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The Maillard reaction is a network of different non-enzymatic reactions between carbonyl groups of reducing sugars and amino groups from amino acids, peptides, or proteins, which progresses in three major stages and originates a very heterogeneous mixture of reaction products. It is also known as non-enzymatic browning, due to the brown macromolecular pigments formed in the final stage of the reaction. The chemistry underlying the Maillard reaction is complex. It encloses not only one reaction pathway, but a whole network of various transformations. As virtually all foods contain both proteins and carbohydrates, Maillard reaction products are present in the daily diet in considerable amounts. The endogenous formation of Maillard reaction products, especially related to ageing and diabetes, aroused intense discussions about the health consequences of the “glycation”, the term that describes the in vivo reaction corresponding to the Maillard reaction in foods. Melanoidins are the final brown products of the Maillard reaction. They are responsible for the color formed during the heat processing of foods like coffee, bread, malt, and beef. Melanoidins are high molecular weight polydisperse polymers containing nitrogen. Their structure is largely unknown. Coffee melanoidins, which are object of the present study, contain thermally transformed polysaccharides, proteins, and phenolic compounds. Since the mechanisms involved on the formation of these macromolecules, and the chemical transformations which take place during the heat treatment are not completely elucidated, key structural features were analyzed. Especially the incorporation of chlorogenic acids in the melanoidin skeleton was object of attention of the present work. Another major aim of this work was to investigate the influence of the Maillard reaction on the inhibitory potential of food components against zinc metalloproteases. The studied enzymes were three human matrix metalloproteases (MMP-1, -2 and -9), which are able to degrade matrix proteins and participate in many physiological processes, including tissue turnover and repair, but also constitute important targets in malignant and degenerative diseases. A microbial collagenase from Chlostridium histolyticum was chosen due to its subtract similarity to MMPs. Furthermore, Angiotensin Converting Enzyme (ACE), which plays a central role in cardiovascular pathologies such as hypertension and cardiac hypertrophy, was investigated. As a prototypical Maillard reaction product, coffee melanoidin was adopted. Due to the roast dependent inhibitory activity of the coffee melanoidin fractions against matrix metalloproteases, the functionalization caused by the non-enzymatic browning was closer investigated. Na-carboxyalkylated derivatives of a sequence of relevant peptides were synthesized, in a variation of the process-induced formation of Nε-carboxymethyllysine, a major advanced glycation end-product (AGE). The inhibitory activity against zinc metalloproteases of the sequence of selected peptides and their Na-carboxymethyl- (CM-) and Na-carboxyethyl- (CE-) derivates was investigated.:LIST OF CONTENTS I LIST OF TABLES IV LIST OF FIGURES V LIST OF ABBREVIATIONS VII 1 INTRODUCTION 1 2 BACKGROUND 3 2.1 Maillard reaction in food 3 2.1.1 Melanoidins 8 2.2 Coffee 11 2.2.1 General aspects 11 2.2.1.1 Coffee production 12 2.2.1.2 General chemical composition 14 2.2.1.3 Coffee and health 20 2.2.2 Coffee melanoidins 24 2.2.2.1 Chemistry of coffee melanoidins 24 2.2.2.2 Properties of coffee melanoidins 29 2.3 Zinc metallopeptidases 32 2.3.1 Matrix metalloproteinases (MMPs) 33 2.3.1.1 Functions of MMPs 35 2.3.1.2 Structure of MMPs 37 2.3.1.3 Inhibition of MMPs 39 2.3.2 Clostridium histolyticum collagenase (ChC) 43 2.3.2.1 Functions of ChC 43 2.3.2.2 Structure of ChC 43 2.3.2.3 Inhibition of ChC 44 2.3.3 Agiotensin converting enzyme (ACE) 45 2.3.3.1 Functions of ACE 45 2.3.3.2 Structure of ACE 46 2.3.3.3 Inhibition of ACE 48 3 EXPERIMENTAL SECTION 50 3.1 Chemicals, materials and equipment 50 3.1.1 Chemicals 50 3.1.2 Material 52 3.1.3 Equipment 52 3.1.4 Solutions 54 3.2 Synthesis of Nα-carboxyalkylated peptides 55 3.2.1 Nα-carboxyalkylation of GP, LL, IA, GA, GL, AP, IP and IPP by reductive alkylation 55 3.2.2 Nα-carboxyalkylation of IW using sodium cyanoborohydride 56 3.3 Purification 57 3.3.1 Ion Exchange Chromatographic purification 57 3.3.1.1 Spotting test 58 3.3.2 HPLC purification of CM-IW 58 3.3.3 Overview of the synthesis and elution conditions 59 3.4 Characterization of carboxyalkylated peptides 61 3.4.1 Mass spectrometry 61 3.4.2 Elemental Analysis 61 3.4.3 Analytical characteristics of carboxyalkylated peptides 62 3.5 Preparation of coffee fractions 65 3.5.1 Roasting conditions 65 3.5.2 Fractionation of coffee samples: Isolation of coffee melanoidins 67 3.6 Structural studies 69 3.6.1 Estimation of the molecular weight 69 3.6.2 C/N ratio 70 3.6.3 Amino acid analysis 70 3.6.3.1 Acid hydrolysis 70 3.6.3.2 General amino acid analysis 70 3.6.3.3 Lysinoalanine 71 3.6.3.4 Pentosidine 72 3.6.4 Total phenols 74 3.6.5 Raman spectroscopy 74 3.7 Study on inhibition of zinc metalloproteases 75 3.7.1 Inhibition of ACE 75 3.7.1.1 General enzymatic assay 75 3.7.1.2 Quantification 78 3.7.2 Inhibition of MMP-1, -2 and -9 79 3.7.2.1 General enzymatic assay 80 3.7.2.2 Effect of zinc addition on the inhibition of MMP-1 by melanoidins 81 3.7.3 Inhibition of ChC 82 3.7.3.1 General enzymatic assay 82 3.7.3.2 Quantification 84 3.7.4 Calculation of IC50 84 4 RESULTS AND DISCUSSION 86 4.1 Coffee melanoidins 86 4.1.1 Isolation of coffee fractions 86 4.2 Inhibition of zinc-dependent peptidases by coffee fractions 89 4.2.1 Inhibition of MMPs 89 4.2.2 Inhibition of other zinc metalloproteases 98 4.2.3 General considerations 99 4.3 Structural studies on coffee melanoidins 101 4.3.1 Gel permeation chromatography 102 4.3.2 Elemental analysis: C/N ratio 113 4.3.3 Amino acid analysis 116 4.3.4 Total phenolics 120 4.3.5 Correlation between total phenols content and C/N ratio in coffee melanoidins 123 4.3.6 Raman spectroscopy 124 4.4 Derivatization of peptides 129 4.4.1 Nα-carboxyalkylation of peptides by reductive alkylation 130 4.5 Preliminary investigations on the inhibitory potential of Nα-carboxyalkyl derivatives of peptides against metalloproteases 133 4.5.1 Inhibition against ACE 134 4.5.2 Inhibition against other zinc metalloproteases 138 5 SUMMARY 141 6 REFERENCES 145 LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS 168 AKNOWLEDGMENTS 169 ERKLÄRUNG 170

Bluthochdruck zählt zu einer der häufigsten Zivilisationskrankheiten und ist der Hauptfaktor für die Entstehung kardiovaskulärer Erkrankungen. Das Präventionspotenzial bei Hypertonie ist sehr hoch, da lebensstilassoziierte Faktoren wie Übergewicht, hoher Kochsalz- und Alkoholkonsum oder Stress die Entstehung eines erhöhten Blutdrucks wesentlich begünstigen. Daher wird eine antihypertensive Therapie meist mit nicht-medikamentösen Maßnahmen eingeleitet. Für die Regulation des Blutdrucks ist die nähere Betrachtung des Angiotensin-Converting Enzymes (ACE) wichtig, da es eines der Schlüsselenzyme des Renin-Angiotensin-Aldosteron-Systems und des Kallikrein-Kinin-Systems darstellt. Die Möglichkeit, dass ACE-inhibierende Peptide aus Lebensmittelproteinen über die Nahrungsmittelaufnahme einen positiven physiologischen Effekt auf den Blutdruck ausüben, ist ein vielversprechender Ansatz zur Unterstützung einer nicht-medikamentösen Therapie bei Hypertonie. In der Literatur sind zahlreiche Peptide beschrieben, welche eine inhibitorische Wirkung auf das ACE in vitro besitzen. Die vorliegende Arbeit beschäftigte sich mit der Klasse der tryptophanhaltigen Dipeptide, die in der Literatur als potente, natürliche ACE-Inhibitoren beschrieben sind. Die tryptophanhaltigen Peptide wurden hinsichtlich ihrer Gewinnung, ihrer Hemmwirkung auf das Zielenzym und bezüglich ihrer Bioverfügbarkeit in vitro und in vivo untersucht.

Herz-Kreislauf-Erkrankungen sind nach wie vor die häufigste Todesursache. Vor allem Bluthochdruck ist in diesem Zusammenhang ein wichtiger Risikofaktor für die Entstehung von koronaren Herzerkrankungen, Myokardinfarkten, Herzinsuffizienz und Schlaganfall. Zur Behandlung der Hypertonie werden unterschiedliche Pharmaka eingesetzt, hauptsächlich Substanzen, die das Renin-Angiotensin-Aldosteron-System (RAAS) hemmen. Dazu gehören synthetische Inhibitoren des angiotensin-converting enzyme (ACE). Für präventive Zwecke können diese ACE-Inhibitoren auf Grund mehrerer Nebenwirkungen nicht eingesetzt werden. Interessant für eine präventive Anwendung sind natürliche ACE-hemmende Peptide, welche in der Sequenz unterschiedlicher Lebensmittelproteine vorliegen und durch enzymatische Hydrolyse freigesetzt werden. Ein besonders potenter ACE-Hemmer ist das Dipeptid Isoleucin-Tryptophan (IW) und damit ein interessanter Kandidat für den Einsatz in einem funktionellen Lebensmittel. Um dies jedoch realisieren zu können, muss IW in einer ausreichenden Menge produziert werden. Durch die enzymatische Hydrolyse ist dies aktuell nicht möglich, da die Peptidsequenz IW sehr selten in Proteinen vorhanden ist. Aus diesem Grund war es Ziel der vorliegenden Arbeit eine innovative biotechnologische Methode zu etablieren, um das ACE-hemmende Dipeptid IW in höheren Mengen und vor allem lebensmittelkonform zu produzieren. Die Produktion des ACE-hemmenden Peptids wurde biotechnologisch mittels rekombinanter DNA-Technologie realisiert. Hierfür wurde eine repetitive IW-Sequenz entworfen (264 bp), welche für ein 10 kDa großes Protein codierte. Dieses IW-Konstrukt enthielt in der Sequenz 16-mal IW. Mit Hilfe von Escherichia coli (E. coli) wurde ein 52 kDa großes Fusionsprotein überexprimiert. Als Fusionstag diente das Maltose Binding Protein (MBP). Dieses rekombinante Fusionsprotein (MBP-IW) lag nach einer Kombination von zwei verschiedenen chromatographischen Verfahren gereinigt vor. Mit dieser Methode war es möglich, 0,52 mg lösliches MBP-IW pro 1 g E. coli Feuchtmasse zu produzieren. MBP-IW wurde enzymatisch mit dem Enzym α-Chymotrypsin hydrolisiert und das Dipeptid IW anschließend chromatographisch isoliert. Nach der Hydrolyse und Isolation lag die Ausbeute des rekombinant produzierten IW (rIW) mit einer Reinheit von ≥ 96 % bei 14 µg. Somit konnten 28 % des möglichen Anteils an rIW vom sauberen MBP-IW gewonnen werden. Die Identifikation von IW erfolgte mit drei unterschiedlichen Methoden, der reversed phase-high performance liquid chromatography-UV-Detektion, der liquid chromatography-electrospray ionisation-tandem mass spectrometry und durch eine N-terminale Derivatisierung des Peptids. Mit diesen Methoden wurde bestätigt, dass es sich bei dem produzierten Peptid um IW handelte. Das rIW wurde im Vergleich zum chemisch produzierten kommerziell erwerblichen L-IW (cIW) und chemisch produzierten kommerziell erwerblichen D-IW (cDIW) auf sein ACE-hemmendes Potential getestet. Um der komplexen und heterogenen Verteilung der ACE-Aktivität im menschlichen Organismus gerecht zu werden, wurde das ACE-hemmende Potential der Dipeptide an verschiedenen ACE-Quellen untersucht. Neben dem nicht-humanen ACE-System (ACE aus der Kaninchenlunge) wurde auch humanes lösliches ACE (aus humanem Plasma) sowie humanes membrangebundenes ACE (aus Human umbilical vein endothelial cells, HUVECs) verwendet. Bei allen getesteten ACE-Systemen zeigte sich kein ACE-hemmendes Potential durch cDIW. Beim Vergleich von rIW mit cIW in Bezug auf deren ACE-hemmendes Potential wurden IC50-Werte von 1,72 ± 0,12 bis 23,30 ± 3,68 µM, abhängig vom getesteten ACE-System, bestimmt. Für alle verwendeten ACE-Quellen konnte gezeigt werden, dass beide unterschiedlich produzierten Dipeptide gleich effektiv waren. Ein weiteres Ziel der Arbeit bestand darin neben einem Peptidmix aus Molkenprotein mit hohem Anteil an IW, noch zwei weitere Peptidmixe pflanzlichen Proteinursprungs hinsichtlich des ACE-hemmenden Potentials zu untersuchen. Auf Grundlage der identifizierten tryptophan- und tyrosinhaltigen Dipeptide in den Hydrolysaten des Molken-, Soja- und Reisproteins wurden drei Peptidmixe hergestellt. Auch hier wurde wieder die Wirkung auf mehrere ACE-Quellen ermittelt. Neben den oben genannten, wurde hier zusätzlich der Einfluss auf membrangebundenes ACE der Rattenaorta untersucht. In allen getesteten ACE-Systemen zeigte der Peptidmix Molke ein signifikant höheres ACE-hemmendes Potential als die Peptidmixe von Soja und Reis. Der Peptidmix Soja war von den getesteten hydrolisierten Pflanzenproteinen der potenteste ACE-Inhibitor. Die IC50-Werte der Peptidmixe lagen, je nach getestetem ACE-System, zwischen 16,60 ± 2,59 und 282,04 ± 18,51 mg/l. Der starke ACE-hemmende Effekt vom Peptidmix Molke wurde mit der hohen Konzentration an IW assoziiert (bis zu 10-fach höher verglichen mit den anderen beiden Peptidmixen). Dies legt nahe, dass das Dipeptid IW hauptverantwortlich für das ACE-hemmende Potential in den getesteten Peptidmixen ist, was nochmals das große Potential des Dipeptids verdeutlicht. Im Rahmen dieser Arbeit konnte gezeigt werden, dass IW aus Molkenprotein im Vergleich mit den bioaktiven Peptidquellen der Proteine aus Soja und Reis, die stärkste ACE-Hemmung aufweist. Des Weiteren ist es erstmals gelungen, das ACE-hemmende Dipeptid IW in hoher Reinheit biotechnologisch mit Hilfe von rekombinanten Proteinen herzustellen. Um den Einsatz als funktionelles Lebensmittel realisieren zu können, müsste im Weiteren die biotechnologische Herstellung von IW optimiert werden, um eine höhere Ausbeute zu generieren. Nach dieser Optimierung könnte in einem Scale-up Verfahren so viel an IW gewonnen werden, dass es industriell einsetzbar wäre. Die ACE-hemmende Wirkung des biotechnologisch hergestellten IWs wurde in dieser Arbeit bestätigt, sodass es in einem innovativen funktionellen Lebensmittel für die tägliche Ernährung eingesetzt werden könnte. Perspektivisch eröffnet sich damit die Möglichkeit IW präventiv zu nutzen, um die Entwicklung von Bluthochdruck und deren Folgeschädigungen zu verzögern oder zu minimieren. / Cardiovascular diseases are still the leading cause of death. Especially hypertension is an important risk factor for the development of coronary heart disease, myocardial infarction, heart failure and stroke. To treat hypertension different drugs are clinically used. This are mainly substances, which inhibit the renin-angiotensin-aldosterone-system (RAAS), such as synthetic inhibitors of angiotensin-converting enzyme (ACE). However these ACE-inhibitors cannot be used for preventive purposes because of several side effects. Therefore natural ACE-inhibitory peptides, which are mostly encrypted in food proteins and released by enzymatic hydrolysis, are of main interest for preventive applications. The dipeptide isoleucine-tryptophan (IW) is a potent ACE-inhibitor and thus an interesting ingredient in functional foods. However, to realize this, IW must be produced in sufficient amounts. This is not possible with the current enzymatic hydrolysis, because the peptide sequence of IW is very rarely present in proteins. For that reason, the aim of the present thesis was to establish an innovative biotechnological method to produce the ACE-inhibitory dipeptide IW in an enlarged amount and especially considering the food-safety. The production of the ACE-inhibitory peptide was realized biotechnologically via recombinant DNA technology. For this, a repetitive IW-sequence (264 bp) was designed, which encoded a 10 kDa protein. In this IW-construct IW was sequenced 16 times. Using Escherichia coli (E. coli) a fusion protein with a size of 52 kDa was overexpressed. The maltose binding protein (MBP) served as fusion tag. This recombinant fusion protein (MBP-IW) was purified by a combination of two different chromatographic methods. It has become possible to produce 0.52 mg of soluble MBP-IW per 1 g wet weight of E. coli. MBP-IW was enzymatically hydrolysed with the enzyme α-chymotrypsin and the dipeptide IW was subsequently isolated by chromatography. After hydrolysis and isolation, the yield of the recombinant produced IW (rIW) with a purity of ≥ 96 % was 14 μg. Thus, 28 % from the possible content of rIW was obtained from the clean MBP-IW. IW was identified by three different methods: reversed phase-high performance liquid chromatography with UV-detection, liquid chromatography-electrospray ionisation-tandem mass spectrometry and N-terminal derivatization of the peptide. These methods confirmed the produced peptide as IW. The ACE-inhibitory potential of rIW was analysed and compared to that of the chemically produced commercially available L-IW (cIW) and of the chemically produced commercially available D-IW (cDIW). To address the complex and heterogeneous distribution of ACE-activity in the human organism, the ACE-inhibitory potential of the dipeptides was investigated in different ACE-sources. Additionally to non-human ACE (from rabbit lung) also human soluble ACE (from human plasma) and human membrane-bound ACE (from human umbilical vein endothelial cells, HUVECs) were used. In all tested ACE-systems cDIW did not show any ACE-inhibitory effect. IC50 values of rIW and cIW ranged from 1.72 ± 0.12 to 23.30 ± 3.68 μM, depending on the investigated ACE-system. In all sources of ACE an equal inhibitory potency of both differently produced dipeptides were determined. The second aim of the thesis was to investigate the ACE-inhibitory effect of two peptide mixes of plant proteins beside the peptide mix of whey protein, containing a high concentration of IW. Based on the identified tryptophan- and tyrosine-containing dipeptides in the hydrolysates of the whey-, soy- and rice-protein, three peptide mixes were prepared. Also here the effect on different ACE-sources was determined. Additionally to the named above, membrane-bound ACE from rat aorta was investigated. In all analysed ACE-systems, the peptide mix of whey showed a significantly higher ACE-inhibitory potential than the peptide mixes of soy and rice. The peptide mix soy was the most potent ACE-inhibitor tested among the hydrolysed plant proteins. The IC50-values of the peptide mixes were between 16.60 ± 2.59 and 282.04 ± 18.51 mg/l, depending on the used ACE-system. The strong ACE-inhibitory effect of the whey peptide mix was associated with the high concentration of IW (10 times higher compared to the other peptide mixes). This indicates that the dipeptide IW is mainly responsible for the ACE-inhibitory potential in the investigated peptide mixes, which demonstrate again the great potential of this dipeptide. It was shown in the present study that IW from whey protein had the strongest ACE-inhibition compared to the bioactive peptides of proteins from soy and rice. Furthermore, for the first time it was possible to produce the ACE-inhibitory dipeptide IW in high purity biotechnologically using recombinant proteins. To use IW as an ingredient in functional foods, the biotechnological production of IW needs to be optimized to receive higher yields. After this optimization, it would be conceivable to increase the production of IW in a scale-up process for industrial application. The ACE-inhibitory effect of the biotechnologically produced IW was confirmed in the present study, thus it could be used in an innovative functional food for daily nutrition. Prospectively, this increases the possibility of using IW preventively in order to delay or minimize the development of hypertension and the consequentially diseases.

Proteinhydrolysate gewinnen als Bestandteil von Lebens- und Futtermitteln zunehmend an Bedeutung. In Abhängigkeit ihrer Sequenz können darin enthaltene Peptide über Wechselwirkungen mit dem Angiotensin-Converting Enzym (ACE), welches eine Schlüsselfunktion bei der Blutdruckregulation einnimmt, physiologisch wirken und damit einen Beitrag zur Gesunderhaltung leisten oder hinsichtlich antioxidativer Eigenschaften einen positiven Effekt auf die Lagerstabilität und damit Qualität von Lebensmitteln ausüben. In der vorliegenden Arbeit wurde zunächst die ACE-inhibierende Aktivität von Pflanzenproteinen im Vergleich zum Molkenprotein hinsichtlich Struktur-Wirkbeziehungen und gegenüber den Domänen des ACEs sowie verschiedenen ACE-Spezies charakterisiert. Des Weiteren wurde eine Verkapselung von in Proteinhydrolysaten enthaltenen Dipeptiden angestrebt und der Einfluss auf deren proteolytische Stabilität untersucht. Dipeptide unterliegen während der gastrointestinalen Verdauung einem Abbau, was einen limitierenden Faktor für deren Bioaktivität darstellt. Zur Charakterisierung von Cyclodextrin-Komplexen mit aromatischen Aminosäuren sowie korrespondierenden Dipeptiden wurden UV- und fluorometrische Methoden sowie NMR-Techniken verwendet. Abschließend wurde das antioxidative Potential von Proteinhydrolysaten zunächst im Modellsystem und anschließend unter Nutzung von Lebensmittelmatrices abgeschätzt. Zusammenfassen konnte für die untersuchten Pflanzenproteinhydrolysate ein ausgesprochen gutes den potenten Milchproteinhydrolysaten vergleichbares ACE-inhibitorisches Potential als auch eine konzentrationsabhängige antioxidative Aktivität abgeleitet werden. Insbesondere Reisproteinhydrolysat erwies sich als potente Quelle physiologisch als auch antioxidativ wirksamer Peptide.

A case study which examines the agreement between prediction and data is performed for three, complex interplanetary shocks which were detected at the Sun-Earth Lagrange point L1 and induced moderate to intense geomagnetic storms. We use model output from previous runs of the coupled coronal-heliosphere WSA-Enlil+Cone model, available through the Community Coordinated Modeling Center (CCMC), and in-situ data from the OMNI data set. Code written in MATLAB is used to compare the model output with the in-situ measurements of the interplanetary magnetic field as well as the density, speed and temperature of the solar wind. In addition, the difference between the predicted and actual shock arrival time is computed and regions of potential temperature depression are identified. A considerable discrepancy is found between data and model for the studied events. The main reason is deemed to be an inadequate representation of the ambient solar wind as well as the complex interactions between interplanetary coronal mass ejections and corotating interaction regions. We suggest future steps to be taken for the further development of the model as well as for the general understanding of space weather and the Sun-Earth connection. / Denna fallstudie undersöker överensstämmelsen mellan modell och data för tre interplanetära chockvågor, som kunde detekteras vid jordens Lagrangepunkt 1, och som orsakade geomagnetiska stormar av måttlig till kraftig styrka. Vi använder oss av tidigare genomförda körningar av den sammansatta WSA-Enlil+Cone modellen, som avbildar fortplantningen av temporära störningar med ursprung i solens korona, såsom koronamassutkastningar, ut i heliosfären. Modellen gjordes tillgänglig av Community Coordinated Modeling Center (CCMC) och datan inhämtades från OMNI. Kod skriven i MATLAB nyttjades för att göra en jämförelse mellan modell och faktiska mätningar av det interplanetära magnetfältet samt solvindens hastighet, densitet och temperatur. Utöver detta, beräknas också skillnaden mellan förväntad och faktisk ankomsttid av respektive interplanetär chock, och tidsperioder med en temperatursänkning utöver det normala identifieras. Vi finner en omfattande avvikelse mellan modell och data, i synnerhet för de fall där på varandra följande koronamassutkastningar förväntas interagera eller rent av slås ihop samt för uppskattningen av den omgivande solvindens egenskaper och det interplanetära fältet under pågående geomagnetisk störning. Interaktionen mellan koronamassutkastningar och närliggande ko-roterande interaktionsregioner har ej heller återskapats väl av modellen ifråga. Slutligen ger vi förslag på möjliga, framtida åtgärder som kan bör tas i åtanke vid konstruerandet av framtida versioner av nämnda modell, liksom för den allmänna förståelsen för rymdvädrets inverkan på Jorden.

Angiotensin Converting Enzyme (ACE) IID, and a actinin 3 (AC1N3) R577X polymorphisms have been linked to the strength and power performance of elite athletes and suggested to influence skeletal muscle function in the general popUlation. This research investigated the association of these two candidate gene polymorphisms with the muscle function of young and older men, and the distribution of these genotypes in frequent fallers compared to controls. Muscle function measurements of young and older men included isometric strength, absolute and relative isokinetic strength at high velocity (ratio of torque at 2400 ·s"; torque at 30°·s") and the time course of an evoked twitch. Additionally body composition was measured by skinfold thickness (young men) and DXA scanning (old men) to estimate fat-free mass, an index of muscularity, and fat mass. ACE and AC1N3 genotypes were determined from whole blood samples using polymerase chain reaction, and serum ACE activity using spectrophotometry. The gemtypes of frequent fallers referred to a Falls Clinic were compared to a control group of healthy men. ACE genotype was not associated with any measure of muscle function, including the time course of an evoked twitch or absolute and relative high velocity torque, or body composition in these populations (ANOVA, 0.12<P<0.97). Serum ACE activity appeared to be weakly associated with knee extensor (R = 0.19, P = 0.07) and elbow flexor (R = 0.20, P = 0.06) isometric strength in older men, and was negatively correlated with the relative torque at high velocity (R = -0.23, P = 0.03). AC1N3 genotype was associated with fat mass in older men (P = 0.04), but was not associated with any measure of muscle function or muscularity (KruskalWaIIis, 0.26<P<0.95). Finally there was no apparent difference in the distribution of ACE IID (r: = 0.54, P = 0.77) and AC1N3 RIX (r: = 0.76, P = 0.68) genotypes between frequent fallers and controls. Any influence of these individual polymorphisms seems unlikely to be of sufficient magnitude to produce genotype related differences in muscle function in young or older free living UK Caucasian men. Serum ACE activity may have a small association with the isometric and dynamic strength of older men. However, AC1N3 genotype was associated with increased fat mass in XX individuals, that suggests this polymorphism may have an association with the accumulation of body fat over the life span of older men.

In der vorliegenden Untersuchung wurde der Einfluss des AT1-R -Antagonisten Valsartan auf die Nierenfunktion bei nierentransplantierten Ratten mit der Fragestellung analysiert, ob eine Langzeittherapie mit diesem Wirkstoff einen positiven Effekt auf die Nierenfunktion entfaltet und sich somit sein Einsatz gegen die Entwicklung einer chronischen Transplantatnephropathie empfiehlt. Die über den gesamten Versuchszeitraum gegenüber der allogenen Kontrollgruppe signifikant erhöhten Urinvolumina stellen allein kein Indiz für eine bessere Nierenfunktion unter Therapie mit Valsartan dar. Dieses Ergebnis ist am ehesten durch Veränderungen der glomerulären Hämodynamik post transplantationem zu erklären. Wie nunmehr in mehreren tierexperimentellen Untersuchungen und klinischen Patientenstudien nachgewiesen worden ist, zeigt sich auch in der Synopsis der eigenen Befunde ein signifikant günstigerer Verlauf des Serumkreatinins, des Serum-BUN, der Kreatinin-Clearance sowie der Proteinurie unter Blutdrucksenkung mit dem AT1-R-Antagonisten Valsartan. An einigen Zeitpunkten der Studie waren die Ergebnisse allerdings statistisch nicht signifikant. Eine positive Wirkung auf die Transplantatfunktion und auf das Langzeitüberleben der Versuchstiere ist anzunehmen, ist aber in dieser Studie nicht weiter verfolgt worden. Eine Untersuchung mit einer größeren Anzahl von Versuchstieren und über einem längeren Versuchzeitraum hin scheint sinnvoll, um signifikante Unterschiede zwischen den Kontrollgruppen und der Versuchgruppe unter Valsartan zu belegen. Die im Vergleich zu den Kontrollgruppen geringere Entwicklung des Körpergewichts hatte bei der o.g. Fragestellung keine Relevanz. Wie in zahlreichen klinischen Studien für die Progredienz des chronischen Nierenversagens seit längerem eindrucksvoll belegt ist, scheint eine pharmakologische Blockade des RAAS auch einen protektiven Effekt auf die Entstehung einer chronischen Transplantatnephropathie zu entfalten. Die eigene Untersuchung liefert hinreichend Belege für diese Vermutung. Auch wenn in einzelnen Studien über negative Auswirkungen einer Blockade des RAAS auf die Transplantatfunktion berichtet worden ist, gibt es genügend Anhaltspunkte für einen günstigeren Verlauf nach Transplantation sowohl in Tierversuchen als auch für den transplantierten Patienten. Das allmähliche Fortschreiten der chronischen Transplantatnephropathie kann damit allerdings nicht ganz aufgehalten werden. Somit bleibt trotz dieser erfolgversprechenden experimentellen Ergebnisse nach Organtransplantation durch diese neuen Therapieansätze (Immunsuppressiva, RAAS-Blockade, Plasmapherese u.a.) die chronische Transplantat-Abstoßung immer noch ein therapeutisch fortbestehendes Problem. Weitere Untersuchungen über die Zusammenhänge immunologischer sowie nicht-immunologischer Ursachen einer chronischen Transplantatnephropathie und eine Optimierung der Immunsuppression sind deshalb auch weiterhin dringend erforderlich. / In spite of the new immunosuppressive drug therapies used in renal transplants, chronic rejection continues to be a major cause of graft dysfunction after the first posttransplant year. This so called chronic allograft nephropathy is characterized by a slow but variable decrease in renal function appearing months or years after transplantation, is often accompanied or proceded by proteinuria ans hypertension and does not respond to immunosuppressive therapy. In our study we could show that posttransplant therapy with the angiotensin-II-receptor antagonist Valsartan improves chronic renal allograft nephropathy in Lewis rats. A similar effect on long-term survival of human kidney transplants can be supposed. Nevertheless further investigations have to be done in oder to understand all immunologigal and non immunological mechanisms of renal chronic rejection and to improve therapies.

Peptídeos com ação inibitória sobre a enzima conversora de angiotensina I (ECA) e com o potencial de reduzir a pressão arterial têm sido obtidos a partir de diferentes tipos de alimentos ou matérias-primas, sendo grande o interesse em aproveitar resíduos da indústria alimentícia como fontes desses peptídeos. Neste aspecto, as amêndoas de cupuaçu (Theobroma grandiflorum S.), que são em sua maioria descartadas pela indústria, apresentam um teor considerável de proteínas e poderiam ser aproveitadas como fontes de peptídeos inibidores da ECA. Assim, o objetivo deste trabalho foi verificar se o concentrado proteico obtido a partir de amêndoas fermentadas de cupuaçu após ser submetido à hidrólise com a enzima pepsina poderia gerar peptídeos com ação inibitória sobre a ECA in vitro. Foi observado que após a hidrólise do concentrado proteico com pepsina por 1h foi obtido um efeito de 50 % de inibição da ECA, em ensaio realizado com o substrato Abz-FRK(Dnp)-P-OH. Posteriormente, esse hidrolisado foi submetido ao fracionamento por cromatografia em fase reversa (RP-HPLC) e resultou em cinco frações (F1-F5), das quais a terceira teve uma subfração (F3.1) com quatro novos peptídeos identificados por LC-MS/ MS com potencial em inibir a ECA. Esses quatro peptídeos (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) foram sintetizados e submetidos aos ensaios para a determinação do IC50 e Ki. Os peptídeos YRLAF e LGYFK, que apresentaram mecanismo de inibição do tipo competitivo e acompetitivo, respectivamente, tiveram valores de IC50 de 4.73 e 11.11&#181;M, e de Ki de 9.14 e 8.15 &#181;M. Dentre os peptídeos identificados merece destaque VTTVVTGLTF que demonstrou ser um inibidor do tipo acompetitivo e apresentou as menores IC50 (0.70 &#181;M) e Ki (2.79 &#181;M). Em contraste, FWVAM atuou como substrato da ECA e não peptídeo inibidor. A partir dos resultados obtidos neste estudo fica demonstrado que as amêndoas fermentadas de cupuaçu podem ser fonte de peptídeos com ação inibitória da ECA, com potencial efeito anti-hipertensivo a ser, futuramente, investigado a partir de estudos in vivo. / Angiotensin converting enzyme (ACE) inhibitory peptides with the potential to reduce blood pressure have been obtained from different types of food or raw materials, and there is a great interest in utilize residues from the food industry as sources of peptides. In this regard, cupuassu almonds (Theobroma grandiflorum S.), which are mostly discarded by the industry, has a considerable protein content and could be used as source of ACE-inhibiting peptides. Thus, the objective of this work was to verify if the protein concentrate obtained from fermented almonds of cupuassu after being submitted to the hydrolysis with the enzyme pepsin could generate peptides with inhibitory activity on ACE in vitro. In this study it was observed that after hydrolysis of the protein concentrate with pepsin for 1 h, a 50% effect of ACE inhibition was obtained in an assay performed with the Abz-FRK (Dnp) -P-OH substrate. Posteriorly, the hydrolyzate was subjected to fractionation by reverse phase chromatography (RP-HPLC) and resulted in five fractions (F1-F5), of which the third had a subfraction (F3.1) with four new peptides identified by LC-MS / MS with the potential to inhibit ACE. These four peptides (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) were synthesized and assayed for IC50 and Ki. The YRLAF and LGYFK peptides, which showed a competitive and uncompetitive type inhibition mechanism respectively, presented IC50 values of 4.73 and 11.11&#181;M, and the values for Ki were 9.14 and 8.15 &#181;M. Among the peptides identified, it is possible to highlight VTTVVTGLTF, which was shown to be an inhibitor of the uncompetitive type and presented the lowest value for IC50 (0.70 &#181;M) and Ki (2.79 &#181;M). While FWVAM acted as a substrate of the ACE and not as an inhibitory peptide. From the results obtained in this study it is demonstrated that cupuassu fermented almonds can be a source of peptides with ACE inhibitory activity with potential antihypertensive effect to be further investigated from in vivo studies.

A ausência (9) e não a presença (+9) de um segmento de nove pares de base do gene codificador do receptor B2 de bradicinina (BDKRB2), está associada com a maior transcrição do gene, com fenótipos cardiovasculares e performance física. Entretanto, os efeitos dessas variantes na reatividade vascular são desconhecidos. Hipotetizamos que, 1) a vasodilatação reflexa em resposta ao exercício de handgrip poderia estar aumenta em sujeitos portadores do genótipo -9/-9, 2) O treinamento físico potencializaria essa resposta, e 3) a atividade da enzima conversora de angiotensina-I (ECA) estaria diminuída. Foram133 homens genotipados para o BDKRB2 (-9/-9 n=30; - 9/+9 n=68; +9/+9 n=35). Freqüência cardíaca (FC, ECG) pressão arterial média (PAM, cuff automático oscilométrico), e fluxo sanguíneo no antebraço (FSA, pletismografia) foram avaliados 3 minutos no repouso e 3 minutos durante o exercício de handgrip (30%CVM). A capacidade funcional foi mensurada por teste cardiopulmonar. E a atividade da enzima conversora de angiotensina I (ECA), foi analisada em soro. As avaliações foram realizadas antes e depois do treinamento físico aeróbio (18 semanas, 90 minutos, 3vezes semanais).No pré-treinamento físico o FSA e a condutância vascular no antebraço (CVA) em resposta ao exercício foram similares entre os genótipos -9/-9, -9/+9 e +9/+9 (FSA: 630±26; 626±22; 585±22 ASC, P=0.71; CVA: 584±21; 592±27; 559±16 UAC, P=0.79, respectivamente). No pós-treinamento físico somente 58 indivíduos mostraram ganho acima ou igual a 10% no VO2pico e, portanto, foram incluídos no estudo. O aumento no FSA e CVA do genótipo -9/-9 foi significantemente maior do que nos genótipos -9/+9 e +9/+9 (FSA: 514±65 vs. 635±44; 672 ±56 vs. 632 ±37; 569±41 vs. 549±45 ASC, P=0.05, respectivamente) (CVA: 512±63 vs. 639±56; 657±101 vs. 622 ±45; 578±49 vs. 549±62 ASC, P=0.05, respectivamente). Ademais a atividade da enzima conversora de angiotensina-I, demonstrou uma redução de 23% em sua atividade no genótipo -9/-9 em comparação aos genótipos -9/+9 e +9/+9 (p<0.008), respectivamente. Finalmente fomos capazes de confirmar em cultura de células musculares lisas vasculares, que o genótipo -9/-9 está associado com aumentos na atividade transcricional do gene BDKRB2 (p=0.03). Esses resultados sugerem que o polimorfismo -9/+9 do gene do receptor B2 de bradicinina, influencia a vasodilatação muscular reflexa durante o exercício em indivíduos treinados. Além disso, a vasodilatação poderia estar aumenta no genótipo - 9/-9, pela menor atividade da enzima conversora de angiotensina-I, e maior biodisponibilidade do substrato bradicinina. / Absence (-9), rather than the presence (+9), of a 9 base pair repeat in the bradykinin type 2 receptor gene (BDKRB2) was associated with higher gene transcriptional activity, cardiovascular phenotypes and physical performance. However, their effects on vascular reactivity are unknown. We hypothesized that vasodilatation response to physical training is modulated by BDKRB2 genotype. hundred and thirty-three healthy volunters were genotyped for the +9/-9 BDKRB2 polymorphism. Heart rate (HR), mean blood pressure (MBP), and forearm blood flow (FBF) were evaluated at baseline. Functional capacity was measured by cardiopulmonary exercise testing. Angiotensin-I converting enzyme (ACE) activity was measured. Aerobic training was performed for 18 weeks, following a standardized protocol. All baseline variables were re-assessed following completion of the training period. Baseline HR, MBP, and forearm vascular conductance (FVC) were not different among -9/+9 genotypes. During handgrip exercise, FBF and FVC increased significantly and similarly among -9/-9, -9/-9 and +9/+9 genotypes. Resting HR, MBP, FBF, and FVC change following, aerobic training completion was not different between genotypes. However, FBF and FVC response to aerobic training were higher in -9/-9 carriers (p=0.05, and p=0.05, respectively). In addition, ACE activity decrease following aerobic training was higher in the -9/-9 group (p <0.008). Finally, we were able to confirm, in primary culture of human vascular smooth muscle cells, that the -9/-9 genotype was associated with increased transcriptional activity of the BDKRB2 gene (p=0.03).These results suggest that reflex muscle vasodilatation response to physical training is modulated by, the +9/-9 bradykinin B2 receptor gene polymorphism in healthy individuals.