@phdthesis{Kilic2002,
  author    = {Kilic, Mehtap},
  title     = {Formation of caspase activating complexes and activation of caspase-12 during apoptosis in AKR-2B mouse fibroblasts},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2002},
  abstract  = {Der Zelltod kann in Dichte-arretierten AKR-2B Mausfibroblasten durch Entzug des Serums oder Behandlung mit Anisomycin induziert werden. Der Zelltod zeigt zwar die f{\"u}r die Apoptose typischen morphologischen Ver{\"a}nderungen der Zelle wie Zellfragmentierung und Chromatinkondensation jedoch fehlen andere apoptotische Charakteristika, wie die oligonukleosomale Fragmentierung der DNA oder {\"A}nderung des Membranpotentials der Mitochondria. W{\"a}hrend der Apoptose wurde eine beachtliche DEVDase Aktivit{\"a}t nach- gewiesen, die einem einzelnen Enzym zugeordnet werden konnte. Dieses Enzym hatte typische Eigenschaften von Effektor-Caspasen, wie die der Caspase-3, besaß jedoch einen ungew{\"o}hnlich hohen KM Wert von 100 µM, und die Molmasse der großen Untereinheit betrug 19 kDa anstelle der erwarteten 17 kDa. Mit Hilfe des rekombinanten mCaspase-3 Proteins wurde dieses Enzym in der vorliegenden Untersuchung als Caspase-3 identifiziert. Die N-terminale Sequenzierung des mCaspase-3 Proteins ergab, daß sich die Spaltstelle seiner Prodom{\"a}ne von der des humanen homologen Proteins unterschied (Asp-9 von Asp-28). Somit betrug die Molmasse der großen Untereinheit der aktiven Caspase-3 19 kDa. Dar{\"u}berhinaus stimmte der KM-Wert der rekombinanter mCaspase-3 von ~100 µM mit der {\"u}berein, die in Zellextrakten bestimmt wurde. Die Affinit{\"a}tmarkierung in Kombination mit einer 2D-Gelelektrophorese best{\"a}tigte, daß tats{\"a}chlich Caspase-3 als Haupt-Effektor-Caspase in AKR-2B-Zellen w{\"a}hrend der Apoptose aktiviert wird. Im Folgenden wurde untersucht, welcher Signalwege in AKR-2B-Zellen, denen Serum aus dem N{\"a}hrmedia entzogen wurden war oder die mit Anisomycin behandelt worden waren, zur Aktivierung der Caspase-3 f{\"u}hrt. Da eine Beteiligunug des Rezeptor-vermittelte Signalweges bereits zuvor ausgeschlossen worden war,wurde {\"u}berpr{\"u}ft, ob der mitochondrial vermittelte Signalweg bei der Aktivierung von Caspase-3 ein Rolle spielt. Gelfiltrations- experimente ergaben, daß Caspase-3 als freies Enzym und nur in geringen Mengen innerhalb unterschiedlich große Komplexe der Molmassen 600 kDa und 250 kDa eluiert wird. Obwohl die apparenten Molmassen der Caspase-3-haltigen Komplexe mit k{\"u}rzlich ver{\"o}ffentlichten Daten {\"u}bereinstimmten, enthielten sie weder Apaf-1 noch Caspase-9. Dies deutet daraufhin, daß der mitochondrial-vermittelte Signalweg ebenfalls nicht an der Caspase-3 Aktivierung beteiligt ist. Dar{\"u}berhinaus wurde ein neuer Caspase-6 enthaltender Komplex (450 kDa) nach Serumentzug in AKR-2B-Zellen gefunden, der sich deutlich von Caspase-3 haltigen Komplexen unterscheidet. Caspase-3 ist f{\"u}r die meisten morphologische Ver{\"a}nderungen w{\"a}hrend der Apoptose verantwortlich. Wahrend der Apoptose in der AKR-2B-Zellen zwar Caspase-3 als Haupt-Effektor-Caspase identifiziert worden war, jedoch keine intranukleosomale Fragmentierung nachgeweisen werden konnen, da bei wurde die intrazellul{\"a}re Lokalisation der Caspase-3 untersucht. Durch {\"U}berexpression eines Caspase-3-GFP Fusionskonstruktes in AKR-2B Zellen wurde die Procaspase-3 im Cytoplasma lokalisiert, w{\"a}hrend die aktive Caspase-3 vorwiegend in membranumh{\"u}llten Vesikeln und zum Teil im Cytoplasma aktiviert wurde. Ebenfalls wurde eine m{\"o}gliche Beteiligung von Caspase-12 und eine ER-Streß vermittelte Signalleitung an der Apoptose in AKR-2B-Zellen untersucht. Kinetische Untersuchungen zeigten, daß Caspase-12 im Fall von Serumentzug oder Behandlung mit Anisomycin zeitgleich mit Caspase-3 aktiviert wurde, was zur Bildung von zwei Spaltprodukte der Molmassen 47 kDa und 35 kDa f{\"u}hrte. Gelfiltrationsexperimente ergaben, daß Caspase-12 als freies Enzym w{\"a}hrend der Apoptose auftritt. Bis zu diesem Zeitpunkt wurde in allen Publikationen beschrieben, daß Caspase-12 in Folge von ER-Streß aktiviert wird. Nach Serumentzug oder Zugabe von Anisomycin zu AKR-2B-Zellen wurde jedoch kein Anstieg der Synthese des Chaperon-Proteins Grp78, einem Marker f{\"u}r ER-Streß, beobachtet. Dies zeigt, daß beide Behandlungen nicht zur ER-Streß f{\"u}hren. Im Gegensatz dazu erzeugten bekannte Indikatoren von ER Streß wie Thapsigargin und A23187 (ionophor) ER-Streß in AKR-2B-Zellen, was zu unspezifischem Abbau der Caspase-12 f{\"u}hrte. Darum ist es unwahrscheinlich, daß in AKR-2B- Zellen Caspase-12 bei ER-Streß aktiviert wird. Zusammenfassend zeigten diese Daten, daß Caspase-12 in AKR-2B-Zellen {\"u}ber Signalwegen aktiviert wird, die ER stress un- abh{\"a}ngig sind und zeigen daß Caspase-3 bei der Aktivierung von Caspase-12 beteiligt ist somit liefern die vorliegenden Untersuchungen einen Hinweis darauf daß neben den klassischen Signalwege weitere Signalwege existieren, die zur Apoptose f{\"u}hrten.},
  subject      = {Apoptosis},
  language  = {en}
}
@phdthesis{Pavel2003,
  author    = {Pavel, Ioana-Emilia},
  title     = {Vibrational spectroscopy and density functional theory calculations, a powerful approach for the characterization of pharmaceuticals and new organometallic complexes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7186},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {In the current work, several well-known pharmaceuticals (1,4-dihydrazinophthalazine sulfate, caffeine, and papaverine hydrochloride) and new organometallic compounds (nickel(II) cupferronato complexes NiL2An, L = PhN2O2-, n = 1, A = o-phenanthroline (1), o,o'-bipyridine (2) and n = 2, A = H2O (3), o-NH2Py (4), o-C6H4(NH2)2 (5); silylene-bridged dinuclear iron complexes [Cp(OC)2Fe]2SiX2 (X = H (6), F (7), Cl (8), Br (9), I (10)); 3-silaoxetane 3,3-dimethyl-2,2,4,4-tetraphenyl-1-oxa-3-silacyclobutane (11) and 3-silathietane 3,3-dimethyl-2,2,4,4-tetraphenyl-1-sila-3-thiacyclobutane (12) compounds), which have successfully been characterized by using vibrational spectroscopy in conjunction with accurate density functional theory (DFT) calculations, are presented. The DFT computed molecular geometries of the species of interest reproduced the crystal structure data very well and in conjunction with IR and Raman measurements helped us to clarify the structures of the compounds, for which no experimental data were available; and this, especially for the new organometallic compounds, where the X-Ray analysis was limited by the non-availability of single crystals (3, 5, 10). Furthermore, a natural population analysis (NPA) and natural bond orbital (NBO) calculations together with a detailed analysis of the IR and Raman experimental as well as calculated spectra of the new organometallic compounds, allowed us to study some special bonding situations (1-12) or to monitor the structural changes observed with the change in temperature during the Raman experiments (11, 12). By combining these two methods (DFT and vibrational spectroscopy), the auspicious results obtained on the organometallic compounds 6-12 and overall in literature, made us confident of the power of theoretical calculations in aiding the interpretation of rich SERS spectra by solving some interesting issues. Consequently, the Raman and SERS spectra of well-known pharmaceuticals (1,4-dihydrazinophthalazine sulfate, caffeine, and papaverine hydrochloride) or new potentially biological active organometallic complexes (1-5), that were synthetized by our coworkers, were discussed with the assistance of the accurate results obtained from DFT calculations (structural parameters, harmonic vibrational wavenumbers, Raman scattering activities), and many previous incomplete assignments have been analyzed and improved. This allowed us to establish the vibrational behavior of these biological compounds near a biological artificial model at different pH values or concentrations (Ag substrate), taking into account that information about the species present under particular conditions could be of great importance for the interpretation of biochemical processes. The total electron density of molecules and the partial charges situated on selected atoms, which were determined theoretically by NPA, allowed us to establish the probability of different atoms acting as an adsorptive site for the metal surface. Moreover, a closer examination of the calculated orbitals of molecules brought further arguments on the presence or absence of the photoproducts at the Ag surface during the irradiation (1,4-dihydrazinophthalazine sulfate). Overall, the results provide a benchmark illustration of the virtues of DFT in aiding the interpretation of rich vibrational spectra attainable for larger polyatomic adsorbates by using SERS, as well as in furnishing detailed insight into the relation between the vibrational properties and the nature of the Ag substrate-adsorbate bonding. Therefore, we strongly believe that theoretical calculations will become a matter of rapidly growing scientific and practical interest in SERS.},
  subject      = {Arzneimittel},
  language  = {en}
}
@phdthesis{Drueppel2003,
  author    = {Dr{\"u}ppel, Kirsten},
  title     = {Petrogenesis of the Mesoproterozoic anorthosite, syenite and carbonatite suites of NW Namibia and their contribution to the metasomatic formation of the Swartbooisdrif sodalite deposits},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6987},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {During the Mesoproterozoic large volumes of magma were repeatedly emplaced within the basement of NW Namibia. Magmatic activity started with the intrusion of the anorthositic rocks of the Kunene Intrusive Complex (KIC) at 1,385-1,347 Ma. At its south-eastern margin the KIC was invaded by syenite dykes (1,380-1,340 Ma) and younger carbonatites (1,140-1,120 Ma) along ENE and SE trending faults. Older ferrocarbonatite intrusions, the 'carbonatitic breccia', frequently contain wallrock fragments, whereas subordinate ferrocarbonatite veins are almost xenolith-free. Metasomatic interaction between carbonatite-derived fluids and the neighbouring and incorporated anorthosites led to the formation of economically important sodalite deposits. Investigated anorthosite samples display the magmatic mineral assemblage of Pl (An37-75) ± Ol ± Opx ± Cpx + Ilm + Mag + Ap ± Zrn. Ilmenite and pyroxene are surrounded by narrow reaction rims of biotite and pargasite. During the subsolidus stage sporadic coronitic garnet-orthopyroxene-quartz assemblages were produced. Thermobarometry studies on amphiboles yield temperatures of 985-950°C whereas the chemical composition of coronitic garnet and orthopyroxene indicate a subsolidus re-equilibration of the KIC at conditions of 760 ± 100°C and 7.3 ± 1 kbar. In the syenites Kfs, Pl, Hbl and/or Cpx crystallized first, followed by a second generation of Kfs, Hbl, Fe-Ti oxides and Ttn. Crystallization of potassium feldspar occurred under temperatures of 890-790°C. For the crystallization of hastingsite pressures of 6.5 ± 0.6 kbar are obtained. In order to constrain the source rocks of the two suites, oxygen isotope analyses of feldspar as well as geochemical bulk rock analyses were carried out. In case of the anorthosites, the general geochemical characteristics are in excellent agreement with their derivation from fractionated basaltic liquids, with the d18O values (5.88 ± 0.19  per mille) proving their derivation from mantle-derived magmas. The results obtained for the felsic suite, provide evidence against consanguinity of the anorthosites and the syenites, i.e. (1) compositional gaps between the geochemical data of the two suites, (2) trace element data of the felsic suite points to a mixed crustal-mantle source, (3) syenites do not exhibit ubiquitous negative Eu-anomalies in their REE patterns, which would be expected from fractionation products of melts that previously formed plagioclase cumulates and (4) feldspar d18O values from the syenites fall in a range of 7.20-7.92  per mille, which, however, is about 1.6  per mille higher than the average d18O of the anorthosites. Conformably, the crustal-derived felsic and the mantle-derived anorthositic suite are suggested to be coeval but not consanguineous. Their spatial and temporal association can be accounted for, if the heat necessary for crustal melting is provided by the upwelling and emplacement of mantle-derived melts, parental to the anorthosites. In order to constrain the source of the 1,140-1,120 Ma carbonatites and to elucidate the fenitizing processes, which led to the formation of the sodalite, detailed mineralogical and geochemical investigations, stable isotope (C,O,S) analyses and fluid inclusion measurements (microthermometrical studies and synchrotron-micro-XRF analyses) have been combined. There is striking evidence that carbonatites of both generations are magmatic in origin. They occur as dykes with cross-cutting relationships and margins disturbed by fenitic aureoles, and contain abundant flow-oriented xenoliths. The mineral assemblage of both carbonatite generations of Ank + Cal + Ilm + Mag + Bt ± Ap ± pyrochlore ± sulphides in the main carbonatite body and Ank + Cal + Mag ± pyrochlore ± rutile in the ferrocarbonatite veins, their geochemical characteristics and the O and C isotope values of ankerite (8.91 to 9.73 and -6.73 to -6.98, respectively) again indicate igneous derivation, with the 18O values suggesting minor subsolidus alteration. NaCl-rich fluids, released from the carbonatite melt mainly caused the fenitization of both, the incorporated and the bordering anorthosite. This process is characterized by the progressive transformation of Ca-rich plagioclase into albite and sodalite. Applying conventional geothermobarometry combined with fluid-inclusion isochore data, it was possible to reconstruct the P-T conditions for the carbonatite emplacement and crystallization (1200-630°C, 4-5 kbar) and for several mineral-forming processes during metasomatism (e.g. formation of sodalite: 800-530°C). The composition and evolutionary trends of the fenitizing solution were estimated from both the sequence of metasomatic reactions within wallrock xenoliths in the carbonatitic breccia and fluid inclusion data. The fenitizing solutions responsible for the transformation of albite into sodalite can be characterised as of NaCl-rich aqueous brines (19-30 wt.\% NaCl eq.), that contained only minor amounts of Sr, Ba, Fe, Nb, and LREE.},
  subject      = {Namibia <Nordwest>},
  language  = {en}
}
@phdthesis{AbdelRahman2003,
  author    = {Abdel Rahman, Faisal Mirghani},
  title     = {Systematic analysis of genes expressed in the retinal pigment epithelium (RPE) and identification of candidates for genetic susceptibility to age-related macular degeneration (AMD)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7053},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {Age related macular degeneration (AMD) is the leading cause of visual impairment in the elderly and the major cause of blindness in the developed world. To date, the molecular mechanisms underlying the disease are not well understood although in recent years a primary involvement of the retinal pigment epithelium (RPE) has become evident. The aim of the present study is to systematically analyse genes which are differentially expressed in the RPE, and to assess their possible association with mechanisms and pathways likely to be related to retinal disease, in particular AMD. Towards this goal, 2379 expressed sequence tags (ESTs) were established from an inhouse generated RPE cDNA library. This library was constructed by using the suppression subtraction hybridization (SSH) technique which normalises redundant sequences and ensures enrichment of rare transcripts. In a first phase, 1002 ESTs were sequenced and subjected to comprehensive alignment with public nucleotide and protein databases. A search of the 1002 ESTs against the human genome draft sequence yielded 168 known genes, 51 predicted genes, 15 unknown transcripts and 41 clones with no significant similarity. Reverse Northern blot hybridization was performed for 318 EST clusters to identify abundantly expressed genes in the RPE and to prioritize subsequent analyses. Representative clones were spotted onto a nylon membrane and hybridized with cDNA probes of driver (heart and liver) and tester (RPE) used in the cDNA library construction. Subsequently, 107 EST clusters were subjected to Northern blot hybridizations. These analyses identified 7 RPE-specific, 3 retina-specific, 7 RPE/retina-specific, and 7 tissue restricted transcripts, while 29 EST clusters were ubiquitously expressed, and evaluation was not possible for another 54 EST clusters. Of the 24 transcripts with specific or restricted expression, 16 clones were selected for further characterization. The predicted gene MGC2477 and 2 novel isoforms of the human transient receptor potential cation channel, subfamily M, member 3 (TRPM3) were cloned and further described in detail. In addition, polymorphic variations for these 2 genes as well as for the human MT-Protocadherin gene were determined. For MGC2477, 15 single nucleotide polymorphisms (SNPs) were identified, with 13 having a frequency of the minor allele greater than 20\%. 10 of the 15 SNPs have not been reported in so far in public SNP repertoires. Partial assessment of the TRPM3 gene yielded 35 SNPs. Of these, 30 (85.7\%) were highly frequent (0.17-0.5\%), and 14 (40\%) were novel. The MT-Protocadherin gene revealed 35 SNPs, including 28 (80\%) with high frequency of the minor allele. 23 (65.7\%) were novel SNPs. These SNPs will be used to construct the most common haplotypes. These will be used in case/control association studies in 400 AMD patients and 200 ethnically and aged matched controls to assess a possible contribution of these genes in the etiology of AMD.},
  subject      = {Senile Makuladegeneration / Pigmentepithel / Genexpression},
  language  = {en}
}
@phdthesis{Nedvetsky2003,
  author    = {Nedvetsky, Pavel I.},
  title     = {Regulation of the nitric oxide receptor, soluble guanylyl cyclase},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7046},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {Soluble guanylyl cyclase (sGC) is the best established receptor for nitric oxide (NO) and regulates a great number of important physiological functions. Surprisingly, despite the wellappreciated roles of this enzyme in regulation of vascular tone, smooth muscle cell proliferation, platelet aggregation, renal sodium secretion, synaptic plasticity, and other functions, extremely little is known about the regulation of sGC activity and protein levels. To date, the only well-proven physiologically relevant sGC regulator is NO. In the present study, some additional possibilities for sGC regulation were shown. Firstly, we evaluated the ability of different NO donors to stimulate sGC. Significant differences in the sGC stimulation by SNP and DEA/NO were found. DEA/NO stimulated sGC much stronger than did SNP. Interestingly, no correlation between the sGC protein and maximal activity distribution was found in rat brain regions tested, suggesting the existence of some additional regulatory mechanisms for sGC. The failure of SNP to stimulate sGC maximally might be one of the reasons why the lack of correlation between the distribution of sGC activity and proteins in brain was not detected earlier. Prolonged exposure of endothelial cells to NO donors produced desensitization of the cGMP response. This desensitization cannot be explained by increased PDE activity, since PDE inhibitors were not able to prevent the NO donor-induced decrease of the maximal cGMP response in endothelial cells. The failure of SH-reducing agents to improve the cGMP response after its desensitization by NO suggests that a SH-independent mechanism mediates NO effects. Demonstration that the potency of the recently described activator of oxidized (heme-free) sGC, BAY58-2667, to stimulate sGC increases after prolonged exposure of the cells to an NO donor, DETA/NO, suggests that oxidation of heme may be a reason for NOinduced desensitization of sGC and decrease in sGC protein level. Indeed, the well-known heme-oxidizing agent ODQ produces a dramatic decrease in sGC protein levels in endothelial cells and BAY58-2667 prevents this effect. Although the mechanism of sGC activation and stabilization by BAY58-2667 is unknown, this substance is an interesting candidate to modulate sGC under conditions where sGC heme iron is oxidized. Very little is known about regulation of sGC by intracellular localization or translocation between different intracellular compartments. In the present study, an increase in sGC sensitivity to NO under membrane association was demonstrated. Treatment of isolated lung with VEGF markedly increased sGC in membrane fractions of endothelial cells. Failure of VEGF to stimulate sGC membrane association in cultured endothelial cells allows us to propose a complex mechanism of regulation of sGC membrane association and/or a transient character of sGC membrane attachment. A very likely mechanism for the attachment of sGC to membranes is via sGCinteracting proteins. These proteins may participate also in other aspects of sGC regulation. The role of the recently described sGC interaction partner, Hsp90, was investigated. Shortterm treatment of endothelial cells with an Hsp90 inhibitor does not affect NO donor or calcium ionophore-stimulated cGMP accumulation in the cells. However, inhibition of Hsp90 results in a rapid and dramatic decrease in sGC protein levels in endothelial cells. These effects were unrelated to changes in sGC transcription, since inhibition of transcription had much slower effect on sGC protein levels. In contrast, inhibitors of proteasomes abolished the reduction in sGC protein levels produced by an Hsp90 inhibitor, suggesting involvement of proteolytic degradation of sGC proteins during inhibition of Hsp90. All these data together suggest that Hsp90 is required to maintain mature sGC proteins. In conclusion, in the present study it was demonstrated that multiple mechanisms are involved in the regulation of sGC activity and its sensitivity to NO. Oxidation of sGC heme by NO seems to be one of the mechanisms for negative regulation of sGC in the presence of high or prolonged stimulation with NO. Another possible means of regulating sGC sensitivity to NO is via the intracellular translocation of the enzyme. It has been also demonstrated here that attachment of sGC to the membrane fraction results in an apparent increase in the enzyme sensitivity to NO. Additionally, Hsp90 was required to maintain sGC protein in endothelial and other cell types. However, we could not find any acute affect of Hsp90 on sGC activity, as reported recently. All these findings demonstrate that the regulation of sGC activity and protein level is a much more complex process than had been assumed earlier.},
  subject      = {Guanylatcyclase},
  language  = {en}
}
@phdthesis{FadlElMola2003,
  author    = {Fadl El Mola, Faisal Mohamed},
  title     = {Bioinformatic and molecular approaches for the analysis of the retinal pigment epithelium (RPE) transcriptome},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6877},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {There is substantial interest in the identification of genes underlying susceptibility to complex human diseases because of the potential utility of such genes in disease prediction and therapy. The complex age-related macular degeneration (AMD) is a prevalent cause of legal blindness in industrialized countries and predominantly affects the elderly population over 75 years of age. Although vision loss in AMD results from photoreceptor cell death in the central retina, the initial pathogenesis likely involves processes in the retinal pigment epithelium (RPE) (Liang and Godley, 2003). The goal of the current study was to identify and characterize genes specifically or abundantly expressed in the RPE in order to determine more comprehensively the transcriptome of the RPE. In addition, our aim was to assess the role of these genes in AMD pathogenesis. Towards this end, a bovine cDNA library enriched for RPE transcripts was constructed in-house using a PCR-based suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996, 1999), which normalizes for sequence abundance and achieves high enrichment for differentially expressed genes. CAP3 (Huang and Madan, 1999) was used to assemble the high quality sequences of all the 2379 ESTs into clusters or singletons. 1.2\% of the 2379 RPE-ESTs contains vector sequences and was excluded from further analysis. 5\% of the RPE-ESTs showed homology to multipe chromosomes and were not included in further assembly process. The rest of the ESTs (2245) were assembled into 175 contigs and 509 singletons, which revealed approximately 684 unique genes in the dataset. Out of the 684, 343 bovine RPE transcripts did not align to their human orthologues. A large fraction of clones were shown to include a considerable 3´untranslated regions of the gene that are not conserved between bovine and human. It is the coding regions that can be conserved between bovine and human and not the 3' UTR (Sharma et al., 2002). Therefore, more sequencing from the cDNA library with reclustering of those 343 ESTs together with continuous blasting might reveal their human orthologoues. To handle the large volume of data that the RPE cDNA library project has generated a highly efficient and user-friendly RDBMS was designed. Using RDBMS data storage can be managed efficiently and flexibly. The RDBMS allows displaying the results in query-based form and report format with additional annotations, links and search functions. Out of the 341 known and predicted genes identified in this study, 2 were further analyzed. The RPE or/and retina specificity of these two clones were further confirmed by RT-PCR analysis in adult human tissues. Construction of a single nucleotide polymphism (SNP) map was initiated as a first step in future case/control association studies. SNP genotyping was carried out for one of these two clones (RPE01-D2, now known as RDH12). 12 SNPs were identified from direct sequencing of the 23.4-kb region, of which 5 are of high frequency. In a next step, comparison of allele frequencies between AMD patients and healthy controls is required. Completion of the expression analysis for other predicted genes identified during this study is in progress using real time RT-PCR and will provide additional candidate genes for further analyses. This study is expected to contribute to our understanding of the genetic basis of RPE function and to clarify the role of the RPE-expressed genes in the predisposition to AMD. It may also help reveal the mechanisms and pathways that are involved in the development of AMD or other retinal dystrophies.},
  subject      = {Senile Makuladegeneration},
  language  = {en}
}
@phdthesis{KittisakSripha2003,
  author    = {Kittisak Sripha,},
  title     = {NOVEL HETEROCYCLIC RING SYSTEMS DERIVED FROM CARACURINE V AS LIGANDS FOR THE ALLOSTERIC SITE OF MUSCARINIC M 2 RECEPTORS},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6841},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {Die vorliegende Arbeit befasst sich mit dem Gebiet allosterischer Modulation des muscarinischen M2 Rezeptors. Allosterische Liganden beeinflussen das Bindungsverhalten eines orthosterischen Liganden (Agonisten oder Antagonisten) an die klassische Bindungsstelle des muscarinischen Rezeptors, indem sie seine Affinit{\"a}t entweder erh{\"o}hen(positive Kooperativit{\"a}t) oder erniedrigen (negative Kooperativit{\"a}t). Die allosterische Bindungsstelle befindet sich extrazellul{\"a}r am Eingang der Rezeptor-Bindungstasche. Sie ist weniger konserviert als die orthosterische Bindungsdom{\"a}ne, die tiefer im Rezeptorkanal zwischen den sieben transmembranalen Dom{\"a}nen lokalisiert ist. Demzufolge ist die Entwicklung subtyp-spezifischer allosterisch wirkenden Liganden leichter als subtypspezifischer Agonisten oder Antagonisten. Die Subtypselektivit{\"a}t kann dar{\"u}ber hinaus {\"u}ber unterschiedliche Kooperativit{\"a}ten zwischen dem orthosterischen und allosterischen Liganden an verschiedenen muscarinischen Subtypen erreicht werden. Ein am M1-Rezeptor mit Acetylcholin positiv kooperativer allosterer Modulator, der sich an anderen muscarinischen Subtypen neutral kooperativ verh{\"a}lt, k{\"o}nnte z.B. f{\"u}r die Therapie von Morbus Alzheimer eingesetzt werden. Bisquart{\"a}re Ammoniumsalze des Strychnos-Alkaloids Caracurin-V geh{\"o}ren zu den potentesten allosterischen M2-Liganden. Die relative Stellung der aromatischen Indolringe und der Abstand zwischen den positiv geladenen Stickstoffatomen (ca. 10) in dem sehr starren Caracurin-V-Ringsystem definieren den Pharmakophor f{\"u}r potente allosterische Modulatoren. Caracurin-V-Salze sind strukturell sehr verwandt mit den starken Muskelrelaxantien Toxiferin-I und Alcuronium und besitzen vermutlich selbst neuromuskul{\"a}r-blockierende Eigenschaften, was ihre Anwendung in der pharmakologischen Forschung einschr{\"a}nken w{\"u}rde. Reduktion des Caracurin-V-Ringsystems auf die wesentlichen Pharmakophorelemente k{\"o}nnte zu allosterisch wirksamen Verbindungen mit vernachl{\"a}ssigbarer muskelrelaxierender Wirkung f{\"u}hren. Ziel dieser Arbeit war die Synthese und pharmakologische Testung von Derivaten eines neuen, von Caracurin V abgeleiteten, heterocyclischen Ringsystems. Das neue gew{\"u}nscht 6,7,14,15-Tetrahydro[1,5]diazocino[1,2-a:6,5-a]-diindole-Ringsystem(6) wurde in einer intermolekularen N-Alkylierung von zwei Molek{\"u}len Bromethylindol 5 aufgebaut. Die Ausgangsverbindung 5 konnte aus dem Indolylessigs{\"a}uremethylester 3 durch Reduktion der Estergruppe zum Alkohol und anschließende Substitution durch Brom dargestellt werden. Der bekannte Ester 3 wurde ausgehend von Tryptamin erhalten. Die dreistufige Synthese umfasste N-Dibenzylierung, Einf{\"u}hrung der Malonestergruppe am C-2 von Indol und anschließende Demethoxycarbonylierung. Die Totalsynthese des neuen Pentacyclus ist im Schema 24 dargestellt. Die 3D-Struktur des neuen Ringger{\"u}stes konnte mit Hilfe von NMR-Spektroskopie und semiempirischen Rechnungen (AM1) aufgekl{\"a}rt werden. Verbindung 6 liegt in L{\"o}sung in einer verdrehten Wanne-Konformation mit unsymmetrisch angeordneten Seitenketten vor. Um den Einfluss der Seitenkettenl{\"a}nge des neuen Ringsystems auf die allosterische Wirksamkeit zu untersuchen, war es geplannt, die Ethylamin-Gruppen durch Methylamin-Einheiten zu ersetzen. Der entsprechende Syntheseplan bestand darin, das unsubstituierte Ringsystem in einer doppelten Mannich-Reaktion zu aminomethylieren. Der Ausgangsstoff f{\"u}r die Dimerisierung, Bromethylindol 32, wurde aus Indol-2-carbons{\"a}ure hergestellt. Die Synthese umfasste folgende Reaktionsschritte: Reduktion der Carboxylgruppe und Benzoylierung des resultierenden Alkohols, nucleophile Substitution mit Kaliumcyanid, alkalische Hydrolyse des Cyanids zu Indolacetessigs{\"a}ure, erneute Reduktion zum Alkohol und abschließende Substitution mit Brom. Da Dimerisierungsversuche von 32 nur zur Bildung des HBr-Eliminierungsproduktes 33 f{\"u}hrten, wurde das entsprechende Tosylat als Ausgangsstoff eingesetzt. {\"U}berraschenderweise entstand nicht das erwartete Diazocinodiindol-Ringger{\"u}st, sondern ausschließlich ein isomeres, noch nicht bekanntes 6,7,14,15-Tetrahydro-15aH-azocino[1,2-a:6,5-b]diindol-Ringsystem 35. Die Bildung des neuen unsymmetrischen Ringsystems ist auf den ambidenten Charakter des Indolylanions zur{\"u}ckzuf{\"u}hren, das entweder am Sticksoff oder an C3 alkyliert werden kann. Umsetzung von 35 nach Mannich lieferte das bisaminoalkylierte Produkt 37, neben einer kleinen Menge der monoalkylierten Verbindung 36. Die Totalsynthese des zweiten Ringsystems ist im Schema 25 dargestellt. Um potentere Verbindungen zu erhalten, wurden beide Endstufen 6 bzw. 37 mit Methyliodid zu 14 bzw. 38 quaternisiert. 37 wurde zus{\"a}tzlich mit Allylgruppen zu 39 substituiert. Die pharmakologische Testung von 14, 37, und 38 erfolgte {\"u}ber Radioligandbindungsstudien an Membransuspensionen der Herzventrikel des Hausschweins. Der allostere Effekt der Testverbindungen wurde {\"u}ber die Hemmung der Dissoziation von [3H]-N-Methylscopolamin([3H]-NMS) von den damit ges{\"a}ttigten Rezeptoren gemessen. Die erhaltenen EC50,diss-Werte geben die Konzentration des allosteren Modulators an, bei der die [3H]-NMS-Dissoziation auf die H{\"a}lfte des Kontrollwertes reduziert ist. Sie sind ein Maß f{\"u}r die Affinit{\"a}t der Testsubstanzen zur allosterischen Bindungsstelle des M2 Rezeptors. F{\"u}r die einzige Verbindung mit dem Diazocinodiindole-Ringsystem 14 wurde ein EC50,diss-Wert von 54 nM gemessen. Da 14 {\"u}ber vier Benzylsubstituenten verf{\"u}gt, kann seine Bindungsaffinit{\"a}t am besten mit der von Dibenzylcaracurinium-Dibromid verglichen werden, die ganz {\"a}hnlich ist (69 nM). Aufgrund der Tatsache, dass die Verkleinerung des NSubstituenten am Caracurin-V-Ger{\"u}st zur erheblichen Steigerung der allosterischen Potenz f{\"u}hrte, ist zu erwarten, dass der Austausch der volumin{\"o}sen Benzylgruppen von 14 durch z.B. Methyl- oder Allylsubstituenten, eine deutliche Affinit{\"a}tssteigerung bewirken w{\"u}rde. Damit scheint die allosterische Potenz des neuen Ringsystems mindestens genauso gut zu sein, wie die von Caracurin V. Die beiden Vertreter des Azocinodiindol-Ringsystems, 38 und 39, sind bereits mit den Gruppen substituiert, die die beste allosterische Potenz bei dem Caracurin-V-Ringsystem zeigten (Methyl- und Allyl). Ihre EC50,diss-Werte (35 nM f{\"u}r 38, 48 nM f{\"u}r 39) sprechen jedoch f{\"u}r eine ca. 4-fach schw{\"a}chere Bindungsaffinit{\"a}t als die der entsprechenden Caracurine, was vermutlich auf einen anderen Abstand zwischen den quart{\"a}ren Stickstoffatomen und eine andere relative Stellung der Indolaromaten in den beiden Ringsystemen zur{\"u}ckzuf{\"u}hren ist. Anders als die entsprechenden Caracurin-V-Salze, sind 38 und 39 negativ kooperativ mit dem Antagonisten [3H]NMS. Zusammenfassend l{\"a}sst sich feststellen, dass von den beiden neu synthetisierten heterocyclischen Ringsystemen das direkt von Caracurin V abgeleitete Tetrahydro- [1,5]diazocino[1,2-a:6,5-a]diindol eine bessere und vielversprechende Leitstruktur f{\"u}r die Entwicklung neuer potenter allosterischen Liganden des M2-Rezeptors darstellt. Weitere synthetische Arbeiten an dem Ringsystem wie z.B. Variation des Sticksstoffsubstituenten und der Seitenkettenl{\"a}nge sollten zu einer Steigerung der Bindungsaffinit{\"a}t in den subnanomolaren Bereich f{\"u}hren. Dar{\"u}ber hinaus sind die Ergebnisse der pharmakologischen Testung an dem muskul{\"a}ren Typ des nicotinischen Acetylcholinrezeptors abzuwarten.},
  subject      = {Muscarinrezeptor},
  language  = {en}
}
@phdthesis{GarciaArguinzonis2003,
  author    = {Garc{\´i}a Arguinzonis, Ma{\´i}sa In{\´e}s},
  title     = {Analysis of signal transduction pathways and the cytoskeleton in VASP-deficient cell lines and mouse models},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6195},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {The mammalian Vasodilator Stimulated Phosphoprotein (VASP) is a founding member of the Ena/VASP family of proteins that includes Drosophila Enabled (ena), the mammalian Ena homologue (Mena) and the Ena-VASP-like protein (Evl). VASP was initially discovered and characterized as a substrate for cGMP- and cAMP-dependent protein kinases (cGKs and cAKs). Ena/VASP proteins are involved in Actin-filament formation, plasma membrane protrusion, acceleration of Actin-based motility of Listeria and the establishment of cell-cell adhesion. Moreover, Ena/VASP proteins have been implicated as inhibitory factors in repulsive axon guidance and inhibition of plasma membrane activity and random motility in fibroblast. In order to study the physiological function of VASP, VASP-deficient mice had been generated in the laboratory by homologous recombination. VASP-/- mice showed hyperplasia of megakaryocytes in the bone marrow and spleen and a two-fold increase in thrombin- and collagen-induced platelet activation. To further investigate the cellular function of VASP, I established cardiac fibroblast cell lines derived from both wild type and VASP-/- mice. Both cell lines presented similar growth rates and normal contact dependent-growth inhibition but showed differences in morphology, migration and adhesion. Adherent VASP-/- cells, despite normal Mena and Evl expression levels, were highly spread. VASP-/- cells covered about twice the substrate surface area as wild type cells, while the cell volumes were unchanged. This shape difference suggests that VASP is involved in the regulation of spreading. Since the small GTPases Rac and Cdc 42 and their effector p21-activated kinase (Pak) are key regulators of lamellipodia formation and cell spreading, I analyzed this signalling pathway in VASP-/- cells stimulated with Platelet Derived Growth Factor-BB (PDGF-BB) or fetal calf serum. In wild type cells Rac and Pak were rapidly and transiently activated by PDGF or serum; however, in the absence of VASP both Rac and Pak activation was dramatically prolonged. The Rac/Pak pathway is known to play an essential role in cell motility. VASP deficient cells showed compromised migration and reorientation in a wound healing assay, probably due to enhanced Rac activity. The spreading phenotype, compromised migration and the effect observed on the Rac and Pak activities were reverted in VASP-/- cells stably transfected with full lenght human VASP, indicating a VASP dependent modulation of the Rac/Pak pathway and Rac/Pak regulated processes. Moreover, adhesion and detachment of VASP-deficient cells were significantly slower when compared to wild type cells. Preincubation of VASP+/+ cells with a cGMP analog accelerated adhesion. This acceleration did not take place in the VASP-/- cells, suggesting a VASP dependent effect. The second part of this work focused on VASP function in platelets. On the one hand I investigated the possibility of VASP-dependent Rac regulation in mouse platelets. Murine platelets are a good model for studying Rac regulation since they express high levels of VASP but not Mena/Evl and since VASP-deficient platelets show an increased platelet activation. Rac was activated by platelet agonists which was inhibited by preincubation with cGMP and cAMP analogs. Initial results which need to be extended showed that the cGMPcaused inhibition of Rac activation was VASP-dependent. Finally, in vivo platelet adhesion (platelet-vessel wall interactions) was studied using VASP-deficient mice. These studies demonstrated in-vivo that VASP down regulates platelet adhesion to the vascular wall under both physiological and pathophysiological conditions.},
  subject      = {Vasodilatator-stimuliertes Phosphoprotein},
  language  = {en}
}
@phdthesis{Jovcic2002,
  author    = {Jovcic, Alexander},
  title     = {Applications of aerobic and anaerobic bacteria in the fields of biological degradation of contaminants and biological wastewater treatment},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6702},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2002},
  abstract  = {In the work here presented four distinctly different problems were investigated. The first problem was an investigation into the degradation of Dichloroethylene (DCE) and 1,1-bis (p-Chlorophenyl)-2-dichloroethylene (DDE) utilising pure bacterial cultures. The second investigation dealt with the degradation of DDE and polychlorinated Biphenyl's (PCB's) utilising anaerobic sediments and soils from New Zealand. The third investigation worked on the Granulation of anaerobic River-sediments in Upflow Anaerobic Sludge Blanket (UASB) Reactors. The last investigation describes the commissioning of an industrial aerobic Wastewater Treatment Plant and the Implementation of biological Nitrogen- and Phosphate removal in this Wastewater Treatment Plant. Since the chemical Structure of DCE and DDE have certain similarities, Bacteria that were capable of degrading DCE, were tested here, whether they would also be able to degrade DDE utilising a co-metabolic pathway. In the experiments the aerobic bacteria Methylosinus trichosporium and Mycobacterium vaccae and the anaerobic bacteria Acetobacterium woodii and Clostridium butyricum were used. Approximately 60\% of the added DCE was degraded by M. vaccae, while M. trichosporium degraded approximately 50\%. A. woodii and C. butyricum degraded 40\% and 30\% respectively of the added DCE. Further experiments with these cultures and DDE lead to a microbial degradation of DDE to an extent of 34.6\% for M. vaccae, 14.1\% for C. butyricum, 2.2\% for A. woodii and 10.5\% for M. trichosporium. Additional experiments, utilising [14C]-DDE, showed that the DDE had not been degraded but were attached to the bacterial cells. The second investigation utilised anaerobic soils and sediments from New Zealand to study the anaerobic co-metabolic degradation of DDE and PCB's. The soils and sediments originated from the River Waikato, from Wastewater Ponds in Kinleith, Marine-Sediments from Mapua, and a variety of soils comtaminated with Pentachlorophenyl (PCP). The cultures from these soils and sediments were raised on a variety of Carbon- and Energy-sources. Beside DDE, Aroclor 1260, and a mix of four pure PCB-Congeneres (one Tetra-, one Hexa, one Hepta- and one Deca-Chlorobiphenyl) were used to test for the reductive dechlorination. The cultivation process of the baceria lasted six months. Samples of the cultures were taken after zero, three and six months. These samples were tested for the increase of cell-protein, the degradation of carbon- and energy-sources, and the removal of the added polychlorinated chemicals. The organochlorines were analysed using reversed phase HPLC and FID-GC. When a change in the Chromatogram was detected the respective cultures were further analysed using ECD-GC and GC-MS. The results showed that the culutres grew under these conditions, but no degradation of DDE and the PCB-Mix could be detected, and only small changes in the composition/chromatograms of Aroclor 1260 were found. The third investigation worked on the Granulation of River-Sediments in UASB-Reactors. Sediments from the River Waikato in New Zealand and the River Saale in Germany were used. In both cases the Granulation process was successful, which was demonstrated by microscopic comparisons of the Sediments and the resulting Granules. The two main bacterial cultures detected were Methanosarcina- and Methanothrix-like cultures. The main carbon- and energy-source was Lactic Acid, which was used at a concentration of 21,8 g COD/L. The Granulation-Process was a combination of using high a COD-Concentration combined with a low Volumetric Loading-Rate. Comparisons of the specific degradation-rates of a variety of carbon- and energy-sources between the Sediments and the Granules, showed no increased degradation rates in regard to the same cell-mass, but the increased bio-mass in the Granules allowed for higher degradation-rates within the UASB-reactors. The fourth investigation describes the commissioning of an industrial Wastewater Treatment Plant for a Dairy-Site in Edendale, Southland, New Zealand. This Plant consists of a DAF-Unit (Dissolved Air Flotation), two Extended Aeration Lagoons with Activated Sludge and two Clarifiers, one for the Activated Sludge and the second for the dosing of Aluminium-Sulphate and the removal of Phosphat-Sulphate. Biological processes for the removal of carbon- and energy-sources were optimised and biological processes for the reduction of Nitrogen- and Phosphate-Concentrations within the wastewater were implemented and optimised. Bilogical removal rates for COD of 95\% and above, for Nitrogen of 85-92\% and Phosphate of 64-83\% were achieved.},
  subject      = {Biologische Abwasserreinigung},
  language  = {en}
}
@phdthesis{Gross2002,
  author    = {Groß, Michaela},
  title     = {Genomic changes in Fanconi anemia: implications for diagnosis, pathogenesis and prognosis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6579},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2002},
  abstract  = {Fanconi anemia (FA) is a genetically and phenotypically heterogenous autoso- mal recessive disease associated with chromosomal instability, progressive bone marrow failure, typical birth defects and predisposition to neoplasia. The clinical phenotype is similar in all known complementation groups (FA-A, FA-B, FA-C,FA-D1, FA-D2, FA-E, FA-F and FA-G). The cellular phenotype is characterized by hypersensitivity to DNA crosslinking agents (MMC,DEB), which is exploited as a diagnostic tool. Alltogether, the FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. FANCA, FANCC, FANCE, FANCF and FANCG interact in a nuclear complex upstream of FANCD2. Complementation group FA-D1 was recently shown to be due to biallelic mutations in the human breast cancer gene 2 (BRCA2). After DNA damage, the nuclear complex regulates monoubiquitylation of FANCD2, result- ing in targeting of this protein into nuclear foci together with BRCA1 and other DNA damage response proteins. The close connection resp. identity of the FA genes and known players of the DSB repair pathways (BRCA1, BRCA2, Rad51) firmly establishs an important role of the FA gene family in the maintenance of genome integrity. The chapter 1 provides a general introduction to the thesis describing the current knowledge and unsolved problems of Fanconi anemia. The following chapters represent papers submitted or published in scientific literature. They are succeeded by a short general discussion (chapter 7). Mutation analysis in the Fanconi anemia genes revealed gene specific mutation spectra as well as different distributions throughout the genes. These results are described in chapter 1 and chapter 2 with main attention to the first genes identified, namely FANCC, FANCA and FANCG. In chapter 2 we provide general background on mutation analysis and we report all mutations published for FANCA, FANCC and FANCG as well as our own unpublished mutations until the year 2000. In chapter 3 we report a shift of the mutation spectrum previously reported for FANCC after examining ten FA-patients belonging to complementation group C. Seven of those patients carried at least one previously unknown mutation, whereas the other three patients carried five alleles with the Dutch founder mu- tation 65delG and one allele with the Ashkenazi founder mutation IVS4+4A>T, albeit without any known Ashkenazi ancestry. We also describe the first large deletion in FANCC. The newly detected alterations include two missense mu- tations (L423P and T529P) in the 3´-area of the FANCC gene. Since the only previously described missense mutation L554P is also located in this area, a case can be made for the existence of functional domain(s) in that region of the gene. In chapter 4 we report the spectrum of mutations found in the FANCG gene com- piled by several laboratories working on FA. As with other FA genes, most muta- tions have been found only once, however, the truncating mutation, E105X, was identified as a German founder mutation after haplotype analysis. Direct compar- ison of the murine and the human protein sequences revealed two leucine zipper motifs. In one of these the only identified missense mutation was located at a conserved residue, suggesting the leucine zipper providing an essential protein-protein interaction required for FANCG function. With regard to genotype-phenotype correlations, two patients carrying a homozygous E105X mutation were seen to have an early onset of the hematological disorder, whereas the missense mutation seems to lead to a disease with later onset and milder clinical course. In chapter 5 we explore the phenomenon of revertant mosaicism which emerges quite frequently in peripheral blood cells of patients suffering from FA. We de- scribe the types of reversion found in five mosaic FA-patients belonging to com- plementation groups FA-A and FA-C. For our single FA-C-patient intragenic crossover could be proven as the mechanism of self-correction. In the remaining four patients (all of them being compound heterozygous in FANCA), either the paternal or maternal allele has reverted back to WT sequence. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensat- ing missense mutation 15 amino acids downstream of the constitutional mutation explaining the MMC-resistance of the lymphoblastoid cell line of this patient. In chapter 6 we report two FA-A mosaic patients where it could be shown that the spontaneous reversion had taken place in a single hematopoietic stem cell. This has been done by separating blood cells from both patients and searching for the reverted mutation in their granulocytes, monocytes, T- and B-lymphocytes as well as in skin fibroblasts. In both patients, all hematopoietic lineages, but not the fibroblasts, carried the reversion, and comparison to their increase in erythrocyte and platelet counts over time demonstrated that reversion must have taken place in a single hematopoietic stem cell. This corrected stem cell then has been able to undergo self-renewal and also to create a corrected progeny, which over time repopulated all hematopoietic lineages. The pancytopenia of these patients has been cured due to the strong selective growth advantage of the corrected cells in vivo and the increased apoptosis of the mutant hematopoietic cells.},
  subject      = {Fanconi-An{\"a}mie},
  language  = {en}
}