146 search hits
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Regulation der Aktivität des Anti-Sigmafaktors RsiX aus Bacillus subtilis
(2011)
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Katharina Schäfer
- Der Gram-positive Modellorganismus Bacillus subtilis besitzt 17 alternative Sigmafaktoren, von denen 7 zur Gruppe der ECF (extra-cytoplasmic function) -Sigmafaktoren gehören. Einer dieser ECF-Sigmafaktoren, SigX, und sein entsprechender Anti-Sigmafaktor, RsiX, sind Gegenstand dieser Arbeit. Dabei handelt es sich bei RsiX um ein Transmembranprotein, während SigX im Cytoplasma lokalisiert ist und von der N-terminalen RsiX-Domäne von einer Interaktion mit der RNA-Polymerase abgehalten wird. SigW, ein weiterer ECF-Sigmafaktor, und RsiW, der dazugehörende Anti-Sigmafaktor, sind bereits sehr gut untersucht. Aufgrund struktureller Ähnlichkeiten zwischen SigX/RsiX und SigW/RsiW wurde angenommen, dass die Aktivierung des SigX-Regulons in vergleichbarer Weise erfolgt, wie es für das SigW-Regulon aufgrund intensiver Analysen sehr gut untersucht ist. Die in dieser Arbeit verwendete Methode der Transposonmutagenese lieferte entscheidende Hinweise über die Regulation des sigX-rsiX-Operons. Es konnte gezeigt werden, dass das sigX-rsiX-Operon einer Regulation durch den Transkriptionsterminator Rho unterliegt. Damit wurde das erst dritte Beispiel für eine Termination der Transkription durch den Faktor Rho in B. subtilis bekannt. Als wichtiges Element für die faktorabhängige Termination der Transkription gilt das Vorhandensein einer rut-site, der Bindestelle des Faktors Rho in der naszierenden mRNA. In dieser Arbeit wurden verschiedene Analysetechniken angewandt, um mit Hilfe eines GFP-RsiX-Reporters erstmals eine rut-site in B. subtilis zu kartieren. Die hier identifizierte rut-site von rsiX ist am 5´-Ende dieses Gens lokalisiert. Bei diesen Analysen wurde außerdem deutlich, dass es im sigX-rsiX-Operon zu einer faktorabhängigen intragenischen Termination der Transkription kommt, bei welcher nicht nur die Transkription von rsiX, sondern auch die Expression des stromaufwärts liegenden sigX beeinflusst wird. Punktmutationen in der Sequenz für die rut-site von rsiX zerstörten den Einfluss des Faktors Rho auf das sigX-rsiX-Operon, so dass keine Termination der Transkription mehr stattfand und die detektierbare Menge an sigX-rsiX-Transkript deutlich anstieg. Nur so war es möglich, einen rsiX-Knockout zu komplementieren. Messungen der beta-Galaktosidase-Aktivität eines lacZ-Reporters belegten zudem die Funktion von RsiX als Anti-Sigmafaktor von SigX. Der Verlust des Einflusses von Rho auf das sigX-rsiX-Operon aufgrund einer mutierten rut site machte auch immunologische Nachweise des Anti-Sigmafaktors möglich. Infolgedessen konnte der Frage einer möglichen RsiX-Proteolyse nach dem Vorbild der RsiW-Proteolyse nachgegangen werden. Für RsiW wurde bereits gezeigt, dass ein bestimmtes Stress-Signal den Abbau des Anti-Sigmafaktors einleitet. Die Proteolyse von RsiW erfolgt dann nach dem Mechanismus der regulierten intramembranen Proteolyse (RIP) unter der Beteiligung mehrerer Proteasen. Neben der Beteiligung der Protease RasP an der RsiW-Proteolyse konnte hier auch die Beteiligung von RasP an der RsiX-Proteolyse nachgewiesen werden. Gleichzeitig wurde aber ausgeschlossen, dass die Protease PrsW, welche auch an der RsiW-Proteolyse beteiligt ist, in die RsiX-Proteolyse involviert ist. Demnach können die einzelnen Schritte der RsiW-Proteolyse nicht einfach auf die RsiX-Proteolyse übertragen werden, obwohl es sich bei beiden Transmembranproteinen um Anti-Sigmafaktoren ihrer jeweiligen ECF-Sigmafaktoren handelt.
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Identification of substrate proteins of FtsH during sporulation of Bacillus subtilis
(2012)
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Hue Bach Thi Nguyen
- FtsH is an ATP- and Zn2+-dependent metalloprotease anchored in the cytoplasmic membrane by two transmembrane segments. It is the unique membrane-bound AAA-protease in bacteria that performs a variety of regulatory functions. In B. subtilis, an ftsH knockout exhibits a pleiotropic phenotype such as filamentous growth, sensitivity towards heat, osmotic shock and cells are unable to sporulate. Recently, it has been shown that ftsH knockout cells fail to entry sporulation stage II due to lack of a sufficient amount of Spo0A~P and the first substrate of FtsH identified in B. subtilis is the Spo0E phosphatase, a negative regulator that dephosphorylates Spo0A~P. However, the sporulation frequency in a spo0E ftsH double mutant strain was only partly restored, we hypothesized that FtsH might degrade additional substrate proteins involved in sporulation. To identify these proteins, two different strategies were applied. By using the 2D gel technique, the proteomes of an ftsH wild-type strain was compared with an ftsH null mutant. Several proteins were identified to be either up- or down-regulated in the absence of FtsH. One of them up-regulated about 4-fold was identified as Spo0M. Since ftsH did not interfere with transcription of spo0M, an in vitro proteolysis assay was established using purified components. It was shown that Spo0M was degraded by FtsH in an ATP- and time-dependent way. In the second strategy, an ftsHtrap mutant was constructed and tested for loss of its proteolytic activity. Protease trap mutants are still able to bind substrate proteins, but are unable to degrade them. By using FtsHtrap fused to a GST-tag, YwnF, a membrane protein, was trapped and identified as a substrate of FtsH by mass spectrometry. However, further experiments will be required to confirm YwnF as a target of FtsH. The last part of this thesis was focused on the eag gene, which forms a bicistronic operon with Spo0E. Construction and analysis of an eag insertion mutant exhibited a slight increase in the sporulation frequency and in the amount of Spo0A. A transcriptional fusion between the promoter of the spo0E-eag operon and the lacZ reporter gene revealed an increase in the beta-galactosidase activity from t0 when the cells were grown in sporulation medium. Since the Eag protein may be an integral membrane protein, it may bind excess Spo0E thereby preventing it from dephosphorylating Spo0A~P. Alternatively, Eag may bind Spo0E and present it as a modulator to FtsH for degradation.
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Molecular detection and identification of phytoplasmas in sugarcane in Hawaii, Thailand, Cuba and Near East
(2012)
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Ziad Soufi
- The Yellow leaf syndrome (YLS) had been first detected and described in Hawaiian sugarcane plantations. The polerovirus Sugarcane yellow leaf virus was identified as a causal agent of the syndrome; however there was no strict correlation between the degree of symptom expression and the virus titre. Therefore several surveys on breeding station sugarcane plants in Hawaiian Islands were done for Sugarcane yellow leaf phytoplasma (SCYLP), a bacterium which had been hypothesized to be also a causal agent of YLS. Two types of phytoplasmas were found in Hawaiian sugarcane cultivars mainly sugarcane white leaf phytoplasma (SCWL) which is a member in rice yellow dwarf group, in addition to aster yellows group. This was also true for sugarcane plants from Hawaiian plantations, which routinely use hot water-treatment for the seed cane cuttings. Sugarcane samples were obtained also from other countries including Cuba, Egypt, Syria and Thailand where sugarcane plants are also showing symptoms of yellowing or whiting. Aster yellows and X-disease phytoplasmas were found in Cuban cultivars whereas one sugarcane cultivar from Egypt contains grassy shoot phytoplasma that is a member in rice yellow dwarf group, but the other two Egyptian ones were phytoplasma-free. Syrian sugarcane was infected by phytoplasma that identified preliminary in rice yellow dwarf group. To our knowledge, this is the first report for the detection and identification of phytoplasma in sugarcane plants from Hawaii, Egypt and Syria. Our investigation on Thai sugarcane plants was in agreement with previous literature where sugarcane white leaf (SCWL) phytoplasma is associated with white leaf disease (Nakashima et al., 1994; Wongkaew et al., 1997). Q-PCR (real-time PCR) offers the opportunity to detect the phytoplasma in a sensitive, specific and quick manner, but that is not true for sugarcane plants with a very low titer of phytoplasma. Therefore, nested-PCR is better than qPCR for low titer phytoplasma detection and that is true for sugarcane yellow leaf phytoplasma disease. A BLAST search for the 16S rRNA gene sequences reported in this study showed that they shared 99 to 100% sequence identity with those of other phytoplasmas in the Aster yellows, X-disease and Rice yellow dwarf groups. However, no one of these identified strains can be described as a new “Candidatus Phytoplasma species”. On the other hand, Hawaiian sugarcane cultivar H78-7750 as a representative of Hawaiian breeding station sugarcane contains phytoplasma clustered to strain sugarcane white leaf (SCWL) phytoplasma, closely together with sugarcane white leaf phytoplasma from Taiwan (AY139874). It is possible to explain the occurrence of (SCWL) phytoplasma in Hawaiian Islands, by insect vectors or by infected stem cuttings which were obtained from other countries. Thai sugarcane contains phytoplasma isolate closely together with sugarcane white leaf phytoplasma from Myanmar. The transmission electron microscopic (TEM) studies revealed the presence of sugarcane white leaf phytoplasma only in phloem sieve tubes of diseased sugarcane leaves but not in adjacent cells to the sieve elements including companion cells and phloem parenchyma as well. According to ultrastructural observations under TEM, parenchymatic cells of bundle sheath and mesophyll tissue of affected leaves showed some alterations including accumulations of starch granules, increase plastoglobuli number and size in disorganized chloroplasts. Insect vectors of phytoplasmas are phloem feeders. Thus far, none of aphid species has been found to be a vector of phytoplasmas. Our tests showed also that black sugarcane aphids (Melanaphis Sacchari) were unable to transmit the phytoplasmas from infected sugarcane into the phytoplasma-free one. Hot water treatment has been proposed to cure plant material from phytoplasmas. Our tests showed that the appropriate hot water treatment, which recommended for phytoplasma elimination, is immersion of the sugarcane stem cuttings at 50°C for 60 min.
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Analysis of the "nurse-tree effect" of exotic shelter trees on the growth of the indigenous Podocarpus falcatus in an Ethiopian montane forest
(2011)
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Simone Strobl
- Ethiopian forests disappear with a rate of 1.1% per year due to the high demand of firewood and timber. To protect the remaining parts of the forests, exotic tree plantations were established 60 years ago. But there are considerable concerns regarding exotic plantations: they have the reputation to cause damage to the ecosystems due to high demand of water and nutrients. Considering the environmental deterioration caused by monotonous plantations of exotic tree species, the chance for indigenous woody plants to rejuvenate naturally in those plantations appears to be very small. But there are observations of indigenous tree species regenerating under the shelter of exotic tree plantations. This enhanced growth of indigenous saplings under the canopy of exotics has been termed “nurse-tree effect”. In the Munessa-Shashamene Forest, a tropical montane forest in Ethiopia consisting of plantations of exotic tree species and remnants of natural forest, regeneration and an enhanced growth of native Podocarpus falcatus saplings under the shelter of exotic tree plantations (Pinus and Eucalyptus) was observed. The focus of this work was to examine the different growth patterns of the saplings in the sites, the effects of the on the photosynthetic performance, and to compare the water relations of the Podocarpus saplings and those of the shelter-trees. The results of the study are summarized as follows: 1. Observations over two years showed that the relative growth rates of the saplings were more than three times higher in the Pinus plantation compared to the natural forest and the Eucalyptus plantation. Relative growth rates during the dry and the rainy season were more or less identical. 2. Investigation of the sub-canopy microclimate proved PAR and VPD as major components with impact on the photosynthetic performance of the saplings. 71% of the variations in photosynthetic carbon uptake could be explained by PAR and 4% by VPD. The Pinus plantation was slightly warmer and drier compared to the other two sites. Also highest PAR values of all sites were recorded in the Pinus plantation. In the Eucalyptus plantation, PAR values were the lowest of the three sites, caused by the two-tired canopy of coppiced and uncoppiced Eucalyptus trees. 3. For an assessment of the photosynthetic efficiency of the light climate, the efficacy of the shares of the irradiation from diffuse light and light flecks were determined from light curves. The time spans and distribution of these shares of the daily accumulated radiation were recorded from the daily courses. In the Pinus plantation, the efficiency of the radiation was relatively low (70%), because of the high intensity of the sunflecks, especially during the dry season. On cloudy days the efficiency was nearly 100% and resulted in an optimum photosynthetic performance of the saplings in the Pinus plantation. In the Eucalyptus plantation, the two-tired canopy resulted in a higher proportion of diffuse radiation and less daily accumulated PAR from sunflecks (46%). Also the efficiency of the actual radiation was the lowest of all sites on cloudy (72%) and sunny (53%) days. Daily accumulated PAR under the canopy of the natural forest was in between the other forest types. Such mid-position was also true for the share of the sunflecks and the CO2 uptake. Efficacy of the radiation was 80% on sunny and 86% on cloudy days. 4. Water relations can substantially affect the photosynthetic performance of plants. Especially in the afternoons of the dry season a decrease of photosynthetic CO2 uptake by the Podocarpus saplings became apparent. Whole-tree water consumption was determined by measuring sap flow with the Granier system. In principle sap flow (and transpiration) followed VPD. Comparison of the daily courses of transpiration and stomatal conductance and sap flow showed an earlier decrease of transpiration by the leaves of the saplings than by the shelter-trees, suggesting slight water shortage especially during the dry season. This interpretation is corroborated by the higher 13C values in the leaf tissue of the saplings from the Pinus plantation. Nevertheless severe drought stress did not occur during the two years of investigation. 5. The literature on the „nurse-tree effect“ mentions in particular Eucalyptus as shelter-tree, a finding which is not in agreement with the data of this study: Neither photosynthesis nor growth was enhanced compared with the control saplings in the natural forest. The discrepancy between this work and the literature can be solved when the management of the Eucalyptus plantation is considered. As long as the Podocarpus saplings grow under the two-tired canopy of the coppiced trees, growth is as slow as in the natural forest. However, after coppicing the light climate for the saplings ameliorates considerably and growth rates increase. Thus, a shelter-tree effect could also be observed under Eucalyptus, but its dynamics is stepwise rather than continuous.
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Climate change impacts on habitats and biodiversity :
From environmental envelope modelling to nature conservation strategies
(2011)
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Torsten Bittner
- Climate change will pose entirely new challenges for nature conservation. A literature study of 852 publications (between 2003 and 2010) illuminates this topic, examines driving research forces as well as focal points and shows recent research gaps. Here could be shown that changes in species distribution, diverse consequences for habitats, changing communities as well as biotic interactions and general aspects of diversity are the major challenges. The potential climatic modifications can alter deeply the distribution of animals and plants. Range changes due to recent climate change already exist and are traceable. In order to quantify such changes, environmental envelope modelling can be used. In addition to individual species, changes in distribution of more complex units are also conceivable. The present work mainly focuses on habitat types listed in the Annex I of the European Habitats Directive. To reveal the potential range changes of habitat types, two principally different modelling approaches have been developed. An indirect approach modelling the distribution of a habitat type using the distribution of its characteristic plant species and a direct approach, using the distribution of the habitat type itself. These two approaches were tested by modelling five grassland habitat types. Looking at the modelled results all habitats are projected to lose between 22% and 93% of their range in the ‘no dispersal’ scenario. Both approaches produce reasonable results. However, modelling an extensive set of habitat types using the indirect approach is currently not possible, because of the required but actually lacking amount of plant distribution data. Therefore, the direct approach is an appropriate instrument for modelling habitat types. Here, all 127 widespread terrestrial habitat types defined in the Annex I of the Habitats Directive were modelled and, resulting from this, a map of terrestrial habitat type diversity was calculated. Several habitat types are projected to lose many of their actually suitable areas, in particular bogs, rocky habitats, grassland and in part forests. Due to their developmental time or rather due to their special abiotic requirements, bogs and rocky habitats even lose under the assumption of a full dispersal scenario. However, most heath and grassland habitats are also projected to lose in the full dispersal scenario. Pooling all modelled results together, terrestrial habitat type diversity is shifting partly to mountain regions and the atlantic biogeographical region is projected to decrease in habitat type diversity. According to the Habitats Directive habitat types listed in Annex I are protected in ‘sites of community interest’ aiming to maintain or restore them at a favourable conservation status. Due to the projected changes a static nature conservation concept could face problems which particularly concern the coherence of the protected area network. This could lead to a loss of protective goods in spite of protected areas. To illustrate the potential problems and difficulties emerging with respect to spatial coherence of habitat types between protected areas, an analysis of spatial coherence under future conditions for a variety of habitat types in Germany was conducted. Here, a combination of environmental envelope modelling and graph theory is presented to assess the coherence of nature conservation networks. The possible incapacity of species to reach all climatically suitable areas is currently debated. Therefore, spatial scales are not only crucial for the coherence of protected areas but also for the question if future projected suitable areas could be colonized. Dispersal movements of species are only infrequently possible in our highly fragmented landscape. To answer this raising question, Odonata listed in the Habitats Directive with known dispersal distances were contemplated. The species Coenagrion ornatum, C. mercuriale and Ophiogomphus cecilia are projected to lose range when incorporating specific dispersal distances, while they are projected to extend their range in the unrestricted dispersal scenario. Furthermore, suitable climatic conditions tend to decline for Leucorrhinia albifrons and L. caudalis, whereas L. pectoralis is projected to gain distribution area assuming either species-specific or unrestricted dispersal. The nature conservation measure of translocation is an at least 100 years old methodology with pros and cons. In this thesis, the emerging problems and opportunities of this species preservation strategy are presented. Further, a new question about the ‘focal unit’ is pointed out as well as the problem of genetic variability and the aspect of pre-adopted subspecies. Moreover, a selective assisted colonisation not by moving species but ecotypes is referred.
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Analyse von Condensin-Komplexen in Drosophila melanogaster Charakterisierung der Untereinheit CapG und Identifizierung von Interaktionen
(2012)
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Sabine Herzog
- Die akkurate Verteilung der Chromosomen während der Mitose ist essentiell für die genetische Stabilität der Zellen. Der heteropentamere Condensin-Komplex ist hierbei an der Strukturierung der Chromosomen und deren physikalischer Stabilität während der Segregation maßgeblich beteiligt. In höheren Eukaryoten konnten zwei dieser Komplexe (Condensin I und Condensin II) identifiziert werden, die ihrerseits eine distinkte subzelluläre Lokalisation während der Interphase und auch Beladung an das mitotische Chromatin aufweisen. Die beiden Komplexe haben die Structural maintenance of chromosomes (SMC) Untereinheiten SMC2 und SMC4 gemein, während sie sich in den nicht-SMC-Untereinheiten unterscheiden: In Condensin I findet man die Komponenten Chromosome associated protein D2 (CapD2), CapG und CapH und in Condensin II die verwandten Proteine CapD3, CapG2 und CapH2. Während in Vertebraten die Rolle beider Condensin-Komplexe während der Mitose klar etabliert ist, zeichnet sich in Drosophila melanogaster ein anderes Bild ab. Mutanten in den Genen CapH2 und CapD3 zeigen keine mitotischen Phänotypen und ein zu CapG2 homologes Protein konnte bisher nicht identifiziert werden. Da CapG aus Drosophila ein ähnliches Lokalisationsverhalten wie die Condensin II-Untereinheiten der Vertebraten aufweist, wird CapG als Komponente beider Condensin-Komplexe diskutiert. Im Rahmen dieser Arbeit wurden die Funktion und die Regulation von CapG in Drosophila melanogaster, primär durch die Analyse von EGFP-fusionierten Proteinvarianten, näher untersucht. Es konnte gezeigt werden, dass der C-Terminus von CapG neben den hauptsächlichen Phosphorylierungsstellen auch Kernimportsequenzen enthält, welche die subzelluläre Lokalisation des Proteins während des Zellzyklus regulieren. Während der Interphase zeigt CapG im Zellkern eine Kolokalisation mit dem Heterochromatin-bindenden Protein HP1, was auf eine direkte Chromatinassoziation von CapG hinweist und mögliche Chromatin-strukturierende Funktionen bestätigen würde. Diese Heterochromatin-spezifische Lokalisation kann vom C-terminalen Drittel von Cap-G vermittelt werden, der andererseits für die generelle Chromatinassoziation während der Mitose, die Interaktion mit den anderen Condensin I-Untereinheiten und für die Funktionalität von CapG während der somatischen Mitosen entbehrlich ist. Da ein Proteinfragment entsprechend der N-terminalen Zwei-Drittel von CapG (CapG-NM) während der Interphase hauptsächlich zytoplasmatisch lokalisiert ist, kann die präferentielle Kernlokalisation von CapG während der Interphase, und auch die Phospho-Regulation des Proteins, für die Entwicklung vom Beginn der embryonalen zygotischen Expression bis hin zum adulten Stadium nicht essentiell sein. Die in dieser Arbeit nachgewiesene Lokalisation von CapG und anderen Condensin Untereinheiten in Geweben der weiblichen und männlichen Keimbahn legt die Vermutung nahe, dass CapG, eventuell im Kontext mit den anderen Condensin-Untereinheiten, eine Rolle bei der Gametogenese zukommt. Konsistent hiermit ist die Beobachtung, dass Weibchen steril sind, die in ihren Keimbahnzellen keine funktionelle CapG-Variante exprimieren. Die Integrität der Ovarien in diesen Tieren spricht für eine Funktion von CapG bei der Reifung des Oozytenkerns oder den meiotischen Teilungen. Die Präsenz von CapG in löslichen Komplexen mit den bekannten Condensin I-Untereinheiten konnte in dieser Arbeit durch Koimmunpräzipitationen mit nachfolgender massenspektroskopischer Bestimmung der assoziierten Proteine bestätigt werden. Eine Assoziation mit Condensin II-spezifischen Untereinheiten wurde dagegen nicht gefunden. Ebenso assembliert eine ektopisch exprimierte, funktionelle Variante von CapH2 nicht in einem löslichen Komplex mit CapG. Auch die Analyse direkter Protein-Protein-Interaktionen in einem in vitro-System ergab keine Hinweise auf eine Assoziation von CapG mit Condensin II-spezifischen Untereinheiten. Dagegen konnten in diesem System die Interaktionen nachvollzogen werden, die für Condensin I und Condensin II aus anderen Systemen publiziert und somit erwartet sind. Experimente zur genetischen Interaktion der Condensin-Untereinheiten offenbarten bei Anwesenheit von CapG-Mutationen eine Suppression des Phänotyps, der nach Überexpression von CapH2 in Speicheldrüsen erhalten wird. In einem reziproken Ansatz führten CapG-Mutationen zu einer Verstärkung des CapH2-mutanten Phänotyps in den Nährzellen von Ovarien. CapH2 und CapG scheinen also auf genetischer Ebene miteinander zu interagieren. Zusammengenommen legen die Ergebnisse ein Modell nahe, in dem CapG zwar nicht in direkter Assoziation mit Condensin II-spezifischen Komponenten vorliegt, aber parallel an der Strukturierung von Chromatin beteiligt ist und funktionell zum Teil mit den Aufgaben der Condensin II-spezifischen Komponenten überlappt.
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untitled document
(2009)
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Cathérine Conradty
- Multimediales Lernen, bspw. mit dem Computer, ist eine wichtige Unterstützung im Unterricht geworden. Wenn auch Wirkung und Nutzen kontrovers diskutiert werden, gehört der Computer inzwischen zum Lehrplan an deutschen Schulen. Computerhandhabung ist eine notwendige Kompetenz für das Berufsleben. Zudem gilt das Medium als motivationsfördernd, was sich positiv auf den Lernerfolg auswirken soll. Ein kritischer Faktor bei Multimedia-Lernen ist neben der Multimodalität die Unterrichtsform: meist wird der Computer in ansonsten eher seltener schülerzentrierter Freiarbeit eingesetzt. Das eine wie das andere kann an sich Schüler kognitiv überfordern und damit das Lernen erschweren. Im Studie A wird der Effekt auf kognitive Lernerfolg und motivationale Variablen bei variierter Lehrer-Anleitung in computerunterstützter Freiarbeit empirisch untersucht. Fehlt die aktive Lehrerbegleitung, war der Lernerfolg im Nachtest geringer. Nach sechs Wochen aber schnitten die völlig selbstständig Lernenden besser ab als die betreuten Schüler. Wissen, das selbstständig erarbeitet wurde, ist bei der computerunterstützten Freiarbeit konsistenter. Der durch während des Unterrichts empfundenen Druck und geringe Selbstkompetenz verursachte negative Effekt auf den Lernerfolg scheint hingegen durch gezielte Anleitung verhindert zu werden. Auch konnten mit Anleitung die Jungen für das Thema gewonnen werden - allerdings ohne bemerkenswerten Effekt auf den Lernerfolg. Wichtigster Faktor der Untersuchung scheint das Vorwissen zu sein, das den Lernerfolg, das Interesse und das Kompetenzgefühl steigert, und gleichzeitig den Stress im Unterricht senkt. Diese Ergebnisse lassen darauf schließen, dass tatsächlich eine völlig selbstständige computerunterstützte Freiarbeit lohnenswert ist, allerdings als Festigungsphase nach einem Vorwissen bildenden, evtl. lehrerzentrierten Vorunterricht. In Studie B wurde ein Medienvergleich zwischen Hypertext und Buchtext durchgeführt. Die computerunterstützte Gruppe variierte nach den Erfahrungen aus Studie I den Grad der Lehrerunterstützung: Ein Teil der Computer-Testgruppe hatte statt der Konsolidierung im Frage-Antworten-Spiel mit dem Lehrer ein computerunterstütztes Quiz. Wenn auch das gewohnte Buch kurzfristig zu besserem Lernerfolg verhalf, vergaßen die Schüler mit der computerunterstützten Einheit mit lehrerzentrierter Konsolidierung weniger. Der Lernerfolg war aber letztlich nicht abhängig vom Medium, sondern von der Lehrerbegleitung. Ohne Betreuung waren die Schüler demotiviert und lernten kaum, wobei besonders die Mädchen litten. In Studie C wurde eine Papier-& Bleistift-Methode des Multimedia-Lernens evaluiert, das Concept Mapping (CMing). Diese „persönlichen Landkarten der Konzepte“ scheinen v. a. als Konsolidierungsphase nach computerunterstütztem Lernen hilfreich zu sein. Hier wurden Concept Maps (CMs) über zwei unterschiedlich schwere Themen erstellt, um Fehler zu typisieren und auf ihre mögliche Ursachen zu untersuchen. Die Teilnehmer, obwohl völlig unerfahren, fühlten sich beim CMing sofort kompetent. Über das einfache Thema erstellten sie komplexe Wissenskarten. Hingegen nahmen bei dem zweiten, schwierigeren Thema die technischen Fehler zu. Dies scheint vor allem ein Problem der Verbalisierungsfähigkeit der Schüler zu sein. In Studie D wurde die Eignung des CMings zur Evaluierung von Schülerwissen untersucht. Bei der Diversität der Qualitätsmerkmale für CMs kann ihre Auswertung im Schulalltag kompliziert werden. Dafür reduzierten wir die Qualitätskriterien auf die Anzahl der richtigen Verbindungen in der Wissenskarte, der „Komplexität“. In den CMs über beide Themen korreliert die Komplexität signifikant mit dem kognitiven Nachtest und dies umso besser, wenn man bei der Korrektur technische Fehler vernachlässigte. Allerdings war der Korrelations-Koeffizient schwach. Da durch CMing ein vernetztes, verstehendes Lernen gefördert wird, rechneten wir damit, dass die Qualität der CM auf den Erfolg im spätere Wissenstest schließen lässt. Entsprechende Korrelation von Komplexität mit spätem Nachtest fanden wir allerdings nur bei dem schweren Thema. Eine Reduzierung der Qualitätsmerkmale auf die Komplexität unterschätzt das abgebildete Wissen der Schüler. Studien C und D zeigen, dass sich CMing sehr gut eignet, um spezifische Wissenslücken und Vorstellungen der Schüler aufzudecken, auf die der Lehrer dann seinen Unterricht adäquat ausrichten kann. Multimediale Lerneinheiten lohnen sich als schülerzentrierte Lernform. Gerade im Wechsel mit lehrerzentrierten Wissensvermittlungs-Stunden unterstützen sie den kognitiven Lernerfolg. Wichtiger noch ist vielleicht: die Vielfalt im Schulalltag fördert die Motivation sowohl der Schüler als auch der Lehrenden. Zusätzlich werden Kompetenzen wie Computerhandhabung, Selbstorganisation und eigenverantwortliches Lernen, Vernetztes Denken und Teamwork gefördert.
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Integrated analysis of relationships between 3D-structure, leaf photosynthesis, and branch transpiration of mature Fagus sylvatica and Quercus petraea trees in a mixed forest stand
(2001)
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Stefan Fleck
- Identifying the relevance of forest structure for stand photosynthesis and transpiration is one of the remaining challenges in plant physiological ecology. This thesis uses the historically new chances for the description of 3D-complexity of beech and oak forests that come along with the rapid developments in information technology: It gives a comprehensive description of measured structural features of both species and evaluates and visualizes them with a branch-oriented polyhedra-based 3D-representation (CRISTO). This allows for the first time the proof of a naturally layered structure of Fagus sylvatica and Quercus petraea canopies, as well as the quantification of gap volumes of horizontal layers. The structure description is combined with the measured variability of physiologically relevant leaf parameters throughout the single canopies. A historical trend towards increasing leaf mass per area (LMA)-values is detected from literature. Using LMA and nitrogen content as drivers, a new nitrogen dependent leaf photosynthesis model is designed and validated from A/Ci curves and daily courses of leaf photosynthesis. These measurements have been perormed on standing trees using a high-lift. The program RACCIA allows for the automated derivation of photosynthesis parameters for this model from A/Ci-curves. Leaf properties and 3D-structure are used in the 3D-light model STANDFLUX-SECTORS, which is capble to use a 3D-simulation of the same stand in an unprecedented high resolution and was validated with the LMA-light relationship from another stand. The combined evaluation of the simulated radiation distribution with sapflow measurements on single branches indicates a typical pattern of light sensitivity of transpiration that is interpreted in terms of species-specific ecological specialization.
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Eukaryotic chromosome segregation: New aspects of separase regulation by securin, Cdk1, PP2A and auto-cleavage
(2011)
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Franziska Böttger
- The universal triggering event of eukaryotic chromosome segregation is the proteolytic cleavage of chromosomal cohesin by separase. The activity of this essential but potentially also very dangerous protease must be tightly controlled. Prior to the onset of anaphase separase is kept inactive by association with either securin or cyclin-dependent kinase 1 (Cdk1) in conjunction with cyclin B1. Only when all chromosomes interact properly with the mitotic spindle apparatus does the anaphase promoting complex or cyclosome (APC/C), a multisubunit E3 ligase, mediate the ubiquitylation of securin and cyclin B1. Their subsequent proteasomal degradation then releases active separase. Murine embryonic stem cells, which lack securin and express a Cdk1-resistant phosphorylation site mutant separase are viable. Thus, additional regulations of sister chromatid separation by separase must exist. It was reported that human separase cleaves not only cohesin but also itself and, furthermore, that it interacts with protein phosphatase 2A (PP2A). However, the functions of separase's auto-cleavage and PP2A-interaction remain enigmatic. Moreover, securin was reported to also interact with PP2A but, strangely, with a different isoform of the phosphatase. Thus, the question needs clarification of whether separase or securin or both interact with which isoform of PP2A. In this study, further insights into the relationship between separase auto-cleavage and PP2A binding are presented. Phosphorylation of a serine residue in close proximity to the major cleavage site of separase was found to stimulate auto-cleavage of separase. Interestingly, a quantitative mass-spectrometric approach (SILAC) identified this serine residue as a substrate of separase-bound PP2A. Furthermore, a point mutation within separase was identified, which totally abolishes PP2A binding and which maps to the immediate vicinity of the auto-cleavage sites. Thus, PP2A prevents the auto-cleavage of separase both catalytically and sterically. It could further be shown that non-cleavable separase exhibits increased association with PP2A and that forced cleavage of separase displaces PP2A. Taken together, these results demonstrate that auto-cleavage and PP2A binding constitute two antagonistic mechanisms of separase regulation. Evidence is provided that the interaction of PP2A with securin is indirect and bridged by separase, and that it is the B56- and not the B55-isoform of PP2A which associates with the separase-securin complex. Moreover, free securin is shown to be degraded in early mitosis in a phosphorylation- and APC/C-dependent manner, while separase-associated securin is kept dephosphorylated and, thus, protected by PP2A. Securin levels are frequently increased in tumors. In normal cells, the early removal of excessive securin might later ensure swift separase activation and anaphase onset, thereby contributing to faithful chromosome segregation.
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Members of the Preprotein and Amino Acid Transporter Family Constitute Components of Novel Protein Import Pathways into Chloroplasts
(2011)
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Claudia Roßig
- In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membranes. Translocons at the outer and inner chloroplast envelope membranes, called TOC and TIC, were identified that mediate the import of proteins. N-terminal transit peptides essential for import of the protein precursors are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursors would enter the chloroplast through the same, jointly acting TIC/TOC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. One example is the chloroplast envelope quinone oxidoreductase homologue, ceQORH. This protein is imported into the inner plastid envelope membrane by a TIC/TOC-independent pathway and without any proteolytic cleavage. In the present study 5 proteins were shown to interact with ceQORH during its import and were designated as ceQORH translocon components (QTC). One of these proteins, QTC24 (also called HP20), is a member of the PRAT family comprising preprotein and amino acid transporters found in chloroplasts, mitochondria and free-living bacteria. Different expression patterns and localization of PRAT proteins suggest that they are functionally diverse beyond their role in protein translocation. QTC24/HP20 is located in the outer plastid envelope membrane of chloroplasts where it establishes a hydrophilic translocation pore. Thus, chloroplasts contain besides TOC75 and OEP16-1 a third translocation channel component in their outer envelope membrane that functions in import of transit sequence-less inner envelope proteins. In vitro import into chloroplasts of corresponding isolated A. thaliana knock-out mutants revealed that the lack of HP20 could not be replaced by its close relative HP22. Athp20 plants had no phenotype when grown under standard green house conditions. However, minor defects during the very early stage of greening of etiolated seedlings were observed as the expression of mainly plastid-encoded proteins was delayed. These effects could be interpreted in terms of an impaired amino acid import at this stage of development. A second protein of the PRAT family, HP30, was further subject of this work. However, its role remains unclear at the moment. Isolated homozygous A. thaliana knock-out mutants of HP30 did not reveal any phenotype under the growth conditions analysed in this work. The preliminary investigation of stable RNA silencing mutants indicated that the function of HP30 and its close relative HP30-2 is important during the early stages of seedling development. Young leaves of respective mutant plants exhibited a chlorotic phenotype. A further member of the PRAT family is OEP16-1 that was initially identified as amino acid-selective protein channel. Other studies revealed its role as translocation pore for the PORA precursor. Analysis of the corresponding A. thaliana knock-out mutant to dissect these two mutually not exclusive functions has led to the description of different phenotypes. During a re-screen of the original seed stock, four independent OEP16-1-deficient mutant lines were isolated that exhibited different cell death properties. Two mutants contained elevated amounts of free protochlorophyllide (Pchlide) in darkness that was caused by a defect in the Pchlide-dependent import of PORA. Etiolated seedlings of these lines died after light exposure due to the production of singlet oxygen. The two other mutants did not accumulate excessive amounts of free Pchlide and greened normally. Two of the four mutant lines with seemingly no correlation between the lack of PORA and cell death were analysed in more detail in this thesis. Moreover, a complemented Atoep16-1 mutant that re-expressed functional OEP16-1 protein was shown to restore the wild-type phenotype including PORA import that prevented the accumulation of an excess of free Pchlide and singlet oxygen production upon light exposure of dark-grown seedlings.
