Refine
Year of publication
- 2014 (86) (remove)
Document Type
- Doctoral Thesis (86) (remove)
Language
- English (86) (remove)
Has Fulltext
- yes (86)
Is part of the Bibliography
- no (86) (remove)
Keywords
- Beschleuniger (2)
- Radio Frequenz Quadrupol (2)
- 1,4-dioxane (1)
- ADHD (1)
- Ageing (1)
- Allergie (1)
- Birkenpollen (1)
- Branch and Bound (1)
- Chikungunya (1)
- Chinese river basins, water withdrawals, dam construction, river flow alteration, flow indicators, fish species richness, fish catch, riparian vegetation cover, quantitative analysis (1)
Institute
- Physik (25)
- Biochemie und Chemie (14)
- Biowissenschaften (13)
- Informatik (7)
- Geowissenschaften (6)
- Mathematik (5)
- Pharmazie (4)
- Institut für Ökologie, Evolution und Diversität (3)
- Biochemie, Chemie und Pharmazie (2)
- Psychologie (2)
The nature of spontaneous brain activity during wakefulness and sleep: a complex systems approach
(2014)
In this thesis we study the organization of spontaneous brain activity during wakefulness and all stages of human non-rapid eye movement sleep using an approach based on developments and tools from the theory of complex systems. After a brief introduction to sleep physiology and different theoretical models of consciousness, we study how the organization of cortical and sub-cortical interactions is modified during the sleep cycle. Our results, obtained by modeling global brain activity as a complex functional interaction network, show that the capacity of the human brain to integrate different segregated functional modules is diminished during deep sleep, in line with an informationintegration account of consciousness. We then show that integration is impaired not only across space but also in the temporal domain, by assesing the emergence of long-range temporal correlations in brain activity and how they are modified during sleep. We propose an encompassing explanation for this observation, namely, that the brain operatsat different dynamical regimes during different states of consciousness. Finally, we gather massive amounts of data from different collaborative projects and apply machine learning techniques to reveal that the \resting state" cannot be considered as a pure brain state and is in fact a mixture containing different levels of conscious awareness. This last result has deep implications for future attempts to develop a discovery science of brain function both in health and disease.
To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor and third leading cause of cancer-related death worldwide. Most cases arise as a consequence of underlying liver disease, e.g. developed from chronic hepatitis B or C infectionsalcohol abuse or obesity, and are most often associated with liver cirrhosis. Hypoxiand the hypoxia inducible factors (HIF)-1α and -2α promote tumor progression of HCC, not only affecting tumor cell proliferation and invasion, but also angiogenesis and lymphangiogenesis and thus, increasing the risk of metastasis.
HCC is characterized as one of the most vascularized solid tumors. While HIF-1α and HIF-2α are frequently up-regulated in HCC only HIF-2α is correlated with high patientlethality. HIF-dependent regulation of HCC angiogenesis is controversially discussed.VEGFA, for example, as the most prominent factor inducing tumor angiogenesis represents not only a HIF-1 target, but also a HIF-2 target gene in HCC. This questions whether both isoforms have overlapping functions in regulating the angiogenic switch in HCC.
Besides angiogenesis also tumor-associated lymphangiogenesis significantly influences patient survival in HCC. Lymphatic spread is an important clinical determinant for the prognosis of HCC, but little is known how lymphangiogenesis is controlled in this context. To date, mainly HIF-1α was positively correlated with olymphatic invasion and metastasis in HCC, while a defined role of HIF-2α is missing. Thus, although HIF-1α and HIF-2α are structurally alike and regulate overlapping but not identical sets of target genes, they promote highly divergent outcomes in cancer progression and may even have counteracting roles. The aim of my work was to characterize the specific role of HIF-1α and HIF-2α in the angiogenic switch and lymphangiogenesis induction during HCC development.
Therefore, I created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids and embryonic bodies derived from embryonic mouse stem cells as an in vitro tumor model mimicking the cancer microenvironment to analyze which HIF isoform has key regulatory functions in HCC (lymph)angiogenesis. In cocultures with a HIF-2α knockdown angiogenesis was attenuated but lymphangiogenesis increased, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1)and insulin-like growth factor binding protein 1 (IGFBP1) as HIF-2 target genes.However, prominent angiogenic and lymphangiogenic factors such as VEGFs, PDGFB, ANG and their receptors were not regulated in a HIF-dependent manner. As PAI-1 was linked to angiogenesis in literature and IGF-signaling, which is negatively regulated by IGFBP-1, was correlated with lymphangiogenesis, I decided to investigate their HIF-2α-dependent influence on HCC (lymph)angiogenesis. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis in PAI-1k/d cocultures similar to the HIF-2α k/d phenotype. PAI-1 as the potent inhibitor of tPA and uPA, both inducing the conversion of plasminogen to plasmin, also inhibits plasmin directly. Therefore, I assumed an increase of plasmin in HIF-2α k/d and PAI-1 k/d cocultures as a result of the reduced PAI-1 levels. Blocking plasmin with aprotinin in HIF-2α k/d cocultures restored angioge nesis, suggesting that HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. In further experiments I could exclude PAI-1 to reduce angiogenesis by inducing plasmin-mediated apoptosis of differentiating stem cells in PAI-1 k/d and HIF-2α k/d cocultures, but demonstrated an increase of VEGFA165 degradation in these cocultures, suggesting plasmin-catalyzed proteolysis of VEGF as an additional layer of regulation required to explain the angiogenic phenotype. Besides the pivotal role of PAI-1 in angiogenesis I also investigated its potentialinfluence in lymphangiogenesis. Indeed, the knockdown of PAI-1 reduced lymphaticstructures and implied an important but opposing role in lymphangiogenesis comparedto induced lymphangiogenesis in HIF-2α k/d cocultures. However, blocking plasmin again with aprotinin in HIF-2α k/d cocultures restored lymphangiogenesis to the level of control virus, which indicates a divergent lymphangiogenic role of plasmin in PAI-1 k/d and HIF-2α k/d cocultures, possibly because of other essential pathways masking the lymphangiogenic effects of PAI-1 in HIF-2α k/d cocultures.
HIF-2α resulting in reduced IGFBP1 expression induced the differentiation of stem cells toward a lymphatic cell type and significantly enhanced the assembly of human dermal lymphatic endothelial cells into tubes. These data point the first time to an important impact of HIF-2 in the regulatin of lymphangiogenesis in vitro by inducing IGFBP1 and thus, scavenging IGF-1. Furthermore, matrigel plug assays to investigate the in vivorelevance of these observations confirmed HIF-2α as a crucial factor in the regulation of lymphangiogenesis in vivo
In conclusion, this work provides evidence that HIF-2α is a key regulator of angiogenesis and lymphangiogenesis in HCC by regulating PAI-1 and IGFBP1. HIF-2α positively influences the angiogenic switch via PAI-1 and negatively affects lymphangiogenesis via IGFBP1 expression. Targeting HIF-2α in HCC to reduce tumor angiogenesis should be approached carefully, as it might be overcome by induced lymphangiogenesis and metastasis.
During this study clumped isotope analysis of carbonates was established at the Goethe University of Frankfurt, Germany. Therefore, preparation protocols and analytical parameters were elaborated to obtain precise and accurate Δ47 data. Briefly, analyte CO2 was cleaned cryogenically using glass extraction lines to remove traces of water that enable re-equilibration of C–O bonds in the gases. Furthermore, analyte CO2 was passed through a gas chromatograph (GC) to clean it from contaminants that produce isobaric interferences with m/z 47. Initially, phosphoric acid digestions of carbonates was conducted at 25 °C in McCrea-type reaction vessels. Afterwards samples were reacted at 90 °C using a common acid bath. Mass spectrometric analyses were performed using a MAT 253 equipped with a dual inlet system. Δ47 values were directly projected to the absolute scale using CO2 gases equilibrated at distinct temperatures.
In cooperation with Stefano Bernasconi and his research group at ETH Zurich we studied the non-linearity that occurs for the measurement of m/z 47. This effect results from secondary electrons created by the m/z 44 beam. These electrons cause a negative background on the m/z 47 collector. A correction procedure was proposed that relies on the determination of the negative background on the m/z 47 Faraday cup. This approach might reduce time-consuming analyses of heated gases which were used so far to account for the observed non-linearity. However, the suggested correction of the negative background on the m/z 47 cup is only applicable if the slit width of the m/z 44 beam is significantly wider than that of the m/z 47 beam.
This thesis, furthermore, presents a comparison of the different phosphoric acid digestion techniques which are commonly used for carbonate clumped isotope analysis. For calcitic and aragonitic material digested at 25 °C in McCrea-type vessels we observed that the sample size has an effect on Δ47 data: higher mean Δ47 values and a larger scatter of data were received for samples <7 mg than for larger aliquots. For carbonate samples digested at 90 °C in a common acid bath no sample size effect was determined. We assume that secondary re-equilibration of CO2 with water preferentially occurs at 25 °C producing the observed differences. However, a sample size effect can be avoided if reaction temperature is increased to 90 °C.
In order to make carbonate Δ47 data obtained from acid digestions at 90 °C comparable to Δ47 data received from reactions at 25 °C the difference of the acid fractionation factores (Δ47*25-90) between both temperatures has to be known. For the determination of the Δ47*25-90 value we have considered Δ47 data made at 25 °C from samples >7 mg only. For calicte and aragonite we obtained differences in fractionation factores of 0.075‰ and 0.066‰, respectively. These Δ47*25-90 values are coincident with the theoretical prediction of 0.069‰ proposed for calcite (Guo et al., 2009).
Moreover, this dissertation comprises a calibration study of the clumped isotope thermometer based on various natural calcites that grew between 9 and 38 °C. The samples include a brachiopod shell, a bivalve shell, an eggshell of an ostrich and foraminifera tests which formed from distinct biomineralizing processes. Furthermore we included an authigenic carbonate crystallized from biological-induced precipitation. The following linear relationship between 1/T2 and Δ47 was determined (with Δ47 in ‰ and T in K):
Δ47 = 0.0327 (± 0.0026) x 106 / T2 + 0.3030 (± 0.0308) (R2 = 0.9915)
This equation differs from the pioneering Ghosh et al. (2006a) calibration. However, our regression line is statistically indistinguishable from that of Henkes et al. (2013) which is based on aragonitic mollusks and calcitic brachiopod shells. Both studies have in common that calibration data were, at first, directly referenced to the absolute scale. In addition, both datasets rely on similar digestion techniques. Furthermore, the two calibrations are conform with the theoretical prediction of Guo et al. (2009).
The calcite calibration of the clumped isotope paleothermometer received in this study was applied to Δ47 data measured for Silurian brachiopods shells from Gotland/Sweden. Prior to isotopic analysis the fossils were intensively investigated for their preservation state (CL, SEM, trace elements). The lowest T(Δ47) values of ca. 28 to 33 °C were estimated from ultrastructurally well-preserved regions of some shells. For these samples also the lowest δ18Ow values of Silurian seawater were determined. These estimates of ca. −1‰ confirm the assumption that the δ18O value of the Silurian ocean was buffered to (0 ± 1)‰.
Nevertheless, most studied shells were characterized by a patchwork of pristine and altered shell portions resulting in elevated T(Δ47) values which plot mostly between 40 and 60 °C. Our results indicate that the clumped isotopic composition of the shells were altered at low water-rock ratios, not affecting the δ18O values. Δ47 and δ18O data of associated diagenetic phases (sparitic and micritic phases of the inner fillings of the fossils) provide evidence that the sparitic cements grew during several diagenetic events which occurred at different temperatures in fluid-buffered systems. We, furthermore, conclude that the micritic phases lithified at a very early diagenetic stage with the δ18O values being most probably close to a Silurian seawater composition
The subject of this thesis is the experimental investigation of the neutron-capture cross sections of the neutron-rich, short-lived boron isotopes 13B and 14B, as they are thought to influence the rapid neutron-capture process (r process) nucleosynthesis in a neutrino-driven wind scenario.
The 13;14B(n,g)14;15B reactions were studied in inverse kinematics via Coulomb dissociation at the LAND/R3B setup (Reactions with Relativistic Radioactive Beams). A radioactive beam of 14;15B was produced via in-flight fragmentation and directed onto a lead-target at about 500 AMeV. The neutron breakup of the projectile within the electromagnetic field of the target nucleus was investigated in a kinematically complete measurement. All outgoing reaction products were detected and analyzed in order to reconstruct the excitation energy.
The differential Coulomb dissociation cross sections as a function of the excitation energy were obtained and first experimental constraints on the photoabsorption and the neutron-capture cross sections were deduced. The results were compared to theoretical approximations of the cross sections in question. The Coulomb dissociation cross section of 15B into 14B(g.s.) + n was determined to be s(15B;14B(g:s:)+n) CD = 81(8stat)(10syst) mb ; while the Coulomb dissociation cross section of 14B into a neutron and 13B in its ground state was found to be s(14B;13B(g:s:)+n) CD = 281(25stat)(43syst) mb: Furthermore, new information on the nuclear structure of 14B were achieved, as the spectral shape of the differential Coulomb dissociation cross section indicates a halolike structure of the nucleus.
Additionally, the Coulomb dissociation of 11Be was investigated and compared to previous measurements in order to verify the present analysis. The corresponding Coulomb dissociation cross section of 11Be into 10Be(g.s.) + n was found to be 450(40stat)(54syst ) mb, which is in good agreement with the results of Palit et al.
The number of multilingual texts in the World Wide Web (WWW) is increasing dramatically and a multilingual economic zone like the European Union (EU) requires the availability of multilingual Natural Language Processing (NLP) tools. Due to a rapid development of NLP tools, many lexical, syntactic, semantic and other linguistic features have been used in different NLP applications. However, there are some situations where these features can not be used due the application type or unavailability of NLP resources for some of the languages. That is why an application that is intended to handle multilingual texts must have features that are not dependent on a particular language and specific linguistic tools. In this thesis, we will focus on two such applications: text readability and source and translation classification.
In this thesis, we provide 18 features that are not only suitable for both applications, but are also language and linguistic tools independent. In order to build a readability classifier, we use texts from three different languages: English, German and Bangla. Our proposed features achieve a classification accuracy that is comparable with a classifier using 40 linguistic features. The readability classifier achieves a classification F-score of 74.21% on the English Wikipedia corpus, an F-score of 75.47% on the English textbook corpus, an F-score of 86.46% on the Bangla textbook corpus and an F-score of 86.26% on the German GEO/GEOLino corpus.
We used more than two million sentence pairs from 21 European languages in order to build the source and translation classifier. The classifier using the same eighteen features achieves a classification accuracy of 86.63%. We also used the same features to build a classifier that classifies translated texts based on their origin. The classifier achieves classification accuracy of 75% for texts from 10 European languages. In this thesis, we also provide four different corpora, three for text readability analysis and one for corpus based translation studies.
Terrestrial climate and ecosystem evolution during ‘Greenhouse Earth’ phases of the early Paleogene remain incompletely known. Particularly, paleobotanical records from high southern latitudes are giving only limited insights into the Paleocene and early Eocene vegetation of the region. Hence, data from continuous well-calibrated sequences are required to make progress with the reconstruction of terrestrial climate and ecosystem dynamics from the southern latitudes during the early Paleogene.
In order to elucidate the terrestrial conditions from the high southern latitudes during the early Paleogene, terrestrial palynology was applied in the present study to two well-dated deep-marine sediment cores located at the Australo-Antarctic region: (i) IODP Site U1356 (Wilkes Land margin, East Antarctica) and (ii) ODP Site 1172 (East Tasman Plateau, southwest Pacific Ocean). The studied sequence from IODP Site U1356 comprises mid-shelfal sediments from the early to middle Eocene (53.9 – 46 million years ago [Ma]). For the ODP Site 1172, the studied succession is characterized by sediments deposited in shallow marine environments of the middle Paleocene to the early Eocene (60.7 – 54.2 Ma).
Based on the obtained pollen and spores (sporomorphs) results from the studied sequences of Site U1356 and Site 1172, this study aims to: (1) decipher the terrestrial climate conditions along the Australo-Antarctic region from the middle Paleocene to the middle Eocene; (2) evaluate the structure, diversity and compositional patterns of forests that throve in the Australo-Antarctic region during the early Paleogene; (3) understand the response of forests from the high southern latitudes to the climate dynamics from the early Paleogene; (4) establish a connection between the generated terrestrial palynomorph data and published Sea Surface Temperatures (SSTs) from the same cores.
To decipher the terrestrial climatic conditions on the Australo-Antarctic region, this study relies on the nearest living relative (NLR) concept that assumes that fossil taxa have similar climate requirements as their modern counterparts. This approach was applied to the sporomorph results of Site U1356 and Site 1172, following mainly the bioclimatic analysis. With regard to the structure and diversity patterns of the vegetation from the same region, the present study presents combined qualitative (i.e., reconstruction of the vegetation based mainly on the habitats of the known living relatives) and quantitative (i.e., application of ordination techniques, rarefaction and diversity indices) analyses of the fossil sporomorphs results.
The overall results from the paleoclimatic and vegetation reconstruction approaches applied in the present study, indicate that temperate and paratropical forests during the early Paleogene throve under different climatic conditions on the Wilkes Land margin and on Tasmania, at paleolatitudes of ∼70°S and ∼65°S, respectively.
Specifically, the sporomorph results from Site U1356, suggest that a highly diverse forest similar to present-day forests from New Caledonia was thriving on Antarctica during the early Eocene (53.9 – 51.9 Ma). These forests were characterized by the presence of termophilous taxa that are restricted today to tropical and subtropical settings, notably Bombacoideae, Strasburgeria, Beauprea, Spathiphyllum, Anacolosa and Lygodium. In combination with MBT/CBT paleotemperature results, they provide strong evidence for near-tropical warmth at least in the coastal lowlands along the Wilkes Land margin. The coeval presence of frost tolerant taxa such as Nothofagus, Araucariaceae and Podocarpaceae during the early Eocene on the same record suggests that paratropical forests were thriving along the Wilkes Land margin. Due to the presence of this kind of vegetation, it is possible to suggest that forests in this region were subject to a climatic gradient related to differences in elevation and/or the proximity to the coastline.
By the middle Eocene, the paratropical forests that characterized the vegetation of the early Eocene on the Wilkes Land margin were replaced by low diversity temperate forests dominated by Nothofagus, and similar to present-day cool-temperate forests from New Zealand. The dominance of these forests and the absence of thermophilous elements together with the lower temperatures suggested by the MBT/CBT and the sporomorph-based temperatures indicate consistently cooler conditions during this time interval.
With regard to the sporomorph results of Site 1172, this study suggests that three vegetation types were thriving on Tasmania from the middle Paleocene to the early Eocene under different climatic conditions. During the middle to late Paleocene, warm-temperate forests dominated by Podocarpaceae and Araucariaceae were the prevailing vegetation on Tasmania. The dominance of these forests was interrupted by the transient predominance of cool-temperate forests dominated by Nothofagus and Araucariaceae across the middle/late Paleocene transition interval (~59.5 to ~59.0 Ma). This cool-temperate forest was characterized by a lack of frost-sensitive elements (i.e., palms and cycads) indicating cooler conditions with harsher winters on Tasmania during this time interval. By the early Eocene, and linked with the Paleocene Eocene Thermal Maximum (PETM), Paleocene temperate forests dominated by gymnosperms were replaced by paratropical rainforests with the remarkable presence of the tropical mangrove palm Nypa during the PETM and the earliest Eocene. The overall results from Site U1356 and Site 1172, provide a new assessment of the terrestrial climatic conditions in the Australo-Antarctic region for validating climate models and understanding the response of high-latitude terrestrial ecosystems to the climate dynamics of the early Paleogene on southern latitudes.
The climatic conditions in the higher latitudes during the early Paleogene were further unravelled by comparing the obtained terrestrial and marine results. The integration of the obtained sporomorph data with previously published TEX86-based SSTs from Site 1172 documents that the vegetation dynamics were closely linked with the temperature evolution from the Australo-Antarctic region. Moreover, the comparison of TEX86-based SSTs and sporomorph-based climatic estimations from Site 1172 suggests a warm-season bias of both calibrations of TEX86 (i.e., TEX86Hand TEX86H), when this proxy is applied to high southern latitudes records of the early Paleogene.
In Reaktion auf zellulären Stress wie etwa Schädigungen der DNA oder die vermehrte Aktivität von Onkogenen aktivieren vorgeschaltete Signalkaskaden den Transkriptionsfaktor (TF) p53. Dieser kann über die Aktivierung der Expression von Zielgenen wiederum die Zellteilung stoppen, die Reparatur von DNA Schäden initiieren oder in schweren Fällen die Eliminierung der Zelle durch Apoptose einleiten. Ist p53 durch Mutationen deaktiviert, können sich entartete somatische Zellen vermehren und in der Folge Krebs entstehen.
In Wirbeltieren finden sich neben p53 mit p63 und p73 zwei weitere TFs, welche während der Evolution aus dem gleichen gemeinsamen Vorläufer durch Genduplikationen hervorgegangen sind. Die drei TFs sind modular aufgebaut und alle Isoformen verfügen jeweils minimal über eine DNA Bindungsdomäne (DBD) und eine Tetramerisierungsdomäne (TD). Werden die p53 ähnlichen TFs aktiviert, lagern sie sich über die TD vermittelt zu Tetrameren zusammen, wodurch ihre DBDs kooperativ an DNA Sequenzmotive binden können. Die DBD ist auch über große phylogenetische Abstände hinweg hoch konserviert, wodurch bereits gezeigt werden konnte, dass auch primitive vielzellige Tiere bereits Homologe dieser TF Familie besitzen. Im Vergleich zur DBD variiert die Proteinsequenz der TD deutlich stärker, was andeutet, dass deren Struktur im Laufe der Evolution erhebliche Veränderungen durchlaufen hat. Diese Veränderungen aufzuklären ist das übergeordnete Forschungsvorhaben zu dem diese Dissertationsschrift beiträgt.
Ciona intestinalis (C.int.) ist eine Spezies aus dem Unterstamm der Manteltiere. Diese sind die engsten lebenden Verwandten der Wirbeltiere und C.int. ist ein populärer Modelorganismus für die Erforschung der Embryonalentwicklung. Sein Genom kodiert für zwei p53 ähnliche TFs, welche mit p53/p73-a und p53/p73-b bezeichnet werden. Die Struktur ihrer TDs wurde im Rahmen der vorliegenden Arbeit mittels Kernspinresonanz (NMR) Spektroskopie untersucht.
Die TD von menschlichem p53 (hp53) ist ein Dimer aus Dimeren. Jedes Monomer formt einen beta-Strang und eine alpha-Helix. Im primären Dimer lagern diese sich so zusammen, dass ein beta-Faltblatt entsteht und die alpha-Helices mit entgegen gesetzter Orientierung der Länge nach aneinander packen. Zwei dieser Dimer lagern sich dann so zum Tetramer zusammen, dass zwischen pol-ständigen beta-Faltblättern ein Bündel aus vier Helices entsteht. Dieses Motiv ist auch in den TDs der Ciona Proteine hochkonserviert und wird im Folgenden als Kern?TD bezeichnet. In den TDs von menschlichem p63 und p73 (hp63 und hp73) verfügt jedes Monomer an seinem C-terminus noch über eine zweite Helix. Die zweiten Helices eines jeden Dimers greifen wie Klammern um das jeweils andere primäre Dimer und stabilisieren so das Tetramer. Entscheidend für die stabile Anbindung an die Kern?TD ist dabei ein charakteristisches Tyrosin-Arginin (YR) Motiv in der zweiten Helix, welches sich auch in der Sequenz der TD von C.int. p53/p73-a wiederfindet. Analysen der Sekundärstruktur auf Basis von NMR Experimenten ergaben jedoch, dass die TD von C.int. p53/p73-a bei 25°C keine zweite Helix ausbildet. Mit Hilfe von chimären TD Peptiden, in denen Teile der Ciona Sequenz gegen die entsprechenden Abschnitte von hp73 ausgetauscht wurden, konnte gezeigt werden, dass die Kern TD von C.int. p53/p73-a fähig ist eine zweite Helix zu stabilisieren und hierfür neben dem YR Motiv auch der Sequenzabschnitt zwischen erster und zweiter Helix entscheidend ist. Stabilisierende Substitutionen in diesem Bereich bewirkten ebenso wie ein Absenken der Temperatur die Ausbildung einer zweiten Helix, welche jedoch im Gegensatz zu jener in hp73 nur transient faltet und auch nicht essentiell für die Bildung des Tetramers ist, wohl aber dessen Stabilität erhöht.
Spezifisch in der Entwicklungslinie von Ciona kam es dazu, dass eine, für eine entsprechende Vorläuferversion von C.int. p53/p73-a kodierende, mRNA spontan zurück in DNA übersetzt und ins Genom eingefügt wurde. Die durch diese Retrotransposition erzeugte neue Genkopie C.int. p53/p73-b muss demnach ursprünglich einmal für die gleiche Proteinsequenz kodiert haben, innerhalb der TD finden sich konservierte Reste jedoch nur im Bereich der Kern TD.
Von der TD von C.int. p53/p73-b wurde die molekulare Struktur in freier Lösung mittels NMR ermittelt. Diese zeigte, dass interessanterweise in der TD von C.int. p53/p73-b jedes Monomer am C-terminus eine stabil gefaltete, zweite Helix besitzt. Obwohl diese zweite Helix sich aus einer Sequenz faltet, die keinerlei Sequenzhomologie zu homologen Proteinen aus Wirbeltieren aufweist, lagert sie sich in einer Position auf die Kern TD, welche der in hp73 sehr nahe kommt. Da die primären Dimere der Kern TD aber anders als in hp63 und hp73 durch Salzbrücken miteinander verbunden sind, ist die zweite Helix jedoch nicht essentiell, um das Tetramer zu stabilisieren. Vermutlich kommt der zweiten Helix von C.int. p53/p73-b vielmehr u.a. die Aufgabe zu die Bildung von Heterotetrameren aus C.int. p53/p73-a und –b zu unterbinden.
Zusammengenommen zeigen die Ergebnisse, dass die Architektur der TD mit zweiter Helix bereits der Prototyp für die TDs aller p53 ähnlichen Proteine der Wirbel- und Manteltiere war und die als eine Art Klammer das Tetramer stabilisierende zweite Helix sich nicht erst während der Evolution der Wirbeltiere entwickelt hat.