Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.

Cieľom tejto diplomovej práce je stanoviť kvalitu a zloženie medu, taktiež stanoviť jeho botanický pôvod na základe profilu sacharidov v mede. Med je prírodný produkt vysokej kvality a vďaka značnej spotrebe a nemalej trhovej hodnote je tiež terčom na falšovanie. S cieľom identifikovať podvody a cudzorodé látky, pochádzajúce z cukrových sirupov, boli analyzované oligosacharidy a polysacharidy na báze maltodextrínov. Teoretická časť popisuje chemické zloženie medu a hlavné princípy aplikovanej inštrumentálnej techniky. Poskytuje literárny prehľad existujúcich analytických metód na stanovenie sacharidov prítomných v mede a na odhalenie jeho falšovania. Boli definované hlavné body falšovania a autenticity, vrátane legislatívnych aspektov a opisu bežných druhov falšovania. Experimentálna časť obsahuje postupy, ktoré vedú k vývoju a optimalizácii chromatografických podmienok a parametrov elektrochemického detektora na stanovenie sacharidov a maltodextrínov (oligosacharidov a polysacharidov) vo vzorkách medu. Použitá analytická technika bola vysoko účinná aniónovo-výmenná chromatografia (HPAEC) spojená s pulzným amperometrickým detektorom (PAD). Vyvinutá a optimalizovaná chromatografická metóda bola taktiež validovaná z hľadiska linearity, dynamického rozsahu, analytických limitov, presnosti a správnosti. Na záver bola táto vypracovaná metóda úspešne zhodnotená analýzou autentických a falšovaných vzoriek medu rôzneho botanického pôvodu pochádzajúcich z členských štátov EÚ.

Background: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. Results: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. Conclusion: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.

L'analyse chimique est indispensable à de nombreuses étapes de la mise au point et dusuivi des procédés de retraitement des combustibles nucléaires usés, de la gestion des déchetsnucléaires, ou encore de l’optimisation des combustibles du futur. Le cycle global d’analysecomprend généralement plusieurs étapes de séparations chimiques longues, manuelles etdifficiles à mettre en oeuvre en raison de leur confinement en boite à gants. Il apparaîtaujourd’hui nécessaire de proposer des solutions innovantes et viables dans le butd’automatiser ces étapes mais aussi de réduire le volume de déchets radioactifs en fin de cycleanalytique. Une solution est alors la conception de plateformes analytiques miniaturiséesautomatisées et jetables.L’objectif de cette thèse est de concevoir un système miniaturisé alternatif à lapremière étape actuelle d’analyse des combustibles usés séparant, par chromatographied’échange d’ions, l’Uranium et le Plutonium des autres éléments constituant l’échantillon. Cesystème doit permettre à la fois de conserver les performances analytiques du processusactuel, de réduire drastiquement l’exposition des expérimentateurs par l’automatisation, ainsique le volume de déchets produits en fin de cycle analytique. Ainsi, la séparation a étéimplantée sur un microsystème jetable en plastique (COC), au design spécialement adapté àl’automatisation : un lab-on-CD.Le prototype développé intègre une micro-colonne séparative d’échange d’anionsremplie d’un polymère monolithique dont la synthèse in-situ ainsi que la fonctionnalisation desurface ont été optimisées spécifiquement pour la séparation voulue. Le développement duprotocole de séparation adapté à ces micro-colonnes a été réalisé à l’aide d’un outil desimulation de l’élution des différents éléments d’intérêts. Cet outil permet de prévoir lagéométrie de la colonne (section et longueur) afin d’obtenir, en fonction de l’échantillon, desfractions de collecte de l’Uranium et du Plutonium pures.Finalement, le prototype actuel est capable de conduire simultanément 4 séparationsde façon automatisée et permet de réduire à la fois le nombre de manipulations, le tempsd’analyse mais aussi de diviser approximativement par 1000 le volume des déchets liquidesgénérés. / The chemical analysis of spent nuclear fuels is essential to design future nuclear fuelscycle and reprocessing methods but also for waste management. The analysis cycle consistsof several chemical separation steps which are time consuming and difficult to implement dueto confinement in glove boxes. It is required that the separation steps be automated and thatthe volume of radioactive waste generated be reduced. The design of automated, miniaturizedand disposable analytical platforms should fulfill these requirements.This project aims to provide an alternative to the first analytical step of the spent fuelsanalysis: the chromatographic separation of Uranium and Plutonium from the minor actinidesand fission products.The goal is to design a miniaturized platform showing analytical performancesequivalent to the current process, and to reduce both the exposure of workers throughautomation, and the volume of waste produced at the end of the analysis cycle. Thus, theseparation has been implemented on a disposable plastic microsystem (COC), specificallydesigned for automation: a lab on a Compact Disk or lab-on-CD.The developed prototype incorporates an anion-exchange monolithic micro-columnwhose in-situ synthesis as well as surface functionalization have been optimized specificallyfor the desired separation. The development of an adapted separation protocol was carried outusing a simulation tool modeling the elution of the various elements of interest. This tool isable to predict the column geometry (length and cross section) suited to obtain pure fractionsof Uranium and Plutonium as a function of the sample composition.Finally, the prototype is able to automatically carry out four separationssimultaneously reducing the number of manipulations, the analysis time and reducing thevolume of liquid waste by a factor of 1000.

Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische Änderung der katalytischen Aktivität bewirkte die Assoziation des Proteasomen-Aktivators. Innerhalb der ersten zwölf Stunden einer IFN gamma Stimulation wurde das Epitop vermehrt mit der Unterstützung des Proteasomen-Aktivators generiert, nach 24 Stunden zunehmend durch freies 20S Proteasom. Die Ergebnisse der vorgestellten Arbeit zeigen, dass Strukturvarianten des Proteasoms zusammen mit PA28 redundant funktionieren und eine hohe proteolytische Plastizität des UPS gewährleisten. / The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces a heterogeneity of 20S proteasomes in the early stage of stimulation, acting in combination with the proteasome activator in a redundant manner; and provides a high proteolytic placticity of the proteasome system.