβολισμό της γλυκόζης και οι κινητικές αναλύσεις έδειξαν ότι είχαν βραδύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων από την κυκλοφορία του αίματος. Θέλοντας να ερευνήσουμε και το ρόλο του υποδοχέα της LDL, πραγματοποιήθηκε μια σειρά ανάλογων πειραμάτων σε LDLr-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 24 εβδομάδες. Τα LDLr-/- ποντίκια είχαν σημαντική συσσώρευση τριγλυκεριδίων στο ήπαρ και NAFLD προτείνοντας ότι η ηπατική συσσώρευση τριγλυκεριδίων μέσω της apoE είναι μια διαδικασία ανεξάρτητη από τον LDLr. Τα ευρήματα μας προτείνουν ένα νέο ρόλο κλειδί για την apoE ως ένας περιφερικός συντελεστής στην ομοιόσταση των ηπατικών λιπιδίων και στην ανάπτυξη της διατροφικά επαγόμενης NAFLD. Επιπλέον, δείχνουν ότι οι διαταραχές στο μεταβολικό μονοπάτι των χυλομικρών σχετίζονται άμεσα με την εμφάνιση της NAFLD. Συμπερασματικά, το μεταβολικό σύστημα λιπιδίων και λιποπρωτεϊνών φέρεται να κατέχει κεντρικό ρόλο στην εναπόθεση ηπατικών τριγλυκεριδίων και στην εμφάνιση της NAFLD. / In the present study, we investigated the contribution of HDL and the clylomicron/VLDL pathways in the development of obesity, glucose metabolism and diet-induced non alcoholic fatty liver disease (NAFLD). Thus, we chose to study apolipoproteins A-I (apoA-I) and E (apoE), as well as the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is the main protein of high density lipoprotein (HDL) and is responsible for it’s synthesis, LCAT esterifies the free cholesterol of plasma lipoproteins and forms mature particles of HDL and apoE participates in the catabolism of chylomicrons, very low density lipoproteins (VLDL) and low density lipoproteins (LDL) and also participates in the de novo biogenesis of HDL. In an attempt to study the role of all these particles in the development of diet-induced NAFLD, apoA-I deficient, LCAT deficient, apoE deficient and control C57BL/6 mice were fed western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5Kcal/g) for 24 weeks and their sensitivity towards NAFLD was assessed by histological and biochemical methods. ApoA-I deficient (apoA-I-/-) mice showed increased body weight, increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN, DGAT-1, and PPARγ mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I-/- mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I-/- mice exhibited enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic VLDL triglyceride secretion. In agreement with these findings, adenovirus-mediated gene transfer of apoA-IMilano in apoA-I-/- mice fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. In order to evaluate the contribution of HDL quality in the development of the metabolic disturbances described above, we studied LCAT-/- mice which have immature discoidal HDL circulating in the plasma. Similarly to apoA-I-/- mice, in the LCAT-/- group we observed increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. In contrast hoewever, these mice gained significantly more body weight, compared to the control group though they did not develop disturbances in their plasma glucose metabolism. Mechanistic analyses indicated that LCAT deficiency was associated with enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic very low density lipoprotein triglyceride secretion. No statistical difference in the average daily food consumption between mouse strains was observed. Adenovirus-mediated gene transfer of LCAT in LCAT-/- mice that were fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and a great improvement of hepatic histology and architecture. Taken together, these data suggested that HDL metabolic pathway is a central modulator of processes associated with diet-induced hepatic lipid deposition and NAFLD development. Furthermore, our results sypport that the the coexistence of reduced and possibly dysfunctional HDL with NAFLD in patients with metabolic syndrome is not a mere coincidence, rather indicates a strong mechanistic link between these two conditions. In order to study the role of the chylomicron metabolic pathway, we employed apoE-deficient mice, which show a very slow catabolism of dietary lipids. Our data indicate that the apoE-/- mice are resistant to obesity and to diet-induced NAFLD compared to control C57BL/6 mice and they don’t reveal disturbances in the glucose metabolism. In an attempt to identify the molecular basis for this phenomenon biochemical and kinetic analyses revealed that apoE-/- mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma. In contrast to apoE-/- mice, LDLr-/- mice fed western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD suggesting that the apoE-mediated hepatic triglyceride accumulation in mice is independent of the LDLr. Our findings suggest a new role of apoE as key peripheral contributor to hepatic lipid homeostasis and the development of diet-induced NAFLD. Furthermore, they show that the disturbances in the metabolic pathway of chylomicron are related, directly, with the development of NAFLD. Overall, our findings reinforce our initial hypothesis that the transport of dietary lipids from the intestine to the liver plays a central role to the deposition of triglycerides in the liver and the development of NAFLD.

Oxidative stress is characterized by the cellular accumulation of reactive oxygen species (ROS). Increased production of ROS, such as the superoxide anion (O2.-), or a deficiency in their clearance by antioxidant defenses, mediates the cellular pathology. Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder commonly manifesting in milieu of the metabolic syndrome. Oxidative stress is an important pathogenic mediator in NAFLD, and in its associated morbidities like atherosclerosis. The objective of my research was to investigate the regulation of oxidative stress and the antioxidant actions of natural health products (NHPs) in the context of NAFLD and its associated disorders. The O2.- generating NADPH oxidase contributes to atherogenesis by facilitating macrophage induced vascular injury. In manuscript I, the plant alkaloid berberine effectively abolished NADPH oxidase mediated O2.- production in lipopolysaccharide stimulated macrophages. Real-time PCR analysis and siRNA transfection studies revealed that berberine mediated its effects through down-regulation of the oxidase’s catalytic subunit gp91phox. Berberine also restored the activity of the O2.- clearing enzyme superoxide dismutase (SOD). High fat diet (HFD) fed rodents are a popular model for investigating NAFLD pathogenesis. In manuscript II, folic acid supplementation significantly reduced HFD-induced hepatic oxidative stress and liver injury in mice. Folic acid decreased NF-kB/DNA binding, down-regulated NADPH oxidase gene expression, and inhibited the oxidase. The antioxidant activities of SOD and catalase were restored and the reduced to oxidized glutathione ratio (GSH:GSSG) was re-established with folic acid supplementation. Folic acid’s hepatoprotective antioxidant effects were associated with a marked improvement in liver histology. Homocysteine (Hcy) levels are perturbed in NAFLD, but the etiology is unclear. In manuscript III, HFD fed mice exhibited decreased Hcy levels. Real-time PCR and Western Immunoblotting analysis revealed that Hcy catabolising enzymes cystathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE) were increased in the liver of these animals. The transsulfuration activities of these enzymes were elevated and coincided with enhanced hepatic hydrogen sulfide biosynthesis. Glutathione was maintained despite increased hepatic oxidative stress. Taken together, NHPs such as berberine and folate, and Hcy catabolising enzymes CBS and CSE, might have therapeutic potential for managing oxidative stress in NAFLD and its associated co-morbidities. / October 2015

Oxidative stress, lipid peroxidation, and endotoxaemia with cytokine-mediated injury have been implicated as factors in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The degree of insulin resistance together with co-existing inadequacies of vital antioxidant defence mechanisms may be important determinants of progression to fibrosis in patients with non-alcoholic steatohepatitis (NASH). Current therapies are targeted at improving insulin sensitivity as well as addressing hepatic repair including anti-inflammatory strategies. Anti-oxidants remedies have also been tested but the role of selenoenzymes with antioxidant action, namely thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPX1) have been ignored. The aim of this thesis is to investigate the role of selenium in the pathophysiology of NAFLD both in vitro and in vivo. The in vitro studies used cell lines representing the cell types involved in the disorder; hepatocytes (C3A line) and hepatic stellate cells (LX-2 line). In order to assess the influence of selenium status and selenoenzymes expression on the pathogenesis of NAFLD it was necessary to develop a culture system which allowed good cell viability in selenium free culture medium. This was achieved by the use of an insulin and transferrin (IT)-supplemented medium which importantly was free of any animal serum additions. Using this IT culture medium, selenium addition (as selenite) produced a significant increase in the expression of GPX1 and TR1 in both C3A and LX2 cells. TR1 and GPX1 were expressed at similar levels in both C3A and LX-2 cells. It was also necessary to develop an in-vitro model for fat loading C3A cells to mimic fatty liver pathophysiology. Two models of fat loading were investigated. One model used lactate, pyruvate, octanoate and ammonium (LPON). LPON has been previously used to increase the functionality of C3A cells but it was observed that fat droplets accumulated in these LPON treated cells. Dissection of the agents in the LPON revealed that octanoate was the factor that increased the triglyceride accumulation. Interestingly, octanoate also increased the expression of TR1 and GPX1, suggesting that it could induce oxidative stress leading to the induction of selenoenzymes to afford a protective defence mechanism. In the second model, oleate and/or palmitate were used to fat-load C3A cells. These cells had significantly higher triglyceride content than the LPON-fat-loaded cells. However, oleate and/or palmitate treatments did not increase the expression of either TR1 or GPX1 in C3A cells suggesting perhaps these cells were not under oxidative stress. LPON and oleate/palmitate were also capable of fat loading LX2 cells. Selenium-supplementation of C3A and LX-2 cells efficiently protected (measured by their lactate dehydrogenase retention) them from oxidative damage induced by t-butylhydroperoxide. This suggests that selenium supplementation through its incorporation into selenoenzymes could protect the cells from the oxidative damage. The role of selenium was also investigated in the regulation of α-1 pro-collagen mRNA expression. In LX-2 cells, the expression of α-1 pro-collagen mRNA was unaffected by the selenium status of the cell. Similarly the selenium status of C3A cells had no effect on modifying α-1 pro-collagen mRNA of LX2 cells when co-culture or conditioned medium experiments were performed. These results suggest that LX-2 cells were already largely activated and at a stage unable to be ameliorated by selenium treatment. In contrast, studies on C3A cells revealed that TGF-β1 (common inducer of α-1 pro-collagen mRNA in hepatic stellate cells) dramatically increased the expression of α-1 pro-collagen mRNA in C3A cells to the levels observed in LX-2 cells. More interestingly, selenium supplementation of C3A cells notably decreased α-1 pro-collagen mRNA expression in response to TGF-1. In the in vivo study, plasma selenium in type 2 diabetics (high risk of developing NAFLD) were inversely related to the body mass index and in most patients selenium levels were below that required to maximally express GPX1 in red cells. Furthermore, type 2 diabetics had lower plasma selenium levels compared to the healthy control group. Collectively, this suggests that in the UK population, obesity is a risk factor for both insulin resistance and decreased selenium status leading to sub-optimal antioxidant protection. In conclusion, this study provides evidence that selenium through increasing the expression of selenoenzymes is beneficial in protecting liver cells from oxidative stress. Furthermore, selenium is capable of suppressing α-1 pro-collagen mRNA expression in hepatocytes although not in activated hepatic stellate cells. Taken together these data support the view that suboptimal selenium intake in the UK may be a risk factor in the pathogenesis of NAFLD.

In pharmacotherapeutics, the term "correct dosing" is based on the concept that too high a systemic concentration will lead to drug toxicity, while drug levels that are too low may not produce the intended therapeutic effect. Often, the factors determining the ability of a patient to manage a given dose rely on their capacity to efficiently metabolize and eliminate drugs from the body. The liver plays a crucial role in the processing of many clinically relevant drugs via three stages of hepatic drug management. Drugs must first be taken into hepatocytes by uptake transporters. Drugs are then metabolized by phase I and phase II enzymes to make them more manageable. Finally, metabolites are removed from the hepatocyte by efflux transporters either into the bile for elimination or reintroduction to systemic blood. Alterations in one or more of the hepatic drug management stages increase the potential for adverse drug reactions (ADRs).In the United States, ADRs account for between 3%-12% of admissions to hospitals, and approximately 5% of deaths each year. While less than 20% of these cases are due to genetic polymorphisms, the vast majority of ADRs are due to environmental factors including disease. Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of conditions progressing from steatosis to non-alcoholic steatohepatitis (NASH) and often leading to cirrhosis. Presently, NASH patients represent the greatest population of candidates for liver transplant, illustrating the severity as well as the incidence of this disease. Patients with NAFLD are typically treated for co-existing conditions of the metabolic syndrome (i.e. hyperlipidemina or type II diabetes) and therefore represent a distinct population at risk for adverse drug reactions.The following studies show that experimental NAFLD affects both the signal transduction pathways regulating hepatic drug management genes as well as the hepatic uptake transporter function. Additionally, patient livers diagnosed with progressive stages of NAFLD, display altered CYP activity and efflux transporter expression similar to those previously reported in experimental NAFLD. Given that changes observed in experimental NAFLD result in functional changes in hepatic drug management, similar changes observed in patients with this disease suggest an increased risk for ADRs.

This study concerned the effect of ethanol on cardiac activity and brain respiration in chick embryos. Ethanol dosages tested ranged from 1.0 mg to 4.0 mg/g weight. Each experiment lasted at least 150 minutes. Cardiac activity in terms of total waveform energies was integrated and printed out for plotting and analysis. The embryonic heart rates were simultaneously determined from physical graph tracings. The embryonic brain respiration was measured using a differential microrespirometer. The effect of ethanol on cardiac activity was one of slight (10 to 13), but statistically significant (p<.05) rate depression. The brain slices exhibited a marked, immediate, and irreversible decrease (39 to 89%) in oxygen consumption at both ethanol dosages. The data indicated that chick embryonic brain tissues were more susceptible to alcohol effects than cardiac tissue. Therefore, the mental abnormalities seen in the offspring of alcoholic mothers may be more cerebral in nature than cardiovascular.

The leading causes of liver disease are Hepatitis C virus infection and chronic alcohol abuse. Alcohol accelerates liver disease in HCV but the mechanisms are poorly understood. The identification of molecular gene expression profiles on human liver tissue was performed using microarrays. Samples were obtained from alcoholic-cirrhotic, HCV-cirrhotic, HCV/alcohol-cirrhotic and control non-cirrhotic liver tissue. Probe set expression summaries were calculated using RMA. Probe set level linear models were fit where probe set expression was modeled by HCV status, alcohol status, and the interaction between HCV and Alcohol. HCV cirrhosis was associated with up-regulation of genes related to viral and immune response, apoptosis and inflammation. There were down-regulation of genes in the ubiquititin-proteasome system in alcoholic cirrhosis. The interaction of HCV and alcohol revealed negative interaction for genes involved in apoptosis and immune response. There was a negative estimate for genes involved in class II restricted antigen presentation.

SLAVÍČKOVÁ, T. Tématicko apercepční test u uživatelů drog. Pedagogická fakulta Univerzity Karlovy v Praze, 2012. 140 s. Diplomová práce. Diplomová práce měla za cíl odhalit specifika použití TAT u problémových a závislých uživatelů drog a na základě srovnání s výsledky, kterých v testu dosáhla kontrolní skupina, přinést evidenci pro zhodnocení diagnostického potenciálu TAT u uživatelů drog. Výzkumné šetření bylo provedeno v Kolíně na klientech nízkoprahového kontaktního centra, které poskytuje služby harm reduction skryté populaci uživatelů nealkoholových drog. Těžiště výzkumu bezprostředně souvisí s metodou analýzy příběhů, jejichž vyprávění bylo u probandů vyvoláno předložením tabulí TAT. K analýze příběhů byl použit psychometricky zakotvený skórovací systém SCORS (Social Cognition - Object Relation Scale) současného amerického autora D. Westena. Použití TAT u uživatelů drog tak bylo zaměřeno na zhodnocení úrovně jejich fungování v sociálních vztazích. Metodologický princip diplomové práce tkví v induktivním usuzování a statistickém zpracování dat. Způsob zpracování dat, který je až na několik oblastí výhradně v režii kvantitativních metod, spolu s dostatečně velkým souborem zkoumaných osob umožňuje zobecnění důležitých rozdílů ve výsledcích TAT u výzkumné a kontrolní skupiny. V rámci analýzy...

This thesis in considered on financial analysis of nine most important companies which are interested in production of non-alcoholic drinks in Czech republic. Most of them are subsidiaries of global corporations. In thesis is processed spatial resolution of this field in years 2008 and 2009. First part deals with methodology of spatial resolution (Spider analysis). Next is about the field of non-alcoholic drinks. In analysis were compared years 2008 and 2009 and the values of indicators and their change during the time and also competitiveness of the companies in 2009. Conclusion is global table and comments to each company and their result in Spider analysis.