Lycopene is a antioxidant found in natural and processed foods. The connection between antioxidants and heart disease has been explored in several observational studies1-4, yet very few investigators have examined the impact of dietary antioxidants in patient with advanced heart disease such as heart failure (HF). A novel strategy for preventing or delaying the complications of HF related to inflammation and oxidative stress may be to increase dietary lycopene. The purpose of this dissertation was to test the impact of dietary intervention consisting of lycopene (V8® juice) on biomarkers of inflammation and oxidative stress in patients with HF; Prior to testing the dietary intervention, preliminary work was conducted: 1) a review of the literature on dietary lycopene interventions in patients with HF and 2) a longitudinal study to examine whether lycopene and sodium intake interact to produce an effect on event-free survival in patients with HF. Forty patients with HF were randomly assigned to one of two treatment groups (intervention and usual care). The intervention group received 24 mg of lycopene by drinking 11.5 ounces of V8®100% vegetable juice daily for 30 days. The usual care group continued their usual diet. Serum levels of uric acid and C-reactive protein were obtained to determine the impact of the lycopene dietary intervention. Patients in the intervention group had higher levels of plasma lycopene after one month drinking V8® juice. We also found a significant decrease in plasma CRP levels among women in the intervention group, while there was no change in CRP levels among men in the intervention group. This dissertation has provided insight about lycopene as a potential nutritional intervention for patients with HF, aimed at reducing inflammation and oxidative stress. This dietary intervention is practical, easy to replicate, cost effective and is safe for patients with HF. Additional research is needed to determine the effects of long-term outcomes of dietary antioxidants in patients with HF.

Stoffwechsel und antioxidativer Status bei Merinofleischschafen im peripartalen Zeitraum Medizinische Tierklinik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im Juni 2010 (99 Seiten, 28 Abbildungen, 38 Tabellen, 357 Literaturangaben) Problem- und Zielstellung: Dem antioxidativen System wird im Rahmen von Stoffwech-seluntersuchungen bei Schafen nur wenig Bedeutung geschenkt, jedoch gewinnt es als Schutz- und Regulationssystem zunehmend an Bedeutung. Besonders die Trächtigkeit und die Frühlaktation bei Mutterschafen sowie die postnatale Entwicklung der Lämmer stellen enorme Anforderungen an die Energieversorgung und an das antioxidative System. Das Ziel dieser Arbeit ist, die Bewältigung dieser Anforderungen darzustellen und zu zei-gen, ob und wie stark Zwillingsträchtigkeiten ein Belastung für Mutterschafe darstellen, und ob Lämmer aus einer Zwillingsträchtigkeit möglicherweise benachteiligt sind. Tiere, Material und Methoden: Untersucht wurden insgesamt 23 trächtige Merinofleisch-schafe und zwölf Merinofleischschaflämmer. Bei 13 Muttertieren wurde sonografisch eine Einlingsträchtigkeit (M1) und bei zehn eine Zwillingsträchtigkeit (M2) festgestellt. Bei den Lämmern stammten sechs Tiere aus einer Einlingsgeburt (L1) und sechs aus Zwillingsge-burten (L2). Den Muttertieren wurden von der 5. Woche a.p. bis 1 Woche a.p. wöchentlich sowie 2 Wochen p.p., den Lämmern eine, vier und zwölf Wochen post natum Blutproben entnommen. Im Serum wurden die Konzentrationen von Betahydroxybutyrat (BHB), Glu-cose, Bilirubin, freien Fettsäuren (FFS), Gesamtprotein und Albumin, die Aktivitäten der Glutamatdehydrogenase (GLDH), sowie im Vollblut die Aktivitäten der Superoxiddismutase (SOD) und der Glutathionperoxidase (GPX) gemessen. Ergebnisse: Die SOD- und GPX-Aktivitäten stiegen gleichermaßen bei einlingsträchtigen und zwillingsträchtigen Muttertieren signifikant an. Die SOD-Aktivitäten stiegen bei M1 von 488,2 ± 28 vier Wochen a.p. auf 524,8 ± 96,1 U/g Hb eine Woche a.p. und bei M2 von 447,4 ± 65,1 fünf Wochen a.p. auf 619,4 ± 141,1 U/g Hb zwei Wochen p.p. an. Die GPX-Aktivitäten bei M1 stiegen von 782,1 ± 220,5 eine Woche a.p. auf 1037,62 ± 382,9 U/ml Hk zwei Wochen p.p.. M2 zeigten eine tendenziell niedrigere Aktivität, welche von 641,8 ± 118,4 auf 911,8 ± 168,5 U/ml Hk anstieg. Es bestanden signifikante Zusammenhänge der SOD mit FFS, BHB und Gesamtprotein sowie der GPX mit Bilirubin, FFS und GLDH. Für die SOD-Aktivität wurde ein Referenzbereich von 328,10 bis 837,25 U/g Hb und für die GPX-Aktivität von 473,38 bis 1259,05 U/ml Hk bei Muttertieren ermittelt. Ausgenommen die Glucosekonzentration eine Woche a.p. bestanden keine signifikanten Differenzen zwi-schen M1 und M2. Die BHB-Konzentrationen zeigten bis zur Lammung einen konstanten Verlauf und stiegen bei M1 eine Woche a.p. auf 0,59 ± 0,27 mmol/l sowie bei M1 und M2 zwei Wochen p.p. auf 0,76 ± 0,29 mmol/l (M1) bzw. 0,77 ± 0,25 mmol/l (M2) signifikant an. Gesicherte Korrelationen bestanden zu FFS, Gesamtprotein und Glucose. Die Gesamtprotein- und Albuminkonzentrationen verliefen über den gesamten Untersuchungszeitraum nahezu konstant. Die Albuminkonzentration sank bei M1 nach einem kontinuierlichen An-stieg von einer Woche a.p. (37,2 ± 2,9 g/l) zu zwei Wochen p.p. (35,3 ± 3,6 g/l) signifikant ab. Gesicherte Korrelationen bestanden zu Glucose, SOD, BHB, Bilirubin und FFS. Die Glucose- Konzentrationen stiegen bei M1 bis eine Woche a.p. auf 4,84 ± 1,17 mmol/l sig-nifikant an, korrelierten neben den Proteinen mit Bilirubin und fallen 2 Wochen p.p. wieder auf ihren Ausgangswert ab, der bei M2 konstant blieb. Die Bilirubinkonzentrationen verlie-fen konstant, die der FFS fielen nach einem Anstieg eine Woche a.p. (M1) signifikant ab. Die GLDH-Aktivitäten zeigten a.p. einen konstanten Verlauf und p.p. einen signifikanten Anstieg auf 20,61 ± 14,11 U/l bei M2. Zwischen L1 und L2 bestanden zu keinem Zeitpunkt signifikante Unterschiede. Die SOD zeigte abnehmende Aktivitäten mit zunehmendem Lebensalter, - bei L1 von 757,7 ± 94,4 eine Woche p.p. auf 597,8 ± 255,0 U/g Hb 4 Wochen p.p. und bei L2 von 487,17 ± 353,3 auf 464,0 ± 330,9 U/g Hb. Sie korrelierte gesichert mit Albumin und Glucose in der ersten Lebenswoche. Die GPX-Aktivitäten stiegen mit zunehmendem Lebensalter bei statistisch gesicherter Korrelation mit Glucose von 724,3 ± 199,8 auf 1011,5 ± 132,9 U/ml Hk bei L1 und von 693,3 ± 120,8 auf 1052,0 ± 146,9 U/ml Hk bei L2. Für Lämmer wurden als Refe-renzwerte 43,00 bis 932 U/g Hb für die SOD bzw. 406,00 bis 1321,00 U/ml Hk für die GPX berechnet. Die Albumin- und BHB-Konzentrationen stiegen zur vierten bzw. zwölften Le-benswoche an, die Glucose-, Bilirubin- und FFS-Konzentrationen im Serum sanken zur 12. Lebenswoche ab. Die Gesamtproteinkonzentration und die Aktivität der GLDH verlie-fen konstant. Es bestanden signifikante Korrelationen zwischen BHB und Gesamtprotein, FFS, GLDH sowie zwischen Gesamtprotein und Glucose, Albumin sowie FFS. Des Weite-ren bestanden gesicherte Korrelationen zwischen Bilirubin und FFS sowie FFS und GLDH. Fazit: Die Aktivitäten der antioxidativen Enzyme SOD und GPX sowie die Konzentrationen der klinisch-chemischen Parameter zeigen mit dem Anstieg der SOD-Aktivität von 447 auf 619 U/g Hb bei M2 einerseits die peripartalen Belastungen und andererseits mit dem An-stieg der GPX-Aktivität von 782 auf 1038 U/g Hk bei M1 die Kompensations- und Regel-mechanismen bei dieser Belastungssituation auf. Die Untersuchungen entsprechen dem Zustand einer ausgeglichen Energiebilanz und gesundheitlich stabilen Bedingungen. Ein Einfluss der Lämmerzahl auf das antioxidative System bzw. den Stoffwechsel kann weder bei Mutterschafen noch bei Lämmern statistisch signifikant belegt werden. / Metabolism and antioxidative status of merino sheep for meat production in the peripartum time period Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig Submitted in June 2010 (100 pages, 28 figures, 38 tables, 357 references) Definition of the problem and objective: Until now, the research regarding the metabol-ism of sheep focussed very little on the importance of the antioxidative system, but the significance of its function as a protective and regulative system is increasing. Especially the pregnancy and the early lactation of the ewes as well as the postnatal development of the lambs places enormous demands upon the energy supply and the antioxidative sys-tem. The objective of this study is to show how these demands are met and to determine whether a twin-pregnancy represents an additional stress for the ewes, and whether lambs from a twin-pregnancy are possibly disadvantaged. Animals, materials and methods: 23 pregnant merino ewes and twelve merino lambs were analyzed. Sonographic tests showed that 13 ewes were pregnant with one lamb (M1), and ten showed a twin-pregnancy (M2). Six of the lambs were born as single lambs (L1), six as twins (L2). Blood samples were taken from the ewes every week from the fifth until the week before the birth, as well as two weeks after birth, the blood samples from the lambs were taken one, four and twelve weeks postpartum. The concentration in the serum of the following components was measured: betahydroxybutyrate (BHB), glucose, biliru-bine, free fatty acids (FFS), total protein and albumine, the activity of the glutamatedehy-drogenasis (GLDH), as well as the activity in whole- blood of superoxidedismutasis (SOD) and glutathionperoxidasis (GPX). Results: The SOD- and GPX-activities rose significantly at the same rate for mother sheep pregnant with one lamb or with twins. The SOD-activity rose for M1 from 488,2 +/- 28 four weeks prepartum to 524 +/- 96,1 U/g Hb one week prepartum, and for M2 from 447,4 +/- 65,1 five weeks prepartum to 619,4 +/- 141,1 U/g Hb two weeks prepartum. The GPX-activity for M1 rose from 782,1 +/- 220,5 one week prepartum to 1037,62 +/- 382,9 U/ml two weeks postpartum, while M2 showed a tendency towards lower activity, which rose from 641,8 +/- 118,4 to 911,8 +/- 168,5 U/ml Hk. There were significant correlations between the SOD and the FFS, the BHB and the total protein as well as the GPX with bili-rubine, FFS and GLDH. For the ewes, an SOD-activity reference range between 328,10 and 837,25 U/g Hb was determined, and a GPX-activity reference range between 473,38 and 1259,05 U/ml Hk. There were no significant differences between M1 and M2 except for the concentration of glucose. The BHB-concentration stayed constant up to birth and then rose significantly to 0,59 +/- 0,27 mmol/l for M1 one week postpartum and for both M1 and M2 to 0,76 +/- 0,29 mmol/l (M1) and 0,77 +/- 0,25 mmol/l (M2) two weeks postpartum. A reliable correlation could be determined with FFS, total protein and glucose. The total protein and the albu-mine concentration stayed almost constant during the whole analyzed time period. After a continuous increase between one week prepartum (37,2 +/- 2,9 g/l) and two weeks post-partum (35,3 +/- 3,6 g/l), the albumine concentration dropped considerably. Here, reliable correlations could be determined with glucose, SOD, BHB, bilirubine and FFS. For the group M1, the glucose concentrations rose significantly until one week prepartum to 4,84 +/- 1,17 mmol/l, correlated with both the proteins and bilirubine, then fell back to their initial level two weeks postpartum, while they did not change for M2. The concentrations of bili-rubine stayed constant, the FFS-concentrations fell significantly after an increase one week prepartum (M1). The GLDH-activities showed a constant trend prepartum, then rose significantly postpartum to 20,61 +/14,11 U/l for M2. There were no significant differences between L1 and L2 at any time. The SOD showed decreasing activity with increasing age, from 757,7 +/- 94,4 one week prepartum to 597,8 +/- 255,0 U/g Hb four weeks postpartum for L1 and from 487,17 +/- 353,3 to 464,0 +/- 330,9 U/g Hb for L2. A reliable correlation was determined with albumine and glucose in the first week after birth. The GPX-activities rose with increasing age with a statistically reliable correlation with glucose from 724,3 +/- 199,8 to 1011,5 +/- 132,9 U/ml Hk for L1 and from 693,3 +/- 120,0 to 1052,0 +/- 146,9 U/ml Hk for L2. The calculated reference val-ues for lambs were 43,00 to 932 U/g Hb for SOD and 406,00 to 1321,00 U/ml Hk for GPX. The concentrations of albumine and BHB rose towards the fourth and respectively the twelvth week of life, the levels of glucose, bilirubine and FFS dropped towards the 12th week. The total protein concentration and the GLDH-activities stayed constant. There were significant correlations between BHB and total protein, FFS, GLDH as well as between total protein and glucose, albumine and FFS. In addition, there were reliable correlations between bilirubine and FFS and FFS and GLDH. Conclusion: The activities of the antioxidative enzymes SOD and GPX as well as the concentrations of the clinical-chemical parameters show the prepartum stress on one side with the rise of the SOD-activity from 447 to 619 U/g for the group M2, and the compensa-tion and regulation mechanisms during this stress period on the other side with the in-crease of the GPX-activity from 782 to 1038 U/g Hk for the group M1. The results of the tests correspond to the status of a well-regulated energy balance and stable health condi-tions. An influence of the number of lambs on the antioxidative system or the metabolism for both lambs and ewes can not be shown with statistical significance.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Paraoxonase (PON) 1 is a high density lipoprotein (HDL) - bound antioxidant enzyme that was originally discovered and better known for its role in protecting against organophosphate (OP) - induced neurotoxicity. In the past two decades, the enzyme has gained prominence as a protective agent against atherosclerosis on account of increasing evidence that it accounts for many of the anti-atherogenic roles attributed to HDL. PON1 is a polymorphic enzyme displaying a high variability in human populations which is associated with a considerable degree of inter-individual differences in enzyme phenotype that translates to differential risk for OP toxicity and cardiovascular disease (CVD). In a series of studies and analyses, this thesis describes investigations regarding the possible involvement of PON 1 in the risk for CVD in a mixed ancestry population from Bellville, Western Cape, South Africa. This was done by evaluating the distribution of PON1 coding region polymorphisms (Q192R and L55M) and their influence on PON1 phenotype as well as the latter‟s relation to CVD risk factors (oxidative stress, inflammation and atherogenic dyslipidemia) and possible involvement in early CVD assessed by measuring intima media thickness of the carotid artery (CIMT). Since PON1 is increasingly measured in samples that have been stored for varied periods of time, the main study was preceded by a pilot study evaluating the influence of baseline conditions on the stability of PON 1 activity and antioxidant status in human sera stored for up to 12 months. It was shown that baseline glycemic status enhances the degradation of antioxidants in stored samples with indications of also accelerating the decline of PON1 levels and activity. Thus baseline glycemic status should be a factor to be considered in analyses involving stored samples. The Q192R polymorphism was found to be the functional variant influencing both concentration and activity of plasma PON1. Contrary to expectation, the L55M was nonfunctional, possibly due to its unusual distribution in this population where the 55M (83%) allele overwhelmingly predominated over the L55 allele. The R allele was the more frequent (60.4%) of the 192 polymorphism. The R allele has previously been associated with less efficient breakdown of lipid peroxides and a subsequent higher risk for atherosclerotic heart disease while the 55M is recognized as a “low concentration/activity” variant. Thus the predominant PON1 genotype distribution in this population constitutes a risk profile that may relate to increased risk for CVD. The risk for CVD was confirmed to be very high in this population indicated by high prevalence of the metabolic syndrome (48%) and its key components (and CVD risk factors) diabetes (28%), obesity (53%) and high blood pressure (57%). Paraoxonase activity associated inversely with indices of inflammation (high sensitive C- reactive protein [hs-CRP] and leptin) and oxidative stress (oxidized low density lipoprotein [LDL]) and directly with adiponectin and markers of systemic antioxidant status. These findings suggest that low paraoxonase-I activity contributes to increased cardiovascular risk possibly via involvement in early atherogenesis. However, only a modest inverse relation was observed between PON1 phenotype and CIMT thus suggesting that PON1 may not play a major role in early atherosclerosis. Taken together, the findings presented in this thesis demonstrate the presence of a risk PON1 genotypic profile and indication that the enzyme may play a role in the enhanced CVD risk in this population possibly via interactions with inflammation and oxidative stress. However, conclusive evidence for the involvement of PON1 in early CVD was not demonstrated indicating a need to explore the participation of PON1 in later stages of CVD. / AFRIKAANSE OPSOMMING: Paraoksonase (PON) 1 is 'n antioksidant ensiem wat aan HDL gebind is. Oorspronklik is dit ontdek en het bekend geword as 'n beskermer teen organofosfaat (OF)-gedrewe neurotoksisiteit. In die afgelope twee dekades het die ensiem belangrik geraak as 'n beskermer teen arterosklerose as gevolg van toenemende bewyse dat dit 'n belangrike rol speel in die beskermende effekte van HDL teen arterosklerose. PON1 is 'n polimorfiese ensiem wat groot variasie toon in verskillende populasies. Daar is ook inter-individuele verskille in ensiem fenotipe wat uitloop op 'n differensiele risiko vir OF toksisiteit en kardiovaskulêre hartsiekte (KVH). Hierdie tesis beskryf 'n reeks analises en ondersoeke betreffende die moontlike betrokkenheid van PON1 in die risiko vir KVH in 'n gemengdeafkoms populasie van Bellville, Wes-Kaap, Suid Afrika. Dit was gedoen deur die evaluering van die verspreiding van die PON-1 koderende omgewing polimorfismes (Q192R en L55M), hulle invloed op PON1 fenotipe en laasgenoemde se verhouding tot KVH risikofaktore (oksidatiewe stress, inflammasie en arterogeniese dislipedimie) en moontlike voorkoms in vroeë kardiovaskulêre siekte bepaal deur die meting van die intima media dikte van die karotied slagaar. Aangesien PON1 al hoe meer gemeet word in monsters wat vir verskeie tydperke gestoor word, was die hoofstudie voorafgegaan deur 'n loodsstudie wat die invloed van basislyn kondisies op die stabiliteit van PON1 aktiwiteit en antioksidant status in menslike sera wat vir tot 12 maande gestoor was, bepaal het. Dis is duidelik aangetoon dat basislyn glisemiese status die afbraak van antioksidante in gestoorde monsters verhoog het, asook aanduidings van die afname van PON1 vlakke en aktiwitetit. Basislyn glisemiese status behoort dus ook as 'n faktor ingereken te word in analises van gestoorde monsters. Die Q192R polimorfisme is aangetoon om 'n funksionele variant te wees wat beide die konsentrasie asook die aktiwiteit van PON1 beïnvloed het. Anders as wat verwag is, was die L55M polimorfisme nie-funksioneel, moontlik as gevolg van sy ongewone distribusie in hiedie populasie waar die voorkoms van die 55M (83%) alleel die L55 alleel oorheers het. Die R alleel was die mees algemene (60.4%) van die 192 polimorfisme. Die R alleel is voorheen reeds geassosieer met minder effektiewe afbraak van lipied peroksides en gevolglike hoër voorkoms van arteriosklerotiese hartsiekte, terwyl die 55M erken word as 'n “lae konsentrasie/aktiwiteit” variant. Die oorheersende PON1 genotipe distribusie in hierdie populasie behels dus 'n risikoprofiel wat betrekkking mag hê op verhoogde KVH. Die risiko vir KVH was bevestig om baie hoog te wees in hierdie populasie, soos aangedui deur 'n hoë voorkoms van die metaboliese sindroom (48%) en die sleutelkomponente daarvan (insluitend KVH risikofaktore), diabetes (28%), obesiteit (53%) en hipertensie (57%). Paraoksinase aktiwiteit was omgekeerd geassosieer met indekse van inflammasie (hoë C-reaktiewe proteïen [hs-CRP] en leptien) en oksidatiewe stres (geoksideerde lae digtheid lipoproteïen [LDL], en direk geassosieer met adiponektien en merkers van sistemiese antioksidantstatus. Hierdie bevindings mag aandui dat lae paraoksonase-1 aktiwiteit bydra tot verhoogde kardiovaskulêre risiko, moontlik via betrokkenheid in vroeë arterogenese. Slegs 'n klein omgekeerde verhouding is egter waargeneem tussen die PON1 fenotipe en karotied intima media dikte, wat mag aandui dat PON1 nie 'n beduidende rol speel in vroeë arterosklerose nie. In geheel, die bevindinge voorgedra in hierdie tesis demonstreer die voorkoms van 'n risiko PON1 genotipiese profiel wat 'n aanduiding mag wees dat die ensiem 'n rol mag speel in die verhoogde KVH risiko in hierdie populasie, moontlik deur interaksies met inflammasie en oksidatiewe stress. Afdoende bewys van die betrokkenheid van PON1 in vroeë KVH was egter nie gedemonstreer nie, wat die nodigheid aandui om die deelname van PON1 in latere stadiums van KVH te ondersoek.

La leptine est une hormone peptidique ayant une action sur de nombreux tissus. Une dérégulation de la sécrétion de cette hormone est observée au cours de l’obésité. L’obésité est fréquemment associée à des troubles de santé dont les principaux sont le diabète de type II, l’hypertension artérielle et les maladies cardiovasculaires. Elle est également un facteur de risque du cancer du sein, particulièrement en post-ménopause favorisant la récidive et augmentant la mortalité. Ces perturbations, associées à un état de stress oxydant défini par un excès de production des espèces réactives de l’oxygène (ERO) par rapport aux systèmes de défense antioxydants, pourraient avoir un impact majeur dans le risque de carcinogenèse chez le sujet obèse. Il est clairement établi aujourd’hui que le statut oxydatif des cellules est directement corrélé aux capacités de prolifération mais aussi de survie des cellules dans leur environnement. A ce jour, très peu de données existent concernant le rôle de la leptine dans la modulation du statut oxydatif des cellules épithéliales mammaires saines et tumorales. L’objectif de cette thèse était d’étudier d'abord les mécanismes d’action et les effets de la leptine sur le statut oxydatif et inflammatoire des cellules épithéliales mammaires saines et néoplasiques ; puis dans un deuxième temps, une étude expérimentale a été conduite pour caractériser in vivo l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et le statut oxydatif et inflammatoire des tumeurs. Le projet avait également pour but de mettre en œuvre une nouvelle technique d’analyse basée sur la détection de fluorescence native induite par excitation laser à 224 nm afin d’évaluer la production de composés bio-actifs de la famille des éicosanoïdes, dont les isoprostanes, impliqués dans le processus inflammatoire. Nous avons exploré in vitro l’impact de la leptine sur le statut oxydatif des cellules épithéliales mammaires. Cette étude nous a permis d’établir que la réponse au signal leptinique varie en fonction du statut néoplasique de la lignée considérée, en fonction du temps de contact et non de la dose testée. Ensuite, nous avons étudié l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et sur le statut oxydatif et inflammatoire des tumeurs à l’aide d’un modèle de souris âgées C57BL/6 nourries avec un régime hyper-lipidique (HL) pendant 14 semaines, et hébergées soit dans un environnement enrichi (EE) pour favoriser l’activité physique et les interactions sociales, soit dans un environnement standard pendant 8 semaines, après quoi des cellules syngéniques de tumeur mammaire EO771 ont été implantées dans les quatrièmes coussinets adipeux mammaires. In vitro, la leptine a stimulé la production de ROS de façon indépendante de la dose et cette augmentation était dépendante de la production d'O2 cytosolique. Les résultats montrent une augmentation significative du poids dans les groupes recevant le régime HL à prise alimentaire journalière identique. La composition corporelle à 8 semaines montre une prise de masse grasse significative sous régime HL, majorée par l’ovariectomie et partiellement limitée par l’activité physique. Après implantation des tumeurs, le régime HL favorise la croissance tumorale et la perte de l’activité locomotrice. Par contre, l’EE prévient la perte d’activité physique des animaux. L’ensemble de ces travaux montre que la leptine contribue à l’apparition d’un stress oxydant en lien avec le statut tumoral des cellules épithéliales mammaires. Ceci peut expliquer en partie l’augmentation du risque de cancer mammaire associée à l’obésité en post-ménopause. Ces résultats permettront d'objectiver le bénéfice d'une intervention nutritionnelle ciblée afin de moduler la réponse des cellules aux stimulations des adipokines. A terme, cette étude doit contribuer à mieux comprendre l’intégration des signaux issus de l’environnement cellulaire. / Obesity is now considered, as a risk factor for developing breast cancer in postmenopausal women and for mortality in response to this pathology. Obesity, which is frequently associated with hyperleptinemia, induces cellular signalling pathways, some of which involving reactive oxygen species (ROS) as intracellular messengers. High levels of ROS contribute to oxidative stress, cellular damages and pathogenesis. Therefore, ROS production associated to obesity could be a major risk factor for mammary carcinogenesis. Furthermore, increased oxidative stress and inflammation characterised by infiltration of immune cells into adipocytes are described. This is associated with a lipid peroxidation and the production of bio-active compounds including isoprostanes.The aim of this study was to determine the impact of leptin in modulating the oxidative and inflammatory status of epithelial mammary cells and in tumor mammary tissue. Moreover, the purpose of this work was to develop a new analysis technique based on native fluorescence detection induced by laser excitation at 224 nm to evaluate the production of bio-active compounds from the family of eicosanoids, involved in the inflammatory process, including isoprostanes.Initially we identified in vitro the leptin effects on ROS production in 3 human epithelial mammary cell models which present different neoplastic status (healthy primary (HMEC) cells, MCF-7 and MDA-MB-231) in presence of two leptin concentrations (10 ng/ml close to physiological values, 100 ng/ml as obesity level). To better understand the potential involvement of adipocyte tumor microenvironment in mammary carcinogenesis, we secondly explored in vivo the impact of high fat diet (HFD) and of enriched environment (EE) on mammary tumor development. Female C57BL/6 mice were fed with a HFD versus a normo-caloric diet (NC) for 14 weeks. After 8 weeks mammary tumor syngeneic cells EO771 were implanted into the fourth mammary fat pads. Before injection, mice were housed in EE or in standard environment (ES) for 8 weeks. In vitro, leptin stimulated ROS production in dose-independent manner and this increase was dependent of cytosolic O2•- production. This ROS production contributed to a different antioxidative response depending of the neoplastic cell status. Leptin induced the antioxidative enzymes expression and activities such as heme-oxygenase or glutathione peroxidase only in HMEC cells. In neoplastic cells, these enzyme activities did not change whatever the leptin concentration used.Thus, high fat diet promoted mammary tumor development associated with a decrease in body fat and an increase in volume and weight of tumors that was not limited by physical activity. This diet induced a decrease of adiponectin and an increase of leptin plasma level compared to NC diet however, leptinemia was not influenced by EE.The native fluorescence isoprostanes determination method, turned out not to be quite sensitive. Therefore, the native fluorescence of these compounds is too low to allow their detection in biological media used. In contrast, the native fluorescence appears to be a potential cellular exploration tool.Through this work, we have shown that leptin contributes to the onset of oxidative stress linked to the status of mammary epithelial tumor cells. This may partly explained the increase of risk of breast cancer recurrence observed in situations of obesity. The results obtained in vivo eventually will support the benefit of a nutrition intervention to modulate cell response to adipokines stimulation. Ultimately, this study contributes to better understand the integration of signals from the cell environment.

A necessidade de criopreservação seminal vem aumentando cada vez mais, concomitantemente à evolução das biotecnologias aplicadas à reprodução animal. Sabe-se que a principal causa da diminuição do desempenho reprodutivo de um touro, criado em clima tropical, é o estresse térmico. Raças de origem européia (Bos taurus taurus) são consideravelmente mais sensíveis à essas condições climáticas quando comparadas aos animais de origem indiana (Bos taurus indicus). Esses animais não adaptados podem apresentar decréscimo na qualidade espermática, como alterações estruturais, funcionais e maior suscetibilidade ao estresse oxidativo, após a descongelação do sêmen. Para avaliar essa hipótese, foram utilizadas amostras criopreservadas de 40 touros, sendo 20 da raça Nelore (Bos taurus indicus) e 20 da raça Simental (Bos taurus taurus), criados em clima tropical, dessas, 10 amostras seminais de cada raça foram coletadas durante inverno e 10 durante o verão. Após a descongelação das amostras, foram realizados os testes espermáticos convencionais como motilidade progressiva, vigor e índice de motilidade espermática, e os testes funcionais tais como integridade de membrana plasmática (Eosina/Nigrosina), integridade acrossomal (POPE), integridade de DNA (Ensaio Cometa) e atividade citoquímica mitocondrial (3\'3 diaminobenzidina DAB). Também foi avaliada a suscetibilidade das células espermáticas à peroxidação lipídica induzida, através da mensuração da concentração de substâncias reativas ao ácido tiobarbitúrico (TBARS). Os dados foram analisados levando em conta as classes: raça (Nelore e Simental) ou estação do ano (verão e inverno), através do programa SPSS 13.0 para Windows. A variável DAB I foi maior (p=0,04) nas amostras dos touros Nelore em comparação com Simentais (17,85 ± 2,38 vs 11,4 ± 1,84). Observou-se o efeito das estações do ano sobre as variáveis motilidade progressiva (26,50% inverno e 13,30% verão; p=0,02), DAB I (22,32% inverno e 13,30% verão; p=0,04) e DAB III (13,88% inverno e 22,10% verão; p=0,01), somente no sêmen dos touros Nelore, e o efeito das raças (p=0,03) sobre as variáveis DAB I (22,34% Nelore e 11,60% Simental) e DAB III (13,88% Nelore e 21,19% Simental), apenas durante o inverno, quanto ao nível de TBARS, não foi verificado diferença entre nenhum grupo. Houve correlação entre alguns testes funcionais e convencionais, porém a variável integridade de DNA não correlacionou com nenhum teste convencional. Esses resultados podem indicar que a peroxidação lipídica sofrida, espontaneamente, pelos touros Europeus, devido ao estresse térmico, selecionou espermatozóides mais resistentes à criopreservação. / Seminal cryopreservation technique has been increasing concomitant reproductive biotechnologies. It is known that heat stress is the main cause that affects bulls reproductive performance under tropical environment. European breeds (Bos taurus taurus) are much more sensible to these conditions than Indian breeds (Bos taurus indicus). These non-adapted animals present a fast decrease in sperm quality as structural and functional parameters and higher oxidative stress susceptibility. In order to test this hypothesis, cryoprotected semen samples of twenty Nelore (Bos taurus indicus) and twenty Simmental (Bos taurus taurus) bulls, raised under tropical conditions, were analyzed; ten samples of each breed were collected during summer and ten during winter. After thawing, semen samples were submitted to following conventional tests: progressive motility, vigor and sperm motility index (IME), and following functional tests: plasmatic membrane integrity (eosin-nigrosin), acrosomal membrane integrity (POPE), DNA fragmentation (Comet Assay) and mitochondrial activity (3\'3 diaminobenzidine - DAB). The spermatozoa susceptibility to the induced lipid peroxidation followed by measurement of thiobarbituric acid reactive substances concentration (TBARS), was used as an oxidative stress index. Statistical analysis were performed for breed (Nelore and Simmental) and season (summer and winter), using SPSS 13.0 for Windows. Comparing Nelore and Simmental, DAB I was higher (p=0,04) in the first group (17,85 ± 2,38 and 11,4 ± 1,84, respectively). Season effect was observed only in Nelore samples on: progressive motility (26,50% winter and 13,30% summer; p=0,02), DAB I (22,32% winter and 13,30% sumer; p=0,04) and DAB III (13,88% winter and 22,10% summer; p=0,01); and breed effect (p=0,03) was observed only during winter on: DAB I (22,34% Nelore and 11,60% Simmental) and DAB III (13,88% Nelore and 21,19% Simmental). TBARS index, did not differ in breed or season. Correlations were observed between some of functional and conventional tests, however, the DNA integrity did not correlate with any conventional test. These results may indicate that probably, the spontaneous peroxidation that European bulls suffer due to the heat stress may have selected sperm more resistant to the cryopreservation.

A espermatogênese é um processo orquestrado e coordenado pelo qual os espermatozoides são produzidos. Ao final deste processo, o excesso de citoplasma presente nas espermátides é fagocitado, permanecendo um resíduo denominado de gota citoplasmática. Os espermatozoides em fase de maturação apresentam gota, que migra da região proximal para regiões mais distais da célula espermática. Estudos relacionam o remanescente citoplasmático à proteção antioxidante e à ativação metabólica de espermatozoides imaturos. Os mecanimos fisiológicos da maturação e os status oxidativo e metabólico em células epididimárias de bovinos são pobremente elucidados. Desta forma, foram realizados dois experimentos com o objetivo de caracterizar o status oxidativo e metabólico de espermatozoides bovinos durante a maturação espermática, e os possíveis papéis da gota citoplasmática envolvidas nesse processo. Para tal, amostras espermáticas foram recuperadas dos três segmentos epididimários (Cabeça, corpo e cauda) e submetidas às avaliações de cinética espermática (CASA) e testes morfofuncionais. Foram avaliadas as atividades das enzimas antioxidantes SOD e GPx e mensurados os níveis de ATP em cada amostra epididimária. Espermatozoides recuperados da cauda do epidídimo apresentaram maior porcentagem de células móveis e progressivas e elevados níveis de ATP em comparação aos demais grupos. As células provenientes do corpo apresentaram maior susceptibilidade à peroxidação lipídica e membranas plasmática e acrossomal lesionadas. Foi nesse segmento que também observamos a migração da gota e o início da aquisição da motilidade, e correlação negativa entre gota distal e susceptibilidade à peroxidação. Em amostras da cabeça, houve correlação positiva entre produção de EROs e níveis de ATP, e alto PMM; e correlação negativa entre atividade da SOD e susceptibilidade à peroxidação lipídica em amostras da cabeça. Não foram observadas diferenças significativas em relação ao potencial de membrana mitocondrial (PMM) e a atividade da SOD e da GPx entre os diferentes segmentos. Por meio dos principais resultados, pudemos sugerir que as células presentes na cauda do epidídimo apresentam o aparato energético ativo, devido à alta porcentagem de células móveis e níveis de ATP. As amostras provenientes do corpo evidenciam que é neste segmento em que a motilidade se inicia e que ocorre os principais remodelamentos de membrana. As correlações observadas nos permitem inferir que as mitocôndrias de espermatozoides bovinos imaturos já estivessem ativas desde o início da maturação, produzindo energia e EROs. Além disso, a correlação nos mostra o papel protetor da SOD contra à peroxidação lipídica. Paralelamente, sugerimos que a gota participe firmemente da ativação da motilidade e desempenhe papel antioxidante contra as EROs. Assim, concluímos que as modificações celulares inerentes ao processo de maturação e a presença da gota citoplasmática sejam cruciais no status oxidativo e metabólico de espermatozoides bovinos imaturos, afim desenvolver a capacidade fecundante do espermatozoide. / Spermatogenesis is an orchestrated and coordinated process by which sperm are produced. In the end of this process, the excess cytoplasm present in the spermatids is phagocytosed, remaining a called cytoplasmic droplet. Spermatozoa in the stage of maturation present cytoplasmic droplet, which migrates from the proximal region to more distal regions in sperm cell. Studies relate the cytoplasmic remnant to the antioxidant protection and to metabolic activation of immature sperma. The physiological mechanisms of maturation and the oxidative and metabolic status of bovine epididymal cells are poorly elucidated. Thus, two experiments aimed to characterize the oxidative and metabolic status of bovine sperm during epididymal maturation, and the possible cytoplasmic droplet roles involved in this process. For this, sperm samples were recovered from the three epididymal segments (Caput, corpus and cauda) and submitted to spermatic kinetics analysis (CASA) and morphofunctional tests. The activities of the antioxidant enzymes SOD and GPx were evaluated and the levels of ATP in each epididymal sample. Sperm cells recovered from the cauda of the epididymis showed a higher percentage of motile and progressive cells and high levels of ATP compared to the other groups. Cells from the corpus showed elevated susceptibility to lipid peroxidation and lesioned plasma and acrosomal membranes. It was in this segment that we also observed the migration of cytoplasmic droplet and the beginning of the acquisition of motility, and negative correlation between distal droplet and susceptibility to peroxidation. In samples from the caput, there was a positive correlation between ROS production and ATP levels, and high PMM; and negative correlation between SOD activity and susceptibility to lipid peroxidation in caput samples. No significant differences were observed in relation to mitochondrial membrane potential (PMM) and SOD and GPx activity among the different segments.By means of the main results, we could suggest that cells present in the cauda of the epididymis present the active energy apparatus, due to the high percentage of mobile cells. Samples from the corpus show that is in this segment that the motility begins and the main membrane remodeling occurs. The observed correlations allow us to infer that mitochondria of immature bovine sperm are already active since the beginning of maturation, producing energy and ROS. In addition, the correlation shows us the protective role of SOD against lipid peroxidation. In parallel, we suggest that cytoplasmic droplet participates firmly in the activation of motility and plays an antioxidant role against ROS. Thus, we conclude that the cellular modifications inherent in the sperm maturation process and the presence of cytoplasmic droplet are crucial in the oxidative and metabolic status of immature bovine spermatozoa, in order to develop fecundant capacity.

A necessidade de criopreservação seminal vem aumentando cada vez mais, concomitantemente à evolução das biotecnologias aplicadas à reprodução animal. Sabe-se que a principal causa da diminuição do desempenho reprodutivo de um touro, criado em clima tropical, é o estresse térmico. Raças de origem européia (Bos taurus taurus) são consideravelmente mais sensíveis à essas condições climáticas quando comparadas aos animais de origem indiana (Bos taurus indicus). Esses animais não adaptados podem apresentar decréscimo na qualidade espermática, como alterações estruturais, funcionais e maior suscetibilidade ao estresse oxidativo, após a descongelação do sêmen. Para avaliar essa hipótese, foram utilizadas amostras criopreservadas de 40 touros, sendo 20 da raça Nelore (Bos taurus indicus) e 20 da raça Simental (Bos taurus taurus), criados em clima tropical, dessas, 10 amostras seminais de cada raça foram coletadas durante inverno e 10 durante o verão. Após a descongelação das amostras, foram realizados os testes espermáticos convencionais como motilidade progressiva, vigor e índice de motilidade espermática, e os testes funcionais tais como integridade de membrana plasmática (Eosina/Nigrosina), integridade acrossomal (POPE), integridade de DNA (Ensaio Cometa) e atividade citoquímica mitocondrial (3\'3 diaminobenzidina DAB). Também foi avaliada a suscetibilidade das células espermáticas à peroxidação lipídica induzida, através da mensuração da concentração de substâncias reativas ao ácido tiobarbitúrico (TBARS). Os dados foram analisados levando em conta as classes: raça (Nelore e Simental) ou estação do ano (verão e inverno), através do programa SPSS 13.0 para Windows. A variável DAB I foi maior (p=0,04) nas amostras dos touros Nelore em comparação com Simentais (17,85 ± 2,38 vs 11,4 ± 1,84). Observou-se o efeito das estações do ano sobre as variáveis motilidade progressiva (26,50% inverno e 13,30% verão; p=0,02), DAB I (22,32% inverno e 13,30% verão; p=0,04) e DAB III (13,88% inverno e 22,10% verão; p=0,01), somente no sêmen dos touros Nelore, e o efeito das raças (p=0,03) sobre as variáveis DAB I (22,34% Nelore e 11,60% Simental) e DAB III (13,88% Nelore e 21,19% Simental), apenas durante o inverno, quanto ao nível de TBARS, não foi verificado diferença entre nenhum grupo. Houve correlação entre alguns testes funcionais e convencionais, porém a variável integridade de DNA não correlacionou com nenhum teste convencional. Esses resultados podem indicar que a peroxidação lipídica sofrida, espontaneamente, pelos touros Europeus, devido ao estresse térmico, selecionou espermatozóides mais resistentes à criopreservação. / Seminal cryopreservation technique has been increasing concomitant reproductive biotechnologies. It is known that heat stress is the main cause that affects bulls reproductive performance under tropical environment. European breeds (Bos taurus taurus) are much more sensible to these conditions than Indian breeds (Bos taurus indicus). These non-adapted animals present a fast decrease in sperm quality as structural and functional parameters and higher oxidative stress susceptibility. In order to test this hypothesis, cryoprotected semen samples of twenty Nelore (Bos taurus indicus) and twenty Simmental (Bos taurus taurus) bulls, raised under tropical conditions, were analyzed; ten samples of each breed were collected during summer and ten during winter. After thawing, semen samples were submitted to following conventional tests: progressive motility, vigor and sperm motility index (IME), and following functional tests: plasmatic membrane integrity (eosin-nigrosin), acrosomal membrane integrity (POPE), DNA fragmentation (Comet Assay) and mitochondrial activity (3\'3 diaminobenzidine - DAB). The spermatozoa susceptibility to the induced lipid peroxidation followed by measurement of thiobarbituric acid reactive substances concentration (TBARS), was used as an oxidative stress index. Statistical analysis were performed for breed (Nelore and Simmental) and season (summer and winter), using SPSS 13.0 for Windows. Comparing Nelore and Simmental, DAB I was higher (p=0,04) in the first group (17,85 ± 2,38 and 11,4 ± 1,84, respectively). Season effect was observed only in Nelore samples on: progressive motility (26,50% winter and 13,30% summer; p=0,02), DAB I (22,32% winter and 13,30% sumer; p=0,04) and DAB III (13,88% winter and 22,10% summer; p=0,01); and breed effect (p=0,03) was observed only during winter on: DAB I (22,34% Nelore and 11,60% Simmental) and DAB III (13,88% Nelore and 21,19% Simmental). TBARS index, did not differ in breed or season. Correlations were observed between some of functional and conventional tests, however, the DNA integrity did not correlate with any conventional test. These results may indicate that probably, the spontaneous peroxidation that European bulls suffer due to the heat stress may have selected sperm more resistant to the cryopreservation.

Stoffwechsel und antioxidativer Status bei Merinofleischschafen im peripartalen Zeitraum Medizinische Tierklinik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im Juni 2010 (99 Seiten, 28 Abbildungen, 38 Tabellen, 357 Literaturangaben) Problem- und Zielstellung: Dem antioxidativen System wird im Rahmen von Stoffwech-seluntersuchungen bei Schafen nur wenig Bedeutung geschenkt, jedoch gewinnt es als Schutz- und Regulationssystem zunehmend an Bedeutung. Besonders die Trächtigkeit und die Frühlaktation bei Mutterschafen sowie die postnatale Entwicklung der Lämmer stellen enorme Anforderungen an die Energieversorgung und an das antioxidative System. Das Ziel dieser Arbeit ist, die Bewältigung dieser Anforderungen darzustellen und zu zei-gen, ob und wie stark Zwillingsträchtigkeiten ein Belastung für Mutterschafe darstellen, und ob Lämmer aus einer Zwillingsträchtigkeit möglicherweise benachteiligt sind. Tiere, Material und Methoden: Untersucht wurden insgesamt 23 trächtige Merinofleisch-schafe und zwölf Merinofleischschaflämmer. Bei 13 Muttertieren wurde sonografisch eine Einlingsträchtigkeit (M1) und bei zehn eine Zwillingsträchtigkeit (M2) festgestellt. Bei den Lämmern stammten sechs Tiere aus einer Einlingsgeburt (L1) und sechs aus Zwillingsge-burten (L2). Den Muttertieren wurden von der 5. Woche a.p. bis 1 Woche a.p. wöchentlich sowie 2 Wochen p.p., den Lämmern eine, vier und zwölf Wochen post natum Blutproben entnommen. Im Serum wurden die Konzentrationen von Betahydroxybutyrat (BHB), Glu-cose, Bilirubin, freien Fettsäuren (FFS), Gesamtprotein und Albumin, die Aktivitäten der Glutamatdehydrogenase (GLDH), sowie im Vollblut die Aktivitäten der Superoxiddismutase (SOD) und der Glutathionperoxidase (GPX) gemessen. Ergebnisse: Die SOD- und GPX-Aktivitäten stiegen gleichermaßen bei einlingsträchtigen und zwillingsträchtigen Muttertieren signifikant an. Die SOD-Aktivitäten stiegen bei M1 von 488,2 ± 28 vier Wochen a.p. auf 524,8 ± 96,1 U/g Hb eine Woche a.p. und bei M2 von 447,4 ± 65,1 fünf Wochen a.p. auf 619,4 ± 141,1 U/g Hb zwei Wochen p.p. an. Die GPX-Aktivitäten bei M1 stiegen von 782,1 ± 220,5 eine Woche a.p. auf 1037,62 ± 382,9 U/ml Hk zwei Wochen p.p.. M2 zeigten eine tendenziell niedrigere Aktivität, welche von 641,8 ± 118,4 auf 911,8 ± 168,5 U/ml Hk anstieg. Es bestanden signifikante Zusammenhänge der SOD mit FFS, BHB und Gesamtprotein sowie der GPX mit Bilirubin, FFS und GLDH. Für die SOD-Aktivität wurde ein Referenzbereich von 328,10 bis 837,25 U/g Hb und für die GPX-Aktivität von 473,38 bis 1259,05 U/ml Hk bei Muttertieren ermittelt. Ausgenommen die Glucosekonzentration eine Woche a.p. bestanden keine signifikanten Differenzen zwi-schen M1 und M2. Die BHB-Konzentrationen zeigten bis zur Lammung einen konstanten Verlauf und stiegen bei M1 eine Woche a.p. auf 0,59 ± 0,27 mmol/l sowie bei M1 und M2 zwei Wochen p.p. auf 0,76 ± 0,29 mmol/l (M1) bzw. 0,77 ± 0,25 mmol/l (M2) signifikant an. Gesicherte Korrelationen bestanden zu FFS, Gesamtprotein und Glucose. Die Gesamtprotein- und Albuminkonzentrationen verliefen über den gesamten Untersuchungszeitraum nahezu konstant. Die Albuminkonzentration sank bei M1 nach einem kontinuierlichen An-stieg von einer Woche a.p. (37,2 ± 2,9 g/l) zu zwei Wochen p.p. (35,3 ± 3,6 g/l) signifikant ab. Gesicherte Korrelationen bestanden zu Glucose, SOD, BHB, Bilirubin und FFS. Die Glucose- Konzentrationen stiegen bei M1 bis eine Woche a.p. auf 4,84 ± 1,17 mmol/l sig-nifikant an, korrelierten neben den Proteinen mit Bilirubin und fallen 2 Wochen p.p. wieder auf ihren Ausgangswert ab, der bei M2 konstant blieb. Die Bilirubinkonzentrationen verlie-fen konstant, die der FFS fielen nach einem Anstieg eine Woche a.p. (M1) signifikant ab. Die GLDH-Aktivitäten zeigten a.p. einen konstanten Verlauf und p.p. einen signifikanten Anstieg auf 20,61 ± 14,11 U/l bei M2. Zwischen L1 und L2 bestanden zu keinem Zeitpunkt signifikante Unterschiede. Die SOD zeigte abnehmende Aktivitäten mit zunehmendem Lebensalter, - bei L1 von 757,7 ± 94,4 eine Woche p.p. auf 597,8 ± 255,0 U/g Hb 4 Wochen p.p. und bei L2 von 487,17 ± 353,3 auf 464,0 ± 330,9 U/g Hb. Sie korrelierte gesichert mit Albumin und Glucose in der ersten Lebenswoche. Die GPX-Aktivitäten stiegen mit zunehmendem Lebensalter bei statistisch gesicherter Korrelation mit Glucose von 724,3 ± 199,8 auf 1011,5 ± 132,9 U/ml Hk bei L1 und von 693,3 ± 120,8 auf 1052,0 ± 146,9 U/ml Hk bei L2. Für Lämmer wurden als Refe-renzwerte 43,00 bis 932 U/g Hb für die SOD bzw. 406,00 bis 1321,00 U/ml Hk für die GPX berechnet. Die Albumin- und BHB-Konzentrationen stiegen zur vierten bzw. zwölften Le-benswoche an, die Glucose-, Bilirubin- und FFS-Konzentrationen im Serum sanken zur 12. Lebenswoche ab. Die Gesamtproteinkonzentration und die Aktivität der GLDH verlie-fen konstant. Es bestanden signifikante Korrelationen zwischen BHB und Gesamtprotein, FFS, GLDH sowie zwischen Gesamtprotein und Glucose, Albumin sowie FFS. Des Weite-ren bestanden gesicherte Korrelationen zwischen Bilirubin und FFS sowie FFS und GLDH. Fazit: Die Aktivitäten der antioxidativen Enzyme SOD und GPX sowie die Konzentrationen der klinisch-chemischen Parameter zeigen mit dem Anstieg der SOD-Aktivität von 447 auf 619 U/g Hb bei M2 einerseits die peripartalen Belastungen und andererseits mit dem An-stieg der GPX-Aktivität von 782 auf 1038 U/g Hk bei M1 die Kompensations- und Regel-mechanismen bei dieser Belastungssituation auf. Die Untersuchungen entsprechen dem Zustand einer ausgeglichen Energiebilanz und gesundheitlich stabilen Bedingungen. Ein Einfluss der Lämmerzahl auf das antioxidative System bzw. den Stoffwechsel kann weder bei Mutterschafen noch bei Lämmern statistisch signifikant belegt werden. / Metabolism and antioxidative status of merino sheep for meat production in the peripartum time period Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig Submitted in June 2010 (100 pages, 28 figures, 38 tables, 357 references) Definition of the problem and objective: Until now, the research regarding the metabol-ism of sheep focussed very little on the importance of the antioxidative system, but the significance of its function as a protective and regulative system is increasing. Especially the pregnancy and the early lactation of the ewes as well as the postnatal development of the lambs places enormous demands upon the energy supply and the antioxidative sys-tem. The objective of this study is to show how these demands are met and to determine whether a twin-pregnancy represents an additional stress for the ewes, and whether lambs from a twin-pregnancy are possibly disadvantaged. Animals, materials and methods: 23 pregnant merino ewes and twelve merino lambs were analyzed. Sonographic tests showed that 13 ewes were pregnant with one lamb (M1), and ten showed a twin-pregnancy (M2). Six of the lambs were born as single lambs (L1), six as twins (L2). Blood samples were taken from the ewes every week from the fifth until the week before the birth, as well as two weeks after birth, the blood samples from the lambs were taken one, four and twelve weeks postpartum. The concentration in the serum of the following components was measured: betahydroxybutyrate (BHB), glucose, biliru-bine, free fatty acids (FFS), total protein and albumine, the activity of the glutamatedehy-drogenasis (GLDH), as well as the activity in whole- blood of superoxidedismutasis (SOD) and glutathionperoxidasis (GPX). Results: The SOD- and GPX-activities rose significantly at the same rate for mother sheep pregnant with one lamb or with twins. The SOD-activity rose for M1 from 488,2 +/- 28 four weeks prepartum to 524 +/- 96,1 U/g Hb one week prepartum, and for M2 from 447,4 +/- 65,1 five weeks prepartum to 619,4 +/- 141,1 U/g Hb two weeks prepartum. The GPX-activity for M1 rose from 782,1 +/- 220,5 one week prepartum to 1037,62 +/- 382,9 U/ml two weeks postpartum, while M2 showed a tendency towards lower activity, which rose from 641,8 +/- 118,4 to 911,8 +/- 168,5 U/ml Hk. There were significant correlations between the SOD and the FFS, the BHB and the total protein as well as the GPX with bili-rubine, FFS and GLDH. For the ewes, an SOD-activity reference range between 328,10 and 837,25 U/g Hb was determined, and a GPX-activity reference range between 473,38 and 1259,05 U/ml Hk. There were no significant differences between M1 and M2 except for the concentration of glucose. The BHB-concentration stayed constant up to birth and then rose significantly to 0,59 +/- 0,27 mmol/l for M1 one week postpartum and for both M1 and M2 to 0,76 +/- 0,29 mmol/l (M1) and 0,77 +/- 0,25 mmol/l (M2) two weeks postpartum. A reliable correlation could be determined with FFS, total protein and glucose. The total protein and the albu-mine concentration stayed almost constant during the whole analyzed time period. After a continuous increase between one week prepartum (37,2 +/- 2,9 g/l) and two weeks post-partum (35,3 +/- 3,6 g/l), the albumine concentration dropped considerably. Here, reliable correlations could be determined with glucose, SOD, BHB, bilirubine and FFS. For the group M1, the glucose concentrations rose significantly until one week prepartum to 4,84 +/- 1,17 mmol/l, correlated with both the proteins and bilirubine, then fell back to their initial level two weeks postpartum, while they did not change for M2. The concentrations of bili-rubine stayed constant, the FFS-concentrations fell significantly after an increase one week prepartum (M1). The GLDH-activities showed a constant trend prepartum, then rose significantly postpartum to 20,61 +/14,11 U/l for M2. There were no significant differences between L1 and L2 at any time. The SOD showed decreasing activity with increasing age, from 757,7 +/- 94,4 one week prepartum to 597,8 +/- 255,0 U/g Hb four weeks postpartum for L1 and from 487,17 +/- 353,3 to 464,0 +/- 330,9 U/g Hb for L2. A reliable correlation was determined with albumine and glucose in the first week after birth. The GPX-activities rose with increasing age with a statistically reliable correlation with glucose from 724,3 +/- 199,8 to 1011,5 +/- 132,9 U/ml Hk for L1 and from 693,3 +/- 120,0 to 1052,0 +/- 146,9 U/ml Hk for L2. The calculated reference val-ues for lambs were 43,00 to 932 U/g Hb for SOD and 406,00 to 1321,00 U/ml Hk for GPX. The concentrations of albumine and BHB rose towards the fourth and respectively the twelvth week of life, the levels of glucose, bilirubine and FFS dropped towards the 12th week. The total protein concentration and the GLDH-activities stayed constant. There were significant correlations between BHB and total protein, FFS, GLDH as well as between total protein and glucose, albumine and FFS. In addition, there were reliable correlations between bilirubine and FFS and FFS and GLDH. Conclusion: The activities of the antioxidative enzymes SOD and GPX as well as the concentrations of the clinical-chemical parameters show the prepartum stress on one side with the rise of the SOD-activity from 447 to 619 U/g for the group M2, and the compensa-tion and regulation mechanisms during this stress period on the other side with the in-crease of the GPX-activity from 782 to 1038 U/g Hk for the group M1. The results of the tests correspond to the status of a well-regulated energy balance and stable health condi-tions. An influence of the number of lambs on the antioxidative system or the metabolism for both lambs and ewes can not be shown with statistical significance.

Thesis (PhD(FoodSc))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Potential beneficial and/or adverse modulatory effects of polyphenol-enriched extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) on the antioxidant homeostasis in the liver were investigated. Phase II metabolism of aspalathin and mangiferin, the major polyphenols of rooibos and honeybush respectively, was assessed for potential glucuronidation and sulphation. Glucuronidation resulted in a loss of antioxidant activity for aspalathin and mangiferin in post-column HPLC-DAD-DPPH• and HPLC-DAD-ABTS•+ assays and also a decreased activity of iron chelating properties of mangiferin in the FRAP assay. Two independent studies for 28 and 90 days with polyphenol-enriched extracts (PEEs) of unfermented rooibos [Aspalathus linearis (PER)] and honeybush [Cyclopia. subternata (PECsub) and C. genistoides (PECgen)] in male Fischer rats were conducted to assess possible beneficial and/or adverse biological effects. PECgen was only included in the 28 day study. PEEs were characterised by in vitro antioxidant assays and HPLC analysis. The importance of detailed chemical characterization of rooibos and honeybush when investigating biological effects in vivo is clear as distinctive biological effects and major differences in compositions were evident. Biological parameters included were serum chemical parameters, activities of selected antioxidant enzymes, levels of glutathione and the modulation of expression of oxidative stress and antioxidant defense related genes in the liver. Sub-chronic (90 days) exposure of rats to PER and PECsub both adversely affected iron absorption significantly (P<0.05) and significantly (P<0.05) and markedly lowered levels of reduced glutathione (GSH) in the liver. The high levels of polyphenol intake were implicated and interaction with glutathione was postulated to occur via catechol o-quinone conjugations with GSH. This was also implicated in the significantly (P<0.05) increased activity of glutathione reductase (GR) following 28 days. These findings suggest that PEEs from rooibos and honeybush have the potential to alter the glutathione homeostasis, which could contribute to oxidative status in the liver. PECsub resulted in alterations in the liver biliary system which was manifested as significantly (P<0.05) increased serum total bilirubin (Tbili) and alkaline phosphatase (ALP), depending on the age of the rats, level of total polyphenols and duration of exposure. The expression of a number of oxidative stress and antioxidant defense related genes were differentially altered by the PEEs of rooibos and honeybush in rat liver and further indicated potential oxidative stress. Modulatory effects of PEEs on expression of 84 of these genes in rat liver were assessed with a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) array and provided additional insights into the possible adverse and protective effects of rooibos and honeybush. Further investigation on total polyphenol dose levels and time of exposure in the application of PEEs of rooibos and honeybush as dietary supplements and functional foods is recommended and will also be of value in anticipated regulatory requirements for future substantiation of safety and efficacy. / AFRIKAANSE OPSOMMING: Die moontlike voordelige en/of nadelige modulerende effekte van polifenol-verrykde ekstrakte van rooibos (Aspalathus linearis) en heuningbos (Cyclopia spp.) op die antioksidant homeostasis in die lewer is ondersoek. Die fase II metaboliete van aspalatien en mangiferin, die hoof verbindings in rooibos en heuningbos onderskeidelik, is ondersoek t.o.v. glukuronidering en sulfonering. Glukuronidasie het gelei tot n verlies in antioksidant aktiwiteit van aspalatien en mangiferin soos bepaal in post-kolom HPLC-DAD-DPPH• en HPLC-DADABTS•+ toetse, asook verminderde interaksie met yster van mangiferin in die FRAP toets. Twee onafhanklike studies van 28 en 90 dae is onderneem met polifenol-verrykde ekstrakte (PVEs) van ongefermenteerde rooibos [Aspalathus linearis (PVR)] en heuningbos [Cyclopia. subternata (PVCsub) and C. genistoides (PVCgen)] in manlike Fisher rotte om die moontlike voordelige en/of nadelige biologiese effekte te ondersoek. PECgen was slegs in die 28 dae studie ingesluit. PVEs is gekarrakteriseer deur in vitro antioksidant en HPLC analises. Die belang van chemiese karaktirisering van rooibos en heuningbos tydens ondersoeke na biologiese aktiwiteit is duidelik aangesien verskeie en variërende biologiese aktiwiteite en verskille in die komposisie in die huidige studie gesien is. Die biologiese parameters wat ondersoek is om die effek van die PVEs te bepaal het serum kliniese parameters, aktiwiteit van geselekteerde ensieme, glutatioon en evaluering van die ekspressie van oksidatiewe en antioksidant verwante gene in die lewer, ingesluit. Sub-kroniese (90 dae) blootstelling van rotte aan PVR en PVCgen het yster absorpsie negatief beïnvloed. Die beduidende (P<0.05) verlaagde vlak van gereduseerde glutatioon in die lewer was toegeskryf aan die hoë vlakke van polifenole ingeneem tydens die studie en word moontlik veroorsaak deur n spesifieke katekol o-konjugasie van GSH. Hierdie interaksie was ook moontlik die oorsaak van n beduidende (P<0.05) toename in die aktiwiteit van glutatioon reduktase. Dié bevindinge het moontlike implikasies t.o.v die glutatioon homeostase en is n moontlike indikase dat PVEs van rooibos kan bydra tot oksidatiewe stres. PVCsub het veranderinge in die lewer gal-sisteem tot gevolg gehad aangesien daar n beduidende (P<0.05) verhoging in die serum totale bilirubin en alkalien fosfaat was. Hierdie veranderinge is beïnvloed deur die ouderdom, vlakke van die totale polifenole en die periode van blootstelling. Die uitdrukking van oksidatiewe en antioksidant verwante gene is op verskillende maniere beïnvloed deur die PVEs van rooibos en heuningbos in rot lewer and dien as n verdere indikasie van onderliggende oksidatiewe stres. Die modulerende effekte van PVEs op geenuitdrukking het gelei tot additionele insig aangaande die moontlike skadelike of beskermende eienskappe van PVEs vir gebruik as kruie produkte of dieet aanvullings. Die indikasies van moontlike oksidatiewe stres was duidelik van biologiese parameters en modulering van geenuitdrukking in die lewer, en vereis verdere ondersoek na die polifenool dosis en periode van toediening voordat PVEs van rooibos en heuningbos as funksionele voedsel produkte gebruik word. Hierdie ondersoek sowel as toekomstige ondersoeke in hierdie verband sal van waarde wees vir regulatoriese vereistes omtrent die veiligheid en effektiwiteit van rooibos en heuningbos kruie produkte.

L’impératif d’une aquaculture durable conduit à orienter l’alimentation des poissons vers la substitution de la farine de poisson par des produits végétaux renouvelables. Toutefois, ce remplacement est souvent limité par des niveaux trop faibles en méthionine dans les matières premières végétales. Ainsi la supplémentation en méthionine de ces nouveaux régimes à base de végétaux est essentielle, mais requière une bonne connaissance de son rôle pour adapter les apports aux conditions physiologiques des poissons tout en prenant en compte les contraintes économiques et environnementales de production. Dans ce contexte, cette thèse avait pour principal objectif de caractériser les effets induits par une carence en méthionine sur le métabolisme mitochondrial de la truite arc en ciel. Les résultats obtenus dans notre première étude montrent que l'alimentation de truites avec un régime déficient en méthionine entraîne une baisse des performances de croissance associée à une baisse de l’intégrité mitochondriale et une diminution du statut oxydatif dans le foie. Nous démontrons également que ces perturbations s’accompagnent de l’induction d’un processus de dégradation des mitochondries par autophagie (appelé mitophagie) ainsi que d’une augmentation du stress du Réticulum Endoplasmique (RE) et de l’apoptose. Ces données originales publiées dans le journal Scientific Reports mettent ainsi en évidence les liens étroits qui existent entre différentes fonctions cellulaires pour faire face à un déséquilibre nutritionnel en méthionine. Outre cet effet à court terme, nous démontrons également, dans un seconde étude, qu’une carence en méthionine alimentaire pendant une courte période (2 semaines) lors des premiers repas des alevins entraîne une induction à long terme de facteurs liés à la mitophagie. Ces résultats, soumis à publication dans Journal of Experimental Biology, démontrent ainsi pour la première fois la mise en place d’un processus de programmation de cette fonction cellulaire par une carence précoce en méthionine. L’enrichissement en H3K4me3 et H3K36me3 des foies des poissons issus d’alevins carencés en méthionine par rapport aux poissons témoins suggère une implication de mécanismes épigénétiques dans ces effets. Enfin, dans une troisième étude qui se détache de la thématique principale de la thèse et qui a fait l’objet d’une publication dans le journal Frontiers in Physiology, nous nous sommes attachés à préciser les interactions existante entre l’autophagie, l’homéostasie du RE et le métabolisme intermédiaire. Dans l’ensemble, ces données approfondissent notre compréhension du rôle de la méthionine alimentaire au niveau cellulaire et soulignent le potentiel de cet acide aminé en tant que levier pour appliquer de nouvelles stratégies alimentaires, comme la programmation nutritionnelle, afin d’optimiser la nutrition et la santé des poissons d'élevage. / The expansion of the aquaculture industry in combination with limited availability and high prices of fishmeal has prompted feed producers to include more plant proteins in the aquaculture feeds. However, replacement of fish meal with plant proteins is often limited by the level of methionine in the alternative plant protein sources. Understanding of the different roles of methionine in fish is therefore essential to develop new diets and/or feeding strategies that are in tune with optimal fish growth, environmental and economic constraints. In this context, the main objective of this thesis was to characterize the effects induced by methionine deficiency on the hepatic mitochondrial metabolism in rainbow trout. The results obtained in our first study show that feeding trout with a methionine deficient diet leads to a decrease in growth performance associated with a decrease in both mitochondrial integrity and oxidative stress in the liver. We also demonstrate that these defects are accompanied by the induction of an autophagy-dependent mitochondrial degradation process (called mitophagy) as well as an increase in Endoplasmic Reticulum (ER)-stress and apoptosis. These original data published in Scientific Reports thus highlight the existence of close interactions between different cellular functions to cope to a dietary methionine deficiency. In addition to this short-term effect, we also demonstrate in a second study (submitted for publication in the Journal of Experimental Biology), that early nutritional stimulus during two weeks with a methionine deficient diet resulted in a long term programming of mitophagy. The enrichment of H3K4me3 and H3K36me3 in the liver of fish from methionine-deficient fry compared to their control counterparts suggests that epigenetic mechanisms are involved in these effects. Finally, in a third and last study, recently accepted for publication in Frontiers in Physiology, we sought to clarify, in primary culture of trout hepatocytes, the existing interactions between autophagy, ER homeostasis and intermediate metabolism under amino acid deprived conditions. Together, the results obtained in the present thesis extended our understanding of the role of dietary methionine at cellular level and emphasize the potential of this amino acid to apply new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.