Renaissance arcades typology of royal architectural works in Bohemia around 1550 A.D. Kostelec nad Černými Lesy, Poděbrady, Brandýs nad Labem. Chateaux owned by ruler Ferdinand I. and situated outside of Prague served as a hunting residences. The buildings became king's property in the 1540th . Various reasons for rebuilding led to conversion from gothic castles to comfortable renaissance chateaux. Renaissance appearance of chateaux with an emphasis on the form of courtyards and arcade's corridors are the main topic of this thesis. Types of arcades are examined on three selected buildings, chateaux in Brandýs nad Labem, Kostelec nad Černými Lesy and Poděbrady. For entire overview of morphology used in renaissance arcades passages it is important to point out noble mansions in whose courts the arcade's corridors were used as well. This thesis specifically attempts to bring the analogy to the selected buildings in the Czech environment and also in foreign countries. The examples in the treatises and the inspiration for certain types of arcades from Antique to the Renaissance period are also reminded.

The aim of this project is a reconstruction of a former kindergarten building in Žďár nad Sázavou to a community center with several functional units. The existing building was bulit in 1960s. The site is located on the border of the old and new buildings – on one side, there is a Stalingrad housing estate from the 1950s built from brick houses in the socialist realism style. On the other side of the site are built new family houses, which are quickly expanding. The smaller part of the object is still used, the rest is abandoned. The house is surrounded by a large garden. The garden is defined by a fence and trees in a way, which creates a barrier, so the building does not communicate with surroundings in any way. The aim of the project is to create a centre of Stalingrad connected with surroundings and creating a meeting place for communities living in the area by using building interventions.

The work deals with the reconstruction and outbuilding of the community center in Zdar nad Sazavou. The existing house was built in the 70s last century. The building served as a day nursery in the past and then as a branch office of primary school. In this time is a part of the building used by the Club in 9 and Day of center for children and youth, part of the building is unused. The building is located in the centre of inaccessible and unkempt gardens and do not communicate with the surrounding. The aim of the diploma work is to change this situation. Using the construction impacts to create from the building a home and garden, which will be open to the surrounding and will be to interact with it. It using the reconstructions and outbuildings, which will respect the original DNA of the house.

CD38 is a transmembrane glycoprotein with both ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase activities; it is also known as a cell surface receptor. CD38 utilizes NAD(P) as a substrate to produce the second messengers, Nicotinic acid adenine dinucleotide phosphate (NAADP) and Cyclic adenosine diphosphate ribose (cADPR). CD38 has been implicated in several diseases. For instance, in chronic lymphocytic leukemia (CLL), it is known as a poor prognostic marker and as a disease modifier. Also, abundant data are available on the receptor functions of CD38 in CLL. However, the aim of the work described in this thesis was to investigate the enzymatic functions of CD38 in leukemia. The work also addresses the question of why CD38+ subset leukemia patients are characterised by poor outcome. It has been postulated that CD38 is the major NADase in cells, and that knocking it down increases NAD levels significantly. Thus, it was hypothesized that NAD levels might be depleted and result in detrimental consequences on cell physiology when CD38 is significantly expressed. Also, it was suggested that a similar linkage might be also present in leukemia, contributing to poor outcome. To test this hypothesis, a human leukaemia cell line (HL60) was used as a convenient model that differentiates into CD38+ cells when stimulated using all-trans retinoic acid (ATRA). It is shown that CD38 is expressed extracellularly and intracellularly in the differentiated cells, as evaluated by qPCR, FACS, Western blotting and the NGD cyclization assay. However, one of the major consequences of the early expression of CD38 (at 3 h) was a substantial depletion of intracellular NAD+ levels that was apparent by 4 h after treatment with ATRA. These novel data suggest a major role for CD38 as a main regulator of NAD during the differentiation. The main role of CD38 in degradation of NAD was confirmed by using a CD38 inhibitor (kuromanin). Interestingly, the drop in NAD+ levels during the differentiation was reversed after treatment with kuromanin. Furthermore, the CD38 homologue, CD157, and other NAD-consuming enzymes (PARP and SIRT) were all investigated, and it was found that there are no substantial roles of all these enzymes on the NAD+ degradation during the differentiation. In contrast, qPCR results for NAD-biosynthesis enzymes during the differentiation process showed a significant rise in indolamine 2,3-dioxygenase (IDO) mRNA expression, with lesser increases in nicotinamide nucleotide adenyltransferase (NMNAT) and nicotinamide phosphoribosyl transferase (NAMPT) mRNA levels. The consequences of low NAD levels on cell metabolism were also assessed; the results show a reduction in lactate production and glutathione levels with an elevation of TBARS levels. However, the NAD+:NADH ratio remained relatively constant. Moreover, the effects of low NAD levels on DNA repair and cell death were also investigated in response to DNA damage caused by UVB. Preliminary findings show that, in CD38+ cells, there is a resistance to apoptotic cell death. Additionally, CD38 expression was also investigated in leukaemia cells, and was found to be regulated in response to hypoxic environment, or the change in NAD+ levels following FK866, kuromanin and NAD+ application. Altogether, these studies raise the possibility that the impact of CD38 enzymatic function on NAD levels and the negative consequences on NAD(P)-dependent processes might play an important role in the poor prognosis in CD38+ leukemia patients.

No description available.

Haemophilus influenzae ist ein fakultativ anaerobes, Gram-negatives Bakterium aus der Familie der Pasteurellacaea. Das physiologische Merkmal von H. influenzae ist die essentielle, aber defiziente Hämin- und Nikotinamid-Adenin-Dinukleotid (NAD+) Biosynthese. Während Hämin für aerobes Wachstum benötigt wird, ist NAD+ sowohl für aerobes, als auch für anaerobes Wachstum essentiell. Als NAD+-abhängiger Organismus fehlen H. influenzae die meisten Enzyme für die NAD+-Biosynthese. Daher kann dieses Bakterium nur eine begrenzte Anzahl an Vorläufermolekülen, wie NAD+(P), Nikotinamid-Mono-Nukleotid (NMN) und Nikotinamid-Ribosid (NR) aus der Umwelt zur NAD+-Synthese nutzen. Andere NAD+-unabhängige Pasteurellacaea-Spezies, wie Haemophilus ducreyi, Pasteurella multocida und Actinobacillus actinomycetemcomitans, können auch kein NAD+ aus der de novo Biosynthese bereitstellen, diese Arten können aber zusätzlich auf Nikotinamid (NAm) wachsen. Die Erforschung des NAD+-Aufnahmesystems kann für die Entwicklung antimikrobieller Therapeutika von großem Interesse sein. Von unserer Arbeitsgruppe wurde die NAD+-Aufnahmeroute aufgeklärt; so werden NAD+(P) und NMN von e(P4) und NadN zu NR degradiert, nachdem NAD+ und NMN durch OmpP2 in das Periplasma gelangt sind. NR wird schließlich, als einziges Substrat, durch einen putativen Transporter in das Zytoplasma aufgenommen. In dieser Arbeit wurde der putative NR-Transporter als das hypothetische Gen HI1077.1 im Genom von H. influenzae identifiziert, welches nur 13,8% Identität zu Escherichia coli pnuC und 43,6% Identität zu P. multocida pnuC aufwies. Es konnten zwei Sequenzierungsfehler im original-annotierten Genom von H. influenzae gefunden werden, die zu einer Leserasterverschiebung geführt haben. HI1077.1 wurde zu pnuCHi reannotiert und weist nun eine 19,7% Identität zu pnuCEc und 71,4% Identität zu pnuCPm auf. Es wurde eine HI1077.1 Mutante konstruiert, die ein Wachstumsdefizit auf BHI-Platten bis zu einer NAD+-Konzentration von 15 mM bzw. unterhalb einer NR-Konzentration von 0,1 mM zeigte. Im Transport-Assay transportierte die HI1077.1 Mutante nur noch etwa 1% des eingesetzten NAD+- bzw. NR-Labels. Im Rattenversuch konnte gezeigt werden, dass das in vitro nicht essentielle pnuC in vivo sehr wohl essentiell ist. Die HI1077.1 Mutante verursachte, im Gegensatz zum Wildtyp und zur pnuC-Komplementante keine Bakteriämie mehr. Bei einer Komplementation mit NadV, kodierend für eine Nikotinamid-Phosphoribosyltransferase von H. ducreyi, wurde ebenfalls eine mit dem Wildtyp vergleichende Bakteriämie verursacht. Da bisher noch keine H. influenzae Isolate bekannt sind, die nadV besitzen, würde sich der hier identifizierte NR-Transporter von H. influenzae gut als antimikrobielles Ziel eignen. Die Protein-Topologie von PnuC wurde hinsichtlich der Membranlokalisation analysiert und PnuC konnte als ein Transmembranprotein bestätigt werden, das in der cytosolischen Membran lokalisiert ist. Durch PhoA und LacZ Analysen konnte die Topologie von PnuC aufgeklärt werden. Demnach besitzt PnuC acht Transmembrandomänen (TMD), wobei die N- und C-Termini im Cytosol lokalisiert sind. Die Analyse der strukturellen Funktion ergab, dass PnuC nur eine Unterbrechung der sechs letzten C-terminalen Aminosäuren (AS) toleriert, während die Proteinfunktion durch einen fusionierten C- und N-terminaler His-Tag nur mäsig beeinflusst wird. Des Weiteren wurde die Substratspezifität von PnuC untersucht. Dabei zeigte sich, dass der lange geglaubte NMN-Transporter von E. coli ebenfalls als NR-Transporter fungiert. Die Substratspezifität wurde auch bei A. actinomycetemcomitans und P. multocida untersucht. Es konnte festgestellt werden, dass A. actinomycetemcomitans und P. multocida ebenfalls als einziges Substrat NR transportieren können, aber im Gegensatz zu H. influenzae NAD+ und NMN nicht als NR-Quellen verwerten können, was wahrscheinlich auf das Fehlen von e(P4) und NadN zurückzuführen ist. Zusätzlich wurde in dieser Arbeit das Gen HI0308 untersucht. Um dem Aspekt der Energetisierung des PnuC-Transporters näher zu kommen, sind die Gencluster HI1078-HI1080 und HI0164-HI0171 in die Untersuchung mit einbezogen worden. Die Na+-NQR-Oxidoreduktase wurde im Hinblick ihrer Dehydrogenase-Aktivität genauer charakterisiert. Die Suche nach möglichen Inhibitoren von PnuC führte zu 3-Aminopyridin-Analoga. Durch eine Mutantenanalyse konnte gezeigt werden, dass 3-AADP, 3-AAD und 3-AmPR der selben Aufnahmeroute folgen wie NADP+, NAD+ und NR, und via PnuC in die Zelle aufgenommen werden. Darüber hinaus konnten wir nachweisen, dass NadR aus 3-AmPR und ATP das 3-AAD synthetisiert, welches als kompetitiver Inhibitor mit NAD+ in den zellulären Redoxreaktionen konkurriert und dadurch den Stoffwechsel hemmt. Des Weiteren wurden mehrere spontan 3-Aminopyridin-resistente H. influenzae Mutanten isoliert. / Haemophilus influenzae is a facultativ anaerobic, Gram-negative bacterium of the family of Pasteurellaceae. A physiological characteristic of H. influenzae is its essential but deficient hemin and NAD+ biosynthesis. While hemin is required for aerobic growth, NAD+ is essential for anaerobic and aerobic growth. As a NAD+ dependent organism H. influenzae lacks the enzymes for NAD+ biosynthesis and that is why it can only incorporate a limited number of precursor molecules, such as NAD+(P), NMN, and NR. Other NAD+-independent Pasteurellaceae such as Haemophilus ducreyi, Pasteurella multocida und Actinobacillus actinomycetemcomitans can grow on NAm, additionally to NR. Hence, the investigation of the NAD+ uptake system harbors some potential to be utilized for the development of antimicrobial therapeutics. Our laboratory has clarified the NAD+ uptake route; NAD+(P) and NMN are degraded by e(P4) and NadN to NR, after NAD+ and NMN reached the periplasm through OmpP2. Finally, NR is taken up in the cytoplasm through a putative transporter. In this study, the putative NR transporter was identified as the hypothetical gene HI1077.1 in the genome of H. influenzae, which showed only 13,8% identity to the pnuC of Escherichia coli and 43,6% identity to the pnuC of Pasteurella multocida. Two sequencing errors were found within the pnuC encoding region in the original annotated genome of H. influenzae, which produced frame shifts. After sequence correction HI1077.1 was re-annotated to pnuCHi now showing 19,7% identity to the pnuC of E. coli, and 71,4% identity to the pnuC of P. multocida. A knock-out mutation was constructed for HI1077.1, which showed a growth deficiency on BHI-plates, if supplemented with NAD+ on concentrations below 15 mM, and NR concentrations below 0,1 mM. In transport assay the HI1077.1 mutant only transported 1% of the provided NAD+ and NR label, respectively. In the rat model it could be shown that the pnuC knock-out mutation was essential. There in respect to bacterial growth in the animal model, the HI1077.1 mutant was not able to cause a bacteremia in contrast to the wild type and the pnuC-complemented mutant. When the mutant was complemented with nadV, encoding a nicotinamide phosphoribosyltransferase of H. ducreyi, bacteremia was maintained that was comparable with the one produced by the wild type. Since, so far there are no H. influenzae isolates known, which carry the nadV gene, hence the NR transporter serves for essential gene function and could be further investigated as a potential antimicrobial target. The topology of PnuC was also analysed. The localisation of PnuC was confirmed at the cytosolic membrane. The membrane topology of PnuC was resolved with the investigation of PhoA and LacZ insertion analysis. According to this, PnuC possesses eight transmembrane domains (TMD) with the N-and C-termini localized within the cytosole. The research of the structural function indicates that PnuC tolerates a truncation of the last six C-terminal amino acids, while the function of the protein was only moderately affected by fusions of a C- or N-terminal His-Tags. In addition, the substrate specificity of PnuC was investigated. Thereby, it was demonstrated that the NMN transporter in E. coli in fact acts as an NR transporter. The substrate specificity of A. pleuropneumoniae and P. multocida was also under examination. It was established that A. pleuropneumoniae and P. multocida can transport NR as the unique substrate. However, NAD+ and NMN could not serve as Substrats to deliver NR, raising the question, whether e(P4) or/and NadN function is present in these bacteria. Additionally, in this study the gene HI0308 was investigated. To approach the aspect of energizing the PnuC transporter and the residual low affinity NR uptake the gene cluster HI1078-HI1080 and HI0164-HI0171 were characterised. The Na+-NQR oxidoreductase was more precisely characterised with regard to the activity of the dehydrogenase. The search for putative inhibitors of PnuC yielded in the analysis of 3-aminopyridine analogs. In this work it was demonstrated, that 3-AADP, 3-AAD and 3-AmPR follow the same uptake route as NADP+, NAD+ and NR, and are taken up via PnuC into the cell. Furthermore, we were able to show that 3-AAD can be synthesized from 3-AmPR and ATP. 3-AAD most likely competes versus NAD+ in metabolic relevant redox reactions and thereby inhibits the biosynthesis rate of the cells. In addition, several 3-aminopyridin resistant H. influenzae mutants were isolated.

I Zusammenfassung Haemophilus influenzae, ein Gram-negatives, Bakterium der Familie Pasteurellaceae, kann beim Menschen eine Vielzahl an Erkrankungen auslösen: Die bekapselte Stämme, v. a. mit Typ b Kapsel können Cellulitis, septische Arthritis, Epiglottitis und Meningitis verursachen. Die nicht-bekapselte Stämme können Otitis media, Sinusitis, Pneumonie und in selteneren Fällen Bakterämie verursachen. Ein besonderes Merkmal des Metabolismus von H. influenzae ist dessen Unfähigkeit Nikotinamid-Adenin-Dinukleotid (NAD+) de novo zu synthetisieren. Daher sind die Enzyme bzw. Transporter, die an NAD+ Aufnahme und Resynthese beteiligt sind, als putative antimikrobielle Ziele von Interesse. In unserer Arbeitsgruppe konnte gezeigt werden, dass NAD+ zu Nikotinamidribosyl degradiert werden muss, bevor es in die Zelle aufgenommen werden kann. Auch Proteine, die an der Degradation des exogenen NAD+ zu Nikotinamidribosyl und dessen anschließender Aufnahme in die Zelle verantwortlich sind, konnten identifiziert und charakterisiert werden. Wie Nikotinamidribosyl im Cytoplasma wiederum zu NAD+ synthetisiert wird, ist auch erst kürzlich geklärt worden: für NadR konnte sowohl eine Ribosyl-Nukleotid-Kinase (RNK) Aktivität als auch eine Nikotinamid-Mononukleotid-Adenylyltransferase (NMNAT) Aktivität in vitro gezeigt werden. Die Kristallstruktur von hiNadR im Komplex mit NAD+ wurde auch aufgeklärt. In dieser Arbeit sollte NadR, insbesondere dessen RNK Domäne, in vivo und in vitro näher charakterisiert werden. Um zu untersuchen, ob beide Domänen in vivo essentiell sind, wurden Deletionsmutanten erzeugt, bei welchen die komplette bzw. der C-terminale Teil der RNK Domäne fehlten. Diese Deletionen konnten im nadV+ Hintergrund erzeugt werden. Die Deletionen konnten in H. influenzae nur zusammen mit dem nadV-Gen transferiert werden oder alternativ nur in die Zellen, die mit pNadRKan Plasmid transformiert wurden. Dies verdeutlicht, dass nicht nur die NMNAT Domäne sondern auch die RNK Domäne bzw. sogar nur wenige C-terminal fehlende Aminosäuren des NadR Proteins essentiell für die Lebensfähigkeit von H. influenzae sind. Gleichzeitig zeigen diese Experimente, dass die RNK-Domäne in Anwesenheit von NadV redundant ist. Ein weiterer Phänotyp der RNK-Deletionsmutante zeigte sich beim Nikotinamidribosyl-Transport. Im Gegensatz zum Wt, welcher ca. 60-80% des 14C-Nikotinamidribosyls aufnahm, konnte für die RNK-Deletionsmutante nur 2-5% Aufnahme gemessen werden. Dies konnte durch das pNadRKan Plasmid komplementiert werden. Weiterhin wurde festgestellt, dass spontan Aminopyridin-resistente H. influenzae Zellen Mutationen im nadR Gen haben, insbesondere im Walker A-Motif (P-Loop) der RNK Domäne. Zusätzlich konnte in dieser Arbeit gezeigt werden, dass NadR aus Aminopyridin und ATP Aminopyridin-Adenin-Dinukleotid synthetisieren kann. Somit konnte gezeigt werden, dass die wachstumshemmende Wirkung eigentlich durch das aus Aminopyridin synthetisierte Aminopyridin-Adenin-Dinukleotid entsteht, welches NAD+ in Redox-Reaktionen verdrängt, wodurch es letztendlich zum Stillstand des Metabolimus kommt. Durch Einführen von gezielten AS-Substitutionen im Walker A und B Motif und in der LID-Domäne von NadR, konnten einige Aminosäuren identifiziert werden, welche essentiell für die Aktivität der RNK Domäne sind. Alle Aminosäuren-Substitutionen führten zum Verlust der RNK Aktivität, die NMNAT Aktivität jedoch war nicht beeinträchtigt. Desweiteren wurden diese NadR Punktmutanten in vivo untersucht. Für alle konnte eine signifikante Defizienz in der Nikotinamidribosyl-Aufnahme beobachtet werden, die gemessene Aufnahme lag im Bereich der RNK-Deletionsmutante. Dadurch konnte eine direkte Korrelation zwischen der RNK Aktivität und der Nikotinamidribosyl-Aufnahme gezeigt werden. In weiteren in vitro Experimenten konnte für NadR eine Feedback-Inhibition durch das NAD+ gezeigt werden, wobei NAD+ in erster Linie die RNK Domäne von NadR inhibiert. Eine graduelle Erhöhung der NAD+ Konzentration führte in den in vitro Assays zu einer graduellen Abnahme der RNK. Bei der NMNAT Aktivität jedoch zeigte sich keine signifikante Inhibition in Anwesenheit von NAD+. Begleitende in vivo Experimente, zeigten eine 2/3 Reduktion der Nikotinamidribosyl-Aufnahme bei den Zellen, die mit NAD+ inkubiert wurden, d. h. höhere intrazelluläre NAD+ Konzentration hatten. Für die genauere Analyse der Feedback-Inhibition durch NAD+ wurden weitere Punktmutanen hergestellt. Bei zwei der Punktmutanten wurde eine Beeinträchtigung der NadR-Aktivität beobachtet, daher wurden diese Punktmutanten von weiteren Analysen im Bezug auf NAD+-Feedback Inhibition ausgeschlossen. Eine Mutante (NadRW256F) jedoch, zeigte ähnliche Aktivität wie das Wt-NadR. In Anwesenheit von NAD+ wurde die RNK Aktivität dieser Punktmutante, im Gegensatz zum Wt-Protein, kaum gehemmt. Dadurch konnte W256 als eine der Aminosäuren identifiziert werden, die an der Vermittlung der NAD+-bedingten Inhibition der RNK-Domäne beteiligt ist. / I Summary Haemophilus influenzae, a gram-negative human pathogen belonging to a family of Pasteurellaceae is a causative agent of several distinct diseases. Whereas capsulated strains, particulary those with tybe b capsule can cause severe invasive infections such as cellulitis, septic arthrithis, epiglottitis and meningitis, non-capsulated strains generally tend to cause localized disease including otitis media, sinusitis, pneumonia and in rare cases bacteremia. The inability to synthesize NAD+ de novo is one of the hallmarks of H. influenzae metabolism, therefore proteins involved in NAD+ uptake and utilization respresent interesting putative targets for development of novel antimicrobial treatment. In our lab we were able to show, that prior to uptake, NAD+ has to be degraded to NR. Several proteins involved in NAD+ degradation and NR uptake were identified and characterized: OmpP2 (an outer-membrane porin), e(P4) (a membrane-bound acid phosphoesterase), NadN (a periplasmatic nucleotidase) and PnuC (a nicotinamidribosyl transporter localized in inner membrane). Enzyme responsible for resynthesis of nicotinamidribosyl to NAD+ was recently found to be NadR: A bifunctional protein containing a nicotinamidribosyl kinase (RNK) and a nicotinamid mononucleotide adenylyltransferase (NMNAT) activity, both of which were confirmed in vitro. Also, the crystal structure of NadR complexed with NAD+ was recently resolved. The aim of this work was to characterize the in vivo function of NadR, particular interest was laid on the characterization of the nicotinamidribosyl kinase domain. To test if both domains of NadR are essential for survival, deletion mutants lacking the entire RNK domain and the C-terminal 58 amino acids were constructed. Initially, these mutants were made in a H. influenzae strain which contains a chromosomal copy of H. ducreyi nadV gene. In following transformation experiments, transfer of the RNK deletion mutants to H. influenzae strain was always accompanied with an nadV transfer as well. Only in strain containing pNadRKan plasmid, no nadV transfer along with RNK-deletions took place. Indirectly, this shows that not only the entire RNK domain is essential for H. influenzae, but also the last 58 amino acids as well. It also shows that in presence of NadV the RNK domain is redundant. RNK deletion mutant displayed a significant deficiency in nicotinamidribosyl transport as well: whereas the Wt strain can accumulate up to 80% of 14C labeled nicotinamidribosyl, RNK mutant is able to accumulate only 2-5%. Introduction of pNadRKan plasmid to RNK mutant restored transport efficiency to Wt level. Studies using spontanous 3-aminopyridine (3-AmPR) resistant H. influenzae isolates, revealed that almost all 3-AmPR resistant isolates have mutations in the nadR gene. A clustering of mutations in Walker A motif of the RNK domain could be observed. Further studies represented in this work, show that 3-AmPR can act as a subtrate for NadR, therefore in ATP consuming reactions aminopyridine-adenindinucleotide can be synthesized. Intracellular aminopyridine-adenindinucleotide replaces NAD+ in redox reactions, which ultimately leads to inhibition of cell metabolism, thereby explaining the mechanism of 3-AmPR based growth inhibition. Using site-directed mutagenesis to introduce amino acid substitutions in distinct parts of the NadR-RNK domain, active sites of the RNK domain were revealed and amino acids essential for the RNK activity were identified. These defined amino acid exchanges resulted in loss of the RNK activity in vitro, but had no effect on the NMNAT activity, which remained intact in these mutant variants of NadR. Following in vivo studies revealed that all mutant NadR proteins caused a severe nicotinamidribosyl uptake deficiency, similar to the one observed in the RNK deletion mutant. Therefore, a direct correlation between the RNK activity and nicotinamidribosyl uptake was shown. Further in vitro studies revealed a feedback inhibition of NadR by NAD+, especially for the RNK domain. In case of RNK domain a gradual increase of NAD+ concentration led to gradual decrease in RNK activity. In contrast, for NMNAT domain no significant inhibition in the presence of NAD+ was observed. Also, in in vivo experiments a 3 fold reduction of nicotinamidribosyl uptake rate was observed when intracellular NAD+ concentrations were higher. To adress the mechanism of NAD+ feedback inhibition, once again, distinct amino acid exchanges were introduced. In vitro, two of the mutant proteins were impaired in their activity, especially if lower protein contrations were used. Therefore, further test concerning inhibtion were not preformed with these mutants. However, a W256F protein displayed activity similar to that of the native protein and furthermore was not inhibited in presence of NAD+. This indicates an involvement of the amino acid W256 in mediating the NAD+ dependent feedback inhibition on NadR activity.

Tesis Magíster en Bioquímica, Área de Especialización en Bioquímica Toxicológica y Diagnóstico Molecular, y Memoria para optar al Título de Bioquímico / dinucleótido de nicotinamida y adenina (NAD+) es una coenzima con múltiples funciones. Participa en el metabolismo redox como molécula transportadora de electrones en procesos metabólicos, actúa como indicador del estado energético celular, y es también sustrato de numerosas enzimas implicadas en la desacetilación de reguladores transcripcionales y la movilización de Ca2+ intracelular, entre otros. Diversas investigaciones sugieren que la disminución de los niveles de NAD+ es un factor importante en la progresión de enfermedades cardiovasculares. En diferentes modelos de miocardiopatías se ha observado una disminución en la expresión de la enzima marcapaso de la síntesis de NAD+, Nampt. También se ha descrito que los niveles de NAD+ están disminuidos en condiciones de riesgo cardiovascular como son el envejecimiento, dislipidemia y diabetes mellitus tipo 2. El objetivo de este trabajo fue determinar los efectos de la disminución de NAD+ en el metabolismo y la capacidad adaptativa de los cardiomiocitos. Se utilizó el inhibidor de Nampt, FK866, para disminuir los niveles de NAD+ en cultivos primarios de cardiomiocitos de ratas neonatas. Se evaluó la viabilidad, el metabolismo mitocondrial y la respuesta adaptativa del cardiomiocito a estímulos de insulina, peróxido de hidrógeno (H2O2) y norepinefrina (NE). Los resultados mostraron que la disminución de NAD+ por FK866 redujo el metabolismo mitocondrial sin afectar la viabilidad de los cardiomiocitos. Además, disminuyó la fosforilación de Akt en respuesta a insulina, la sobrevida frente a H2O2 y previno el aumento del área celular y sarcomerización en respuesta a NE. Para evaluar la relación de causalidad entre la disminución de NAD+ y estos efectos, se rescataron los niveles de NAD+ mediante la administración de nicotinamida mononucleótido (NMN). Se observó la recuperación de los parámetros metabólicos y la sobrevida a H2O2, pero no se restableció la respuesta hipertrófica. En conclusión, este trabajo muestra que NAD+ es esencial para el metabolismo mitocondrial del cardiomiocito, y sugiere su participación en la vía de señalización de insulina y en la respuesta adaptativa a H2O2 y NE. / The nicotinamide adenine dinucleotide (NAD+) is a coenzyme with multiple functions. In redox metabolism participates as an electron carrier in metabolic processes, is a cellular energetic status indicator and it is also substrate of many enzymes involved in transcription factor deacetylation and Ca2+ mobilization, among others. Several reports suggest that NAD+ levels are an important factor in cardiovascular diseases progression. In different cardiomyopathy models the expression of the NAD+ synthesis rate-limiting enzyme, Nampt is diminished. Also NAD+ levels are decreased in cardiovascular risk conditions such as aging, dyslipidemia and type 2 diabetes mellitus. The aim of this work was to determine the effects of NAD+ decrease on cardiomyocytes’ metabolism and adaptive capability. The Nampt inhibitor, FK866, was used to reduce NAD+ levels in primary cultures of neonatal rat cardiomyocytes, and viability, mitochondrial metabolism, and cardiomyocytes adaptive response to insulin, hydrogen peroxide (H2O2) and norepinephrine (NE) stimuli were assessed. The results showed that the NAD+ reduction induced by FK866 decreased mitochondrial metabolism without affecting cardiomyocytes viability. Insulin-stimulated Akt phosphorylation and H2O2-survival were also diminished and cellular area increase and sarcomerization induced by NE was prevented. The causality between NAD+ decrease and those effects was assessed through NAD+ levels recovery by nicotinamide mononucleotide (NMN) administration. Both metabolism and H2O2-survival were reestablished, but not the hypertrophic response. In conclusion this work reveals that NAD+ is essential for cardiomyocyte mitochondrial metabolism, and suggest its participation on insulin signaling pathway and adaptive responses to H2O2 and NE. / Fondap; Fondecyt

Tesis presentada a la Universidad de Chile para optar al grado de Magíster en Bioquímica área de Especialización en Proteínas Recombinantes y Biotecnología y Memoria para optar al Título de Bioquímico / Los dinucleótidos de adenina nicotinamida (NAD(P)(H)) cumplen un rol fundamental como cofactores enzimáticos, principalmente en reacciones de oxido-reducción. Sus concentraciones intracelulares determinan el estado fisiológico celular, en especial la razón NAD(P)H/NAD(P)+, por lo que es necesario tener métodos que permitan una cuantificación confiable de estas moléculas. Los métodos in vitro e in vivo comúnmente utilizados no permiten determinar el estado redox intracelular con precisión dadas las dificultades analíticas que poseen. El diseño de Sensores Fluorescentes Codificados Genéticamente (GEFS, por su sigla en inglés) ayuda a superar esas dificultades, ya que, estos biosensores permiten la cuantificación in vivo y en tiempo real de moléculas específicas, sin dañar las células estudiadas. Estos sensores se diseñan a partir de la fusión de una proteína fluorescente permutada circularmente con un dominio sensor proteico capaz de generar un cambio conformacional en respuesta a la unión de un ligando específico. Utilizando esta estrategia, muchos GEFS han sido diseñados para la cuantificación in vivo de dinucleótidos. Entre los sensores de dinucleótidos publicados a la fecha de inicio de esta tesis, el sensor Peredox era el único GEFS capaz de detectar la razón NADH/NAD+ intracelular. Peredox utiliza como dominio sensor al represor transcripcional sensible al estado redox T-Rex del organismo Thermus aquaticus. Aunque T-Rex es capaz de unir tanto NADH como NAD+, sólo la unión del dinucleótido reducido induce un cambio conformacional de una forma abierta a una cerrada. Este fenómeno permite a Peredox detectar la razón NADH/NAD+. Usando Peredox y la información estructural de T-Rex como punto de partida, el objetivo de esta tesis fue estudiar los determinantes estructurales de especificidad de dinucleótido con el fin de avanzar hacia la generación de un GEFS capaz de detectar la razón NADPH/NADP+, del cual no hay sensores diseñados a la fecha. Para esto se utilizaron estrategias de Diseño Racional mediante aproximaciones in silico e in vitro. Se determinó experimentalmente que el loop β4-β5 de T-Rex contiene determinantes estructurales de la especificidad por dinucleótido. Mediante análisis de Potenciales Estadísticos, comparación de motivos de especificidad basados en homología y simulaciones de Dinámica Molecular, se determinó los residuos clave en la especificidad por NAD(H) y las mutaciones necesarias para obtener una variante NADPH preferente. Se evaluó el efecto de estas mutaciones en la especificidad por NAD(P)H a través de ensayos in vitro de fluorescencia, obteniéndose un sensor preferente por NADPH. Sin embargo, el sensor no presentó un mecanismo de unión mutuamente excluyente a NADPH y NADP+, condición sine qua non para que un sensor de cuenta de la razón NADPH/NADP+ / Nicotinamide adenine dinucleotides (NAD(P)(H)) play a fundamental role as enzymatic cofactors, mostly on oxidation-reduction reactions. The intracellular concentrations of these dinucleotides determine the cellular physiological state, especially the NAD(P)H/NAD(P)+ ratio, so it is necessary to have methods that allow a reliable quantification of these molecules. The in vitro and in vivo methods commonly used do not allow to determine the intracellular redox state with accuracy, given the analytical difficulties they show. The design of Genetically Encoded Fluorescent Sensors (GEFS) aids to overcome these difficulties, since they can perform real-time in vivo detection of specific molecules, without damaging the cells studied. These sensors are designed from the fusion of a circularly permuted fluorescent protein with a protein sensor domain capable of generating a conformational change in response to the binding of a specific ligand. Using this strategy, many GEFS have been designed for in vivo quantification of dinucleotides. Among the dinucleotide sensors published at the start date of this thesis, the sensor Peredox was the only GEFS capable of detecting intracellular NADH/NAD+ ratio. Peredox uses the redox-sensing transcriptional repressor T-Rex, from Thermus aquaticus, as a sensor domain. Although T-Rex is capable of binding both NADH and NAD+, only the binding of the reduced dinucleotide induces a conformational change from an open to a closed form. This phenomenon allows Peredox to detect the NADH/NAD+ ratio. Using Peredox and the structural information of T-Rex as a starting point, the goal of this thesis was the study of the structural determinants of dinucleotide specificity, with aim to achieve to a GEFS capable of detecting the NADPH/NADP+ ratio. There is no sensor designed for this parameter to date. To achieve this goal, we used Rational Design strategies, through in silico and in vivo aproximations. We determined experimentally that β4-β5 loop of T-Rex contains structural determinants of dinucleotide specificity. Through statistical potential analysis, homology-guided comparison of specificity motifs and Molecular Dynamics simulations, a triple mutant of T-Rex was proposed. The effect of these mutations on the specificity for NAD(P)H was evaluated through in vitro fluorescence assays, obtaining a Peredox variant with NADPH preference. However, the sensor did not show a mutually-exclusive binding fashion of NADPH and NADP+, a sine qua non condition for a sensor of the NADPH/NADP+ ratio / Fondecyt

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.