Per primer cop, el nostre grup va identificar un enzim de la superfamilia de les aldo-ceto reductases (AKR) actiu amb retinoides. Atès que l'AKR1B12 presenta una identitat seqüencial pròxima al 70% amb l'AKR1B1 i l'AKR1B10 humanes, es va decidir ampliar l'estudi de l'activitat retinoide oxidoreductasa entre les AKR. En la present Tesi Doctoral, hem aprofundit en l'estudi de l'activitat retinal reductasa de membres de la superfamilia de les aldo-ceto reductases (AKR). El treball es va iniciar amb la caracterització in vitro de l'activitat amb retinoides d'AKR humanes de la família AKR1. En col·laboració amb el grup de la Dra. Natalia Kedishvili (actualment en el Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, EUA) es va realitzar una anàlisi comparativa de l'activitat retinol oxidoreductasa d'enzims de les superfamílies de les deshidrogenases/reductases de cadena mitjana (MDR), deshidrogenases/reductases de cadena curta (SDR) i AKR. Es va emprar un nou mètode basat en la solubilització de retinoides amb BSA, i anàlisi mitjançant HPLC. Així, es van poder determinar els valors de les constants cinètiques per a enzims humans de les tres superfamílies sense l'efecte d'inhibició del Tween-80, detergent utilitzat tradicionalment per a l'estudi cinètic de les MDR i AKR (Capítol I). Aquests resultats van posar de relleu que el valor de Km per a retinoides és similar (0,1-1 μM) en els enzims estudiats. Les principals diferències es centren en el valor de kcat, el qual va variar fins a tres ordres de magnitud entre els diferents enzims. Un altre resultat destacable, va ser la desmostració que cap dels enzims era capaç d'utilitzar com a substrat retinol, o retinal, unit a la proteïna cel·lular unidora de retinol (CRBPI). Fins el present treball, s'utilitzaven dos arguments per defensar un paper principal de les SDR en l'oxidació de retinol a retinal, primera etapa de síntesi de l'àcid retinoic: 1) Valors de Km inferiors de les SDR per als retinoides (fins a dos ordres de magnitud respecte les ADH). 2) Capacitat de reconèixer l'holoCRBPI com a substrat, una propietat aparentment exclusiva d'algunes SDR. El nostre estudi, a part de descartar aquests dos arguments, ha introduït les AKR com a tercer grup enzimàtic implicat en el metabolisme de retinoides. La segona part de la Tesi es centra en l'estudi estructural i funcional de l'AKR1B10, l'AKR que presenta una major eficiència catalítica per a la reducció del retinal. Aquest enzim és sobrexpressat en diferents tipus de càncers humans, el que va fer augmentar el nostre interès per determinar el seu paper en la ruta de síntesi de l'àcid retinoic. A part de l'estudi in vitro de l'activitat retinoide oxidoreductasa de l'enzim, també es va dur a terme un estudi in vivo. Utilitzant cèl·lules COS-1 com a model, i mitjançant transfecció transitòria, es va poder observar que AKR1B10 també participava en la reducció de retinal in vivo, però que en canvi no era capaç d'oxidar retinol. Finalment, i en col·laboració amb el grup de cristal·lografia de proteïnes del Dr. Ignasi Fita (Parc Científic de Barcelona-CSIC), es va obtenir l'estructura tridimencional del complex ternari AKR1B10-NADP+-tolrestat. El tolrestat és un inhibidor d'AKR1B1 àmpliament estudiat com a fàrmac en el tractament de les complicacions de la diabetis, però que també inhibeix AKR1B10 tant in vitro com in vivo. Tot i l'elevada identitat seqüencial entre AKR1B1 i AKR1B10, aquests enzims presenten constants cinètiques molt diferents per a la reducció del retinal. Així, gràcies a la resolució de l'estructura d'AKR1B10, esperem poder estudiar els determinants estructurals que confereixen a cada enzim les seves propietats cinètiques característiques, així com establir les bases per al disseny de nous inhibidors específics per a cadascun d'ells. / Our group was the first to identify an enzyme from the aldo-keto reductase (AKR) superfamily, AKR1B12, active with retinoids (Crosas et al., 2001). Due to the fact that AKR1B12 shows a 70% sequence identity with the human enzymes AKR1B1 and AKR1B10, we decided to study more deeply the retinoid oxidoreductase activity within the AKRs.Here, we extended the study on the retinal reductase activity of AKR superfamily members. The work was initiated with the in vitro characterization of retinoid activities of human AKRs from the AKR1 family. In collaboration with Dr Natalia Kedishvili's group (Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, USA) we carried out a comparative analysis of retinol oxidoreductase activity of the enzymes from the medium chain dehydrogenases/reductases (MDR), short chain dehydrogenases/reductases (SDR) and AKR. A new method based on a retinoid solubilisation with BSA and analysis by HPLC was employed. Kinetic constant values were determined for human enzymes from the three superfamilies, without the inhibitory effect of Tween-80, a detergent traditionally employed for MDR and AKR kinetic studies. The Km values obtained with retinoids (0,1-1 μM) were similar for all the enzymes tested. Main differences were found in the kcat values, which varied up to 10000-fold between the studied enzymes. Furthermore, none of the studied enzymes was able to use retinol, nor retinal, bound to cellular retinol binding protein (CRBPI). So far, a major role for SDR in retinol oxidation was supported by: 1) SDR had lower reported Km values with retinoid (up to 100-fold lower than ADHs). 2) SDR were the only enzymes with reported ability to recognize holoCRBPI as a substrate. Our group refused both arguments and introduced AKRs as the third enzymatic group implicated in retinoid metabolism.The second part of the thesis was focused on the structural and functional study of AKR1B10, which is the AKR with the highest catalytic efficiency reducing retinal. The enzyme is overexpressed in different human cancers, which increased our interest to determine its role in the retinoic acid synthesis pathway. In addition to the study on the retinoid oxidoreductase activity in vitro, we performed an in vivo study. COS-1 cells were used as a model, and transitional transfection experiments were carried out. We observed that AKR1B10 participates in the retinal reduction in vivo, but it was unable to oxidate retinol.Finally, in collaboration with Dr. Ignasi Fita's group (Parc Científic de Barcelona-CSIC), the threedimensional structure of the AKR1B10-NADP+-tolrestat complex was obtained. Tolrestat is an AKR1B1 inhibitor widely studied as a drug for diabetes treatment. It also inhibits AKR1B10 in vitro and in vivo. Despite the high sequential identity shared between AKR1B1 and AKR1B10, the two enzymes showed extremely different kinetic constant values for retinal reduction. The AKR1B10 structure resolution might help us to study structural features for the retinoid specificity showed, and to propose new fundamentals in order to design new specific inhibitors for each enzyme.

It seemed desirable to investigate further the possible role of complement and its components in the protective action of serum against transplanted neoplasms. It is the purpose of this thesis to present data which suggest that the C'4 component plays an active part in this process though the mechanism is not disclosed.

In der vorliegenden Projektarbeit wurde ein erster Design-Vorschlag für eine Superspiegel-Test-Anlage am Ausbildungskernreaktor 2 der TU Dresden erstellt. Mit dieser soll die Reflektivität von Neutronensuperspiegeln bestimmt werden. Die Funktionalität des erarbeiteten Vorschlags ist mit Hilfe der Software McStas überprüft worden. McStas ist ein Monte-Carlo Programm, das Neutronenstrahlen für neutronenoptische Anwendungen simuliert. Basierend auf der Arbeit an einer existierenden Anlage am Paul-Scherrer-Institut, NARZISS, wurde außerdem eine Software entwickelt. Die Software dient der Auswertung der am Instrument gewonnenen Daten.:1. Einleitung 2. Neutronenoptik 2.1. Reflexion und Transmission 2.2. Reflexion an dünnen Schichten 2.3. Superspiegel 2.4. Polarisierende Spiegel 2.5. Monochromatoren 2.6. Detektoren 3. McStas 3.1. Überblick 3.2. Source 3.3. Mirror 3.4. Monitor 4. NARZISS am PSI 4.1. Aufbau 4.2. Justage und Versuchsdurchführung 4.3. Anwendungen am Beispiel eigener Messungen 4.3.1. Neutronensuperspiegel 4.3.2. Wafer 4.4. McStas-Modell NARZISS 5. Simulationen für ein Superspiegeltest-Instrument an der TU Dresden 5.1. Randbedingungen 5.2. Aufbau 5.3. Quelle 5.4. Simulationsergebnisse 5.4.1. Wellenlänge 5.4.2. Kollimation, Divergenz 5.4.3. Abschätzung Messzeit 5.5. Ergebnis und Verbesserungspotentiale 6. NOA - Ein Programm zur Auswertung von NARZISS-Messergebnissen 6.1. Zielstellung 6.2. Architektur 6.3. Berechnungen und Bearbeitungsablauf 6.3.1. Teil: Reflektivität 6.3.2. Teil: Polarisierung 6.4. Ergebnis 7. Zusammenfassung und Ergebnis sowie Ausblick / In the current project there was developed a first design-suggestion for a super-mirror-teststation at the nuclear training reactor Ausbildungskernreaktor 2 at the TU Dresden. It should be used to test the reflectivity of neutron super mirrors. The usability was tested with the software McStas. McStas is a Monte-Carlo ray-tracing simulation tool for neutron optical simulations. Based on the work at an existing reflectometry-station at the Paul-Scherrer-Institut, NARZISS, it was also part of the project to develop a software. The software enables the analysis of the NARZISS data.:1. Einleitung 2. Neutronenoptik 2.1. Reflexion und Transmission 2.2. Reflexion an dünnen Schichten 2.3. Superspiegel 2.4. Polarisierende Spiegel 2.5. Monochromatoren 2.6. Detektoren 3. McStas 3.1. Überblick 3.2. Source 3.3. Mirror 3.4. Monitor 4. NARZISS am PSI 4.1. Aufbau 4.2. Justage und Versuchsdurchführung 4.3. Anwendungen am Beispiel eigener Messungen 4.3.1. Neutronensuperspiegel 4.3.2. Wafer 4.4. McStas-Modell NARZISS 5. Simulationen für ein Superspiegeltest-Instrument an der TU Dresden 5.1. Randbedingungen 5.2. Aufbau 5.3. Quelle 5.4. Simulationsergebnisse 5.4.1. Wellenlänge 5.4.2. Kollimation, Divergenz 5.4.3. Abschätzung Messzeit 5.5. Ergebnis und Verbesserungspotentiale 6. NOA - Ein Programm zur Auswertung von NARZISS-Messergebnissen 6.1. Zielstellung 6.2. Architektur 6.3. Berechnungen und Bearbeitungsablauf 6.3.1. Teil: Reflektivität 6.3.2. Teil: Polarisierung 6.4. Ergebnis 7. Zusammenfassung und Ergebnis sowie Ausblick

A model for the study of the relationship of immunity to cancer is found in AKR mice which harbor Gross virus. This genetically transmitted virus is present in a latent form for months before it spontaneously induces leukemia. Many investigators have demonstrated near normal humoral responses, but abnormal cellular immunity in the preleukemic animal. With increasing age, pathology of the disease is expressed, reflecting diminished immunity. In this study, the ontogeny of humoral antibodies of AKR/J and SWR/J mice was assayed by microagglutination techniques in response to thymus-independent, thymus-dependent, and solubilized antigens. Simultaneous injections of thymusdependent and -independent antigens provided data suggesting an impaired humoral response in the AKR mouse.

För att bibehålla elnätet stabilt är det viktigt för elproducenterna attkunna möta samhällets behov av elkraft. Detta behov varierar beroendepå tid på dygnet och även av årstid. Att kunna samla energi då behovetär lågt, för att sedan utvinna och distribuera energi då behovet ökar ärdärför viktigt. Vattenkraft är en av de energikällor som är enklast attreglera. Denna energi är dessutom relativt miljövänlig. Att ha en stabiloch säker vattenkraft är därför viktigt för samhället.I detta examensarbete har vissa problem som kan uppstå i ettvattenkraftverkets studerats, närmare bestämt alkali-silikatreaktion ibetong. Denna reaktion framträder genom att betongen sväller. Tillföljd av detta kan konstruktionen spricka. Detta beror på att en gelbildas när alkalier och kisel reagerar med varandra. Denna gel kan taupp vatten och då svälla.En specifik vattenkraftstation har i detta examensarbete studeratsnärmare, nämligen Malgomaj kraftverk. Detta är en anläggning somligger i ett område där, till skillnad från övriga Sverige, det finnsbergarter som har en medelsnabb reaktion med avseende på alkalikiselreaktion.Att denna geografiska placering blir ett problem beror påatt det stenmaterial som finns att tillgå i vattenkraftstationens närhethar använts som ballast i anläggningens betongkonstruktion.I den vattenkraftstation som studeras har problem iakttagits på grundav svällningar av betongkonstruktionen kring turbinen. För att få enuppskattning om hur vattenkraftstationens deformationer i framtidenkommer att utbildas har en modell av problemområdet byggts uppmed hjälp av finita elementmetoden, en så kallad FEM-modell. Dennamodell kalibreras mot mätdata och ska sedan ligga till grund för enuppskattning av vattenkraftstationens livslängd.Resultatet från undersökningen i detta examensarbete visar attdeformationerna är små men betydande för vattenkraftstationensmöjlighet till att fortsätta sin energiproduktion. / To maintain a stable power grid, it is important for electricity producers to meetsociety's need for electricity. This need will vary depending on time of day and eventhe season. Being able to accumulate energy when demand is low, and regain energywhen demand increases, is therefore important. Hydropower is one of the energysources that are easiest to regulate. Having a stable and secure hydropower istherefore important for society.In this thesis one problem that can occur in a hydroelectric plant has been studied,namely alkali-silica reaction (ASR) in concrete. This reaction causes the concrete toswell, due to a formation of gel when alkali and silicon react together.A specific hydropower station has been studied in detail, namely Malgomajhydropower plant. This is a facility that is located in an area where, unlike the rest ofSweden, there are stone materials that have a moderately rapid reaction with respectto the ASR.Problems for this hydroelectric power station have been observed because of swellingof the concrete structure surrounding the turbine. To get an estimate prognosis ofhow the hydropower plant will deform in the future, a finite element method-model(FEM-model) has be created of the problem area. This model is calibrated againstmeasured data and will then form the basis for an appreciation of the hydropowerstation's remaining lifetime.The results in this thesis show that the deformations are small but significant for thehydropower station's opportunity to continue its energy production.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...

This work is concerned with a study of the role of complement and antibodies in the serum of rabbits and of a non-susceptible strain of mice in the protection of Akr mice injected with active Akr tumor cells.

This thesis has been an attempt to observe the role antibodies play in extending the life span of tumor infected Akr mice.

Alkali-kiselsyrareaktionen (AKR) är en kemisk reaktion som medför att spänningar uppstår i betongen då den bildade silikatgelen expanderar. Reaktionen kräver tillräcklig hög alkalihalt, reaktiv ballast samt vatten. På grund av de AKR-inducerade spänningarna är det av intresse att få kunskap i hur reaktionen påverkar betongens böjmomentkapacitet, förankring samt skjuvnings- och genomstansningskapacitet. För att besvara frågeställningen har därför en omfattande litteraturinventering tillsammans med tre kompletterande intervjuer utförts. Resultat som har erhållits, påvisar att två huvudsakliga effekter fås av AKR. Utöver en reducerad hållfasthet på grund av expansioner och sprickbildningar, erhålls även en gynnsam förspänningseffekt när armering motverkar expansioner. Med hänsyn till dessa huvudeffekter påverkas betongs bärighet i olika stor utsträckning beroende på expansionens storlek samt armeringens läge och typ i tvärsnittet. Resultaten tyder på att draghållfastheten reduceras till 40 % medan tryckhållfastheten reduceras till 60 % vid 5 mm/m expansion. För böjkapaciteten har ingen större reduktion uppmäts då expansioner understiger 6 mm/m. Emellertid har en reduktion på 25 % observerats vid expansioner större än 6 mm/m. Skjuvkapaciteten i AKR-skadad betong beror till stor del av byglars närvaro samt expansionens omfattning. I balkar utan byglar reduceras skjuvkapaciteten med 15-25 % för slät armering och 20-30 % för räfflad armering. Genomstansningskapaciteten i ett dubbelarmerat betongtvärsnitt reduceras obetydande till dess att expansionen överstiger 6 mm/m varpå en mer påtaglig reduktion fås. Hållfastheten för vidhäftning minskar med ca 40 % då täckande betongskikt understiger 1.5Æ och/eller att inga byglar är närvarande. Om byglar är närvarande samt om täckande betongskikt överstiger 4Æ visas inga tecken på försämrad vidhäftningshållfasthet. Utöver en minskad bärighet, öppnar AKR upp betong och skapar gynnsammare förutsättningar för rost-och frostangrepp. / Alkali-silica reaction (ASR) is a chemical reaction that causes stresses in concrete when the produced alkali silica gel expands. The reaction requires sufficiently high alkali content, reactive aggregates and water. Due to ASR-induced stresses it is of interest to gain insight in how ASR affects the concrete’s bending capacity, anchoring together with shear- and punching shear capacity. An extensive literature review was therefore carried out together with three complementary interviews in order to answer the question at issue. Obtained results show two main effects of ASR. In addition to a reduced strength because of cracking and expansion, an advantageous pre-stress is gained due to restraint to expansion. Thus, the load-carrying capacity of concrete is affected in various extents depending on the size of expansion and location and type of the reinforcement. The results indicate that the tensile strength is reduced to 40 % whereas the compressive strength is reduced to 60 % at 5 mm/m expansions. While expansions lesser than 6 mm/m has not shown a greater reduction of the bending capacity, a reduction of 25 % has been observed in concrete with expansions greater than 6 mm/m. The shear capacity of an ASR-affected concrete structure depends mainly on the presence of links and the extent of expansions. In beams without links, shear capacity is reduced to 15-25 % for smooth bars and 20-30 % for ribbed bars. The punching strength of a concrete structure reinforced in both faces is not reduced until expansions exceed 6 mm/m, whereas a more significant reduction is obtained. The bond strength decreases by about 40 % if the concrete cover is less than 1.5 Æ and/or if no links are present. Meanwhile if links are present and if concrete cover is more than 4Æ, no signs of reduction of the bond strength has been observed. Additionally, to a reduced load-carrying capacity, ASR also opens up the concrete and consequently creates beneficial circumstances for corrosion and frost attacks.

This work is concerned with an attempt to clarify the role of cortisone in both the immune complement response and the progression of mouse leukemic tumor.