Refine
Is part of the Bibliography
- yes (48) (remove)
Year of publication
- 2014 (48) (remove)
Document Type
- Doctoral Thesis (48) (remove)
Keywords
- Epigenetik (4)
- Maus (4)
- Bioinformatik (3)
- Genexpression (3)
- dSTORM (3)
- Bestäuber (2)
- Biodiversität (2)
- Chromatin (2)
- Fluoreszenzmikroskopie (2)
- Hochauflösendes Verfahren (2)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (48) (remove)
Sonstige beteiligte Institutionen
Toll-like receptors (TLR) are pattern recognition receptors (PRR) by which macrophages (MØ) sense pathogen-associated molecular patterns (PAMPs). The recognition of lipopolysaccharide (LPS), the PAMP of gram negative bacteria, by TLR4 triggers signaling cascades and leads to the pro-inflammatory activation of the cells. A recent quantitative and kinetic analysis of the phosphoproteome of LPS-activated primary macrophages highlighted the cytoskeleton as a cell compartment with an enriched protein phosphorylation. In total 44 cytoskeleton-associated proteins were regulated by this post-translational modification and thus might be involved in the control and regulation of key macrophage functions like spreading, motility and phagocytosis.
To investigate the control of cytoskeleton-associated cell functions by TLR4 activation, we first developed a method to quantitatively measure the spreading response of bone marrow MØ after stimulation with LPS. Fluorescence microscopy was used for cell imaging and visualisation of the MØ contact area. In collaboration with the Fraunhofer Institute Erlangen, we developed and validated a software tool for the semi-automated segmentation and quantitation of MØ fluorescence microscopy data, which allowed fast, robust and objective image analysis. Using this method, we observed that LPS caused time-dependent spreading, which was detectable after 1-2 h and maximal after 24 h. Next, the impact of genetic or pharmacological inhibition of known TLR signaling components was investigated. Deficiency in the adapter protein MYD88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of ERK1/2 signaling, indicating that ERK1/2 mediates MYD88-dependent MØ spreading. In contrast, MØ lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The genetic deletion of the MAPK phosphatases DUSP1 and DUSP16 resulted in impaired late spreading, corroborating the essential role for functional MAPK signaling in TLR4-driven MØ spreading.
To identify the contribution of other cytoskeletal phosphoproteins to MØ spreading, siRNA knockdown of selected candidate genes in primary murine MØ was employed and combined with automated quantitative image analysis. These experiments revealed a functional role for the Myosins MYO1e and MYO1f in MØ spreading. These motor proteins are strongly phosphorylated in LPS-activated MØ. Because of their ability to simultaneously bind to actin filaments and cell membrane or other proteins, we investigated their role in phagocytosis, cytokine production and antigen presentation. Phagocytosis and killing of bacteria were not affected in Myo1e-/- macrophages. However, MYO1e plays a role in chemokine secretion and antigen presentation processes. MCP1 (CCL2) release was selectively increased in Myo1e-deficient MØ and dendritic cells (DC), while cytokine secretion was unaffected. Furthermore, macrophages and DCs lacking MYO1e showed lower levels of MHC-II on the cell surface. However, mRNA levels of CCL2 and of MHC-II were unaltered. These data suggest a role for MYO1e in the transport of selected chemokines and of MHC-II molecules to the cell surface. MHC-II-restricted antigen presentation assays revealed an impaired capacity of macrophages and DC lacking MYO1e to stimulate antigen-specific T cells, suggesting that the reduced MHC-II expression is functionally relevant.
Taken together, in this study first a quantitative image analysis method was developed which allows the unbiased, robust and efficient investigation of the macrophage spreading response. Combination of this method with siRNA knockdown of selected cytoskeleton-associated phosphoproteins led to the identification of MYO1e and MYO1f as regulators of macrophage spreading. Furthermore, we identified MYO1e in MØ and DC to be essential for the intracellular transport of CCL2 and MHC-II to the cell surface and for optimal stimulation of antigen-specific CD4 T cells.
The cytokine Interleukin-4 (IL-4) plays a crucial role in the pathophysiology and progression of asthma and other atopic diseases. Its activities are signaled into the cells upon binding to and signaling through a shared receptor complex composed of the subunits IL-4Rα and common γc. Another cytokine, Interleukin-13 shares many functions with IL-4. This can be explained by the fact that both, IL-4 and IL-13, can signal via a shared receptor complex comprising the IL-4R and the IL-13R1 subunit.
Therefore, the IL-4Rα receptor subunit has become a highly promising drug target, since it mediates IL-4 and IL-13 responses and blocking IL-4Rα will abrogate IL-4 as well as IL-13 effector functions. Currently, an IL-4 based mutein (Pitrakinra), acting as a dual IL-4/IL-13 receptor antagonist is in clinical development.
This work describes the generation and production of biologically active IL-4 muteins, which contain a single additional engineered cysteine. The introduction of a free thiol group allows site-specific chemical modification. The muteins were expressed in E. coli in insoluble form, refolded and purified. The thiol group of the mutein was protected as mixed disulfide with the tripeptide glutathione.
A first attempt to chemically reduce the engineered cysteine residue failed, because the three native disulfide bonds of IL-4 exhibit a similar reactivity and chemical reduction of the native disulfide resulted in full deactivation and precipitation of the IL-4 protein. Therefore, an enzymatic approach was developed which specifically reduces the mixed disulfide bonds with an attached glutathion moiety and thus leaves the native structurally essential disulfide bonds unaltered. For optimization, four different IL-4 cysteine muteins with four cysteine residues introduced at positions close to the IL-4Rα binding site were tested and their reduction rates by glutaredoxin was determined. The enzymatic reduction occured at different rates for all four muteins indicating that accessibility is an important influence and must be determined individually for each mutant protein. After optimization of the pH value and particularly the reaction time, all muteins could be prepared with the engineered thiol group being released in reasonable yield. The proteins exhibiting the free thiol group were then modified by
N-ethylmaleimide (NEM) or maleimido-PEG. The effects of these modifications at different positions on binding to IL-4R were measured employing SPR biosensor technology.
In the second project of this study, foldamers, which represent a new class of stable, compactly folded biomolecules and can specifically interact with proteins and nucleic acids, were examined to identify their potential as new drugs to interfere with IL-4 activities.
Fragment-based drug discovery offers great promise for providing new starting points for drug discovery and facilitates the lead optimization. As foldamers equipped with a thiol-group for tethering could not to be produced; only the effect of foldamers present in a synthesized foldamer library on the binding to IL-4R could be tested. Two libraries containing different foldamers based on aromatic amide were synthesized by Michael Grotz and Dr. Michael Deligny and tested in our lab for their capability to disrupt the ligand-receptor interaction of IL-4 and its receptor IL-4Rα [ECD] using surface plasmon resonance technology. None of the studied foldamers could specifically inhibit the IL-4/IL-4Rα interaction. Some foldamers showed non-specific binding.
The study presented here shows the design and production of a potentially new type of IL-4 antagonists, which employ site-specific chemical modification to exert their antagonistic function.
Die Identifikation der Bindungsspezifitäten von Proteininteraktionsdomänen und damit letztlich auch die Fähigkeit potentielle Bindungspartner dieser in vivo vorherzusagen bildet ein grundlegendes Element für das Verständnis der biologischen Funktionen dieser Domänen. In dieser Arbeit wurde untersucht, inwieweit solche Vorhersagen bezüglich der SH3-Domäne – als Beispiel für eine Proteininteraktionsdomäne – mithilfe von Support-Vector-Machines (SVMs) möglich sind, wenn diesen als Informationsquelle ausschließlich die innerhalb der Aminosäuresequenz der Domäne konservierten Informationen zur Verfügung stehen. Um den SVM-basierten Klassifikator zu trainieren und zu validieren, wurde ein Satz aus 51 SH3-Domänen verwendet, die zuvor entsprechend ihrer Ligandenpräferenz in ein System aus acht verschiedenen Klassen eingeteilt worden waren. Da die innerhalb der Aminosäuresequenzen konservierten Informationen in abstrakte Zahlenwerte konvertiert werden mussten (Voraussetzung für mathematisch basierte Klassifikatoren wie SVMs), wurde jede Aminosäuresequenz durch ihren jeweiligen Fisher-Score-Vektor ausgedrückt. Die Ergebnisse erbrachten einen Klassifikationserror, welcher weit unterhalb des Zufallsniveaus lag, was darauf hindeutet, dass sich die Bindungsspezifität (Klasse) einer SH3-Domäne in der Tat von seiner Aminosäuresequenz ableiten lassen dürfte. Mithilfe klassenspezifisch emittierter, artifizieller Sequenzen, implementiert in den Trainingsprozess des Klassifikators, um etwaigen nachteiligen Auswirkungen von Overfitting zu entgegenzuwirken, sowie durch Berücksichtigung taxonomischer Informationen des Klassensystems während Training und Validierung, ließ sich der Klassifikationserror sogar noch weiter senken und lag schließlich bei lediglich 35,29% (vergleiche Zufall: 7/8 = 87.50%). Auch die Nutzung von Feature Selections zur Abmilderung Overfitting-bedingter, negativer Effekte lieferte recht vielversprechende Ergebnisse, wenngleich ihr volles Potential aufgrund von Software-Beschränkungen nicht ausgenutzt werden konnte.
Die Analyse der Positionen im Sequence-Alignment, welche für den SVM- basierten Klassifikator am relevantesten waren, zeigte, dass diese häufig mit Positionen korrelierten, von denen angenommen wird auch in vivo eine Schlüsselrolle bei der Determination der Bindungsspezifität (Klasse) zu spielen. Dies unterstreicht nicht nur die Reliabilität des präsentierten Klassifikators, es gibt auch Grund zur Annahme, dass das Verfahren möglicherweise auch als Supplement anderer Ansätze genutzt werden könnte, welche zum Ziel haben die Positionen zu identifizieren, die die Ligandenpräferenz in vivo determinieren. Informationen, die nicht nur für ein besseres Verständnis der SH3-Domäne (und möglicherweise auch anderer Proteininteraktionsdomänen) von grundlegender Bedeutung sind, sondern auch aus pharmakologischer Sicht von großem Interesse sein dürften.
Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis.
Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm.
Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels.
At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites.
Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation.
Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation.
The consequences of habitat change for human well-being are assumed to be especially extreme in Burkina Faso. The country is located in a highly drought-sensitive zone of West Africa, and small‐scale subsistence farmers may be especially affected if losses of biodiversity lead to changes in ecosystem functioning; many depend on more or less degraded lands for agricultural production.
The overall aim of the present thesis consequently was to characterize the functional traits of soil-organisms which are crucial for a productive and balanced soil environment in the study region – termites and ants. They are true ecosystem engineers whose activity alters the habitat. Through soil-turnover in the course of constructing biogenic structures of varying size and nature (mounds, nests, galleries, soil-sheetings, foraging-holes), they bioturbate huge amounts of soil masses and exert massive effects on soil structure, positively influencing the fertility, stability, aeration and water infiltration rate into soils; and they provide habitats for other species. In sub-Saharan Africa, ants and termites are the only active soil macrofauna during the long dry season; in the sub-Sahel zone of Burkina Faso, termites even represent the only active, quantitatively remarkable decomposers all year round. Since no information was available about the actual diversity of the focal arthropods, I divided the thesis in two main parts: In the first part, a baseline study, I assessed the local termite and ant fauna, and investigated their quantitative and qualitative response to changing habitat parameters resulting from increasing human impact (‘functional response traits’). In the second and applied part, I addressed the impact of the biogenic structures which are important for the restoration of degraded soils (‘functional effect traits’).
Two traditional agricultural systems characteristic for the study region were selected. Each system represented a land-use intensification gradient comprising four distinct habitats now differing in the magnitude of human intervention but formerly having the same initial state. The first disturbance gradient, the temporal cross-section of a traditional soil water conservation technique to restore degraded heavily encrusted, barren soil named Zaï in Ouahigouya (Yatenga province, sub-Sahel zone); the second disturbance gradient, an agriculture type using crop rotation and fallow as nutrient management techniques near Fada N’Gourma (Gourma province, North-Sudanese zone).
No standard protocol existed for the assessment of termite and ant diversity in semi-arid (agro-) ecosystems; two widely accepted standard protocols provided the basis for the newly revised and combined rapid assessment protocol ‘RAP’: the ALL protocol for leaf litter ants of Agosti and Alonso (2000), and the transect protocol for termites in tropical forests of Jones and Eggleton (2000). In each study site, three to four replicate transects were conducted during the rainy seasons (2004—2008).
The RAP-protocol turned out to be very effective to characterize, compare and monitor the taxonomic and functional diversity of termites and ants; between 70% and 90% of the estimated total species richness were collected on all levels (transects, habitats, regions). Together in both regions, 65 ant species (25 genera) and 39 termite species (13 genera) were collected. These findings represent the first records for Burkina Faso. The data indicate a high sensitivity of termites and ants to land-use intensification. The diversity strongly decreased with increasing anthropogenic impact in the North-Sudan region. In total, 53 ant species (23 genera) and 31 termite species (12 genera) were found. Very promising results concerning the recovery potential of the soil-arthropods’ diversity were gathered in the Zaï system. The diversity of both taxa strongly increased with increasing habitat rehabilitation – in total, 41 ant species (16 genera) and 33 termite species (11 genera) were collected. For both taxa significant differences could be noted in the shape of the density variations along the gradient. For instance termites: Fungus-growers showed the greatest adaptability to different management practices. The greatest variations between the habitats were observed in soil and grass-feeding termites. Whole functional groups were missing in heavily impacted habitats, e.g. soil-, grass-, and wood-feeders were absent in the degraded site in the sub-Sahel zone. Several environmental parameters could be identified which significantly explained a great part of the variations in the composition of the arthropods’ communities; they indicate the importance of the habitats’ structural complexity (vegetation structure) and concomitant effects on diurnal temperature and moisture fluctuations, the availability of food sources, and the soil-structure. The diversity of termites in the sub-Sahel region was strongly correlated with the crown-cover percentages, the topsoils’ sand-content, and the availability of litter; in the North-Sudan region with the cumulated woody plant basal area, the topsoils’ clay- and organic matter-content. The parameters identified for ant communities in the Zaï system, were the height of trees, the topsoils’ clay-content and air humidity; in the North-Sudan region the habitats’ crown-cover percentages, the quantity of litter and again the height of trees.
In the second part of the thesis, I first rapidly assessed the (natural) variations in the amount of epigeal soil-structures along the two disturbance gradients in order to judge the relative importance of termites and ants for soil-turnover. The results illustrated impressively that a) in all study sites, termites were the main bioturbators while ant structures were of minor importance for soil turn-over; b) earthworms and grass-feeding termites contributed significantly to soil turn-over in the more humid North-Sudan region; and c) the bioturbated soil mass varied between seasons and years, however, the relative importance of the different taxa seemed to be fairly constant. In the sub-Sahel zone, fungus-growing Odontotermes and Macrotermes species fully take over the important function of bioturbation, leading to the transport of huge amounts of fine-textured soil material to the surface; with increasing habitat restoration, coarse fragments decreased in the upper horizons and became concentrated deeper along the soil profile.
Consequently, in the applied part, I concentrated on the bioturbation activity of fungus-growing termites in the four main stages of the Zaï system: crusted bare soil (initial stage), millet field, young and old forest. In each of the four Zaï sites nine experimental blocks (each comprising four plots of 1m2) were used to stimulate the foraging activity of fungus-growing termites with different, locally available organic materials (Aristida kerstingii hay, Bombax costatum wooden blocks, compost and a control without any organic amendment). The experiment was conducted twice for the duration of four weeks (rainy season 2005, dry season 2006). The plots were regularly checked and the increase of the area covered by sheetings chronologically followed. After four weeks a) all sheeting-soil was collected, air dried and separately weighed according to the different genera, and b) the foraging-holes were counted and their diameter measured. Additionally, c) ponded water infiltration was measured in selected plots, and d) the physicochemical properties of sheeting-soil were analyzed. In case of complete consumption of the offered hay during the experimental 4-weeks-duration, the same procedure (a, b) was followed before adding new hay to the respective plot.
The comparison between the different plots, sites and seasons revealed clearly that hay was the most attractive bait; for each gram of hay removed, Odontotermes brought about 12 g soil to the surface, Macrotermes 4 g. Odontotermes was the only genus attracted by organic material to the degraded area, and was therefore the decisive primary physical ecosystem engineer in the Zaï system, initiating the restoration process. The mass of soil bioturbated in the course of foraging increased strongly from the degraded, barren towards the most rehabilitated reforested site. Combining all 36 experimental plots per Zaï stage, Odontotermes bioturbated 31.8 tons of soil per hectare and month dry season in the degraded area, and 32.4 tons ha-1 mon-1 in the millet fields; both genera moved 138.9 tons ha-1 mon-1 in the young and 215.5 tons ha-1 mon-1 in the old Zaï forest. Few comparable figures were found in the literature. In northern Burkina Faso, both genera constructed 20 tons of sheetings ha-1 mon-1 after mulching with a straw-wood mixture (Mando & Miedema 1997), and in Senegal, around 10 tons ha-1 mon-1 were moved in heavily foraged plots (Rouland et al. 2003). Within a site, soil turn-over and the number of foraging holes created was always highest in hay, followed by compost, then by wood and in the end control. The fungus-growers’ foraging-activity was leading to an enormous increase in surface pore space – after one month of induced foraging activity in hay-plots, the median number of foraging-holes increased from 142 m-2 in the degraded site up to 921 m-2 in the old Zaï forest. The creation of subterranean galleries and macropores significantly increased the water infiltration rate by a mean factor 2–4.
Laboratory analyses revealed that sheeting-soil differed strongly from the respective control soil as well as between the seasons, the food-type covered, and the two genera. Odontotermes-sheetings differed in more parameters than Macrotermes-sheetings, and dry season sheetings differed in more parameters (and more strongly) than rainy season sheetings. In the present study, soil organic matter, carbon and nitrogen contents were significantly increased in all dry season sheetings; in the rainy season mainly in those built on compost. Texture analysis pointed out that both genera used topsoil and soil from deeper horizons in varying mixture ratios, thereby supporting findings of Jouquet et al. (2006).
To summarize, the present thesis contributes to a better understanding of the functional response traits of termites and ants to changing environmental parameters resulting from increasing human impact. The RAP-protocol represents an easy-to-learn and very effective method to representatively characterize, compare and monitor the taxonomic and functional diversity of termites and ants. The experiment has provided conclusive evidence of the importance of the consideration of fungus-growing termites (particularly Odontotermes and Macrotermes species) when aiming to restore infertile, degraded and crusted soils and to maintain a sustainable agricultural production in the Sahel‐Sudanese zone of West Africa.
The contribution of botanical gardens to out-of-school education should be larger than it is currently in Germany. In the curricula of all school types botany plays only a minor role, although plants form the base for all animal life on earth. To increase the attractiveness of botanical gardens for teachers, offers and programs should be created and conducted in didactically sensible manners and allow students an emotional approach towards the topics through trial and experiments. Therefore it is insufficient to conduct guided tours, which are still most common. Student-centered methods, like learning at workstations, or experimental courses, can lead to an improved retention of the contents learned at the out-of-school learning setting. There are, however, methodological differences even within learning at workstations.
In the first part of my study I compared a student- (S) and a teacher-centered (T) type of learning at workstations (chapter III). My intention was to find out, which of both methods results in more positive emotions at the out-of-school learning location and a higher sustainable knowledge increase. Like in all three parts of my study, 8th grade students from so-called “Mittelschulen” and “Realschulen” from Lower Franconia participated in the programs. I evaluated them by using multiple-choice tests assessing the students' knowledge regarding the topic 'plants and water' (see Appendix), following a before-after / control-impact study design. The students' emotions were assessed using the intrinsic motivation inventory directly after the garden visit. Using generalized linear mixed models, I did not find a significant difference between either of the two approaches. A reason for this could be that the students could be practically active in both methods, which made them fairly similar. Given that there was a significant knowledge increase in both methods, and the effort to develop the teacher-centered learning at workstations was much lower, I would suggest to follow that method for educational work in botanical gardens.
Students already have many predefined concepts regarding many topics, especially when these are important in everyday life. These concepts do often not match the scientific state-of-the-art. Still, students bring their so-called 'alternative conceptions' into visits to the botanical garden. According to theory, confronting them with their own conceptions in the light of scientific facts, should foster updating their concepts with scientifically correct additions. To investigate this method regarding my topic 'plants and water', I developed an intervention with experiments on the lotus effect, which also plays a role in everyday life (chapter IV). Topics like the surface tension of the water, which is also found in 6th grade curricula in German schools, were included. Prior to the intervention, I assessed the students' conceptions using questionnaires and used the three most frequent alternative conceptions to develop a multiple-choice test, which was also used in a before-after / control-impact design. A group of students was also confronted with their conceptions during an introductory talk (AC), whereas another was not (NAC). This was conducted in a way, that likely led to dissatisfaction of the students with their own concepts. The analysis of the questionnaires with the Mann-Whitney U test showed, however, no difference between the two groups directly following the treatment. Over longer time, however, the NAC group retained significantly more knowledge. Probably the students confronted with the alternative conceptions remembered the illustrations of these more easily than the scientifically correct view. For some botanical topics it is certainly helpful to include this conceptual change approach, but apparently not for the lotus effect. In this case it is most sensible to focus on the surface structure of water-repellent leaves and fruits, as we describe it in a publication in 'Unterricht Biologie'. For the practical work in botanical gardens I would suggest to rather assess the students' concepts and assumptions in the beginning of an intervention in a botanical garden, especially with respect to feasibility.
In the third part of my study I concentrate on the application of concept maps (chapter V). This method of cross-linking old and newly acquired knowledge is effective, but not very common in Germany, neither in schools, nor in botanical gardens. One group of students followed exclusively a teacher-centered learning at workstations regarding 'plants and water' (NCM), a second group created concept maps directly after the treatment and a second directly before the retention test (CM). The first map was intended to be a means of consolidation, whereas the late map was rather focused on recapitulation of what was learned about six weeks ago. To evaluate that I used the same multiple-choice tests as I did for the first part. The CM group showed a significantly higher knowledge increase, over short and long time-scales, although these students did significantly worse in the pretest than those of the NCM group. Regarding genders, female students profited especially from the first concept map (consolidation), males rather from the second (recapitulation). From the results one can conclude that prominently weaker students benefit from this method. Additionally the gender-related results show that using concept maps multiple times can be beneficial for different types of learners.
In every study there also was a control group (C), which only had to fill out the questionnaires at the same time as the participating students, to account for external factors (like media, etc.).
Especially learning at workstations and concept maps are very appropriate to be conducted at the out-of-school learning location botanical garden and are likely to strongly increase learning success. It is beneficial to mix several methods to achieve the best results in different types of learners. Additionally, when methods in school are mixed with those of out-of-school learning, the education gets more open, practical and colorful. That all resulted in a substantial long-term knowledge gain of all participating students.
Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a vacuole, the so-called inclusion. During replication by a biphasic life-cycle Chlamydia secrete via their type 3 secretion system various effector proteins into the inclusion lumen, the inclusion membrane or the host cell cytosol to form their favored replication niche. Chlamydia-infected cells are highly resistant against apoptosis since the replicative form of Chlamydia is non-infectious and premature cell death would cause complete loss of one Chlamydia generation. The bacteria block apoptosis by preventing mitochondrial outer membrane permeabilization. Various proteins with anti-apoptotic function are enriched in Chlamydia-infected cells such as Mcl-1, cIAP2, Survivin or HIF1α. The accumulation of these proteins is a result of increased gene expression and direct protein stabilization. However, the molecular mechanisms and involved bacterial effector proteins are mostly unknown.
With this work the molecular mechanisms of Mcl-1 stabilization and the participation of chlamydial factors were investigated. Mcl-1 is a member of the Bcl-2 protein family and has an extremely short half-life causing its permanent ubiquitination and subsequent degradation by the 26S proteasome under normal homeostasis whilst Mcl-1 accumulation results in apoptosis inhibition. It was shown that during C. trachomatis infection Mcl-1 ubiquitination is reduced causing its stabilization albeit no cellular ubiquitin-proteasome-system components are involved in this process. However, C. trachomatis express the two deubiquitinases ChlaDUB1 and ChlaDUB2 which are mostly uncharacterized. With this work the expression profile, subcellular localization, substrates and function of the deubiquitinases were investigated. It was shown that ChlaDUB1 is secreted to the surface of the inclusion where it interacts with Mcl-1 which is accumulated in the proximity of this compartment. By utilization of infection experiments, heterologous expression systems and in vitro experiments a direct interaction of ChlaDUB1 and Mcl-1 was demonstrated. Furthermore, it was shown that Mcl-1 is deubiquitinated by ChlaDUB1 causing its stabilization. During replicative phase of infection, ChlaDUB2 seems to be accumulated in the chlamydial particles. However, ChlaDUB2 substrates could not be identified which would give an indication for the physiological role of ChlaDUB2.
Since 2011, a protocol to transform C. trachomatis with artificial plasmid DNA is available. As part of this work the transformation of C. trachomatis with plasmid DNA suitable for the permanent or inducible protein overexpression on a routinely basis was established. In addition, the first targeted homologous recombination into the chlamydial genome to replace the ChlaDUB1 gene by a modified one was performed and validated. The targeted homologous recombination was also used to create a ChlaDUB1 knock-out mutant; however deletion of ChlaDUB1 seems to be lethal for C. trachomatis. Due to the fact that ChlaDUB1-lacking Chlamydia could not be obtained an inhibitor screen was performed and identified CYN312 as a potential ChlaDUB1 inhibitor. Application of CYN312 during infection interfered with chlamydial growth and reduced Mcl-1 quantity in infected cells. Furthermore, CYN312 treated Ctr-infected cells were significantly sensitized for apoptosis.
Taken together, C. trachomatis secretes the deubiquitinase ChlaDUB1 to the surface of the inclusion where it deubiquitinates Mcl-1 causing its accumulation in infected cells resulting in apoptosis resistance. Application of the ChlaDUB1 inhibitor CYN312 interferes with Mcl-1 stabilization sensitizing infected cells for apoptosis.
WISP3 is a member of the CCN family which comprises six members found in the 1990’s: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes.
WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block
caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis.
Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.
Zytotoxische CD8+ T-Lymphozyten spielen in vielen inflammatorischen, aber auch primär neurodegenerativen Erkrankungen eine wichtige Rolle. Daher besitzt die Fragestellung inwiefern CD8+ ZTL Neurone direkt schädigen und ggf. welche mechanistischen Aspekte dieser Schädigung zugrunde liegen, eine hohe Relevanz. Um diese Fragestellung eingehender zu beleuchten, wurde mit dem OT-I-System gearbeitet. Dieses gut vorcharakterisierte CD8+ T-Zell-Modell besitzt den Vorteil, dass diese transgenen Zellen nur eine Peptidsequenz des Ovalbumin (OVA) Protein als spezifisches Antigen erkennen.
Zunächst wurden in der vorliegenden Arbeit Co-Kultivierungs-Experimente durchgeführt. Hierzu wurden akut isolierte murine Hippokampus-Neurone unter verschiedenen Bedingungen mit OT-I Lymphozyten co-kultiviert. Hierbei konnte gezeigt werden, dass unter Antigenpräsentation der Neurone signifikant mehr Neurone in die Apoptose/Nekrose geführt werden, als unter Kontroll-Bedingungen, in denen entweder kein Antigen oder ein Antigen, das nicht von OT-I Lymphozyten erkannt wird, präsentiert wird.
Nachdem die Antigen-abhängigen zytotoxischen Effekte auf Neurone gezeigt werden konnten, wurde mithilfe elektrophysiologischer Techniken die mechanistischen und funktionellen Konsequenzen des direkten neuronalen/OT-I-vermittelten Zellkontakts untersucht. Bei diesem experimentellen Ansatz wurde durch elektrisches Auslenken eines Neurons nach Kontakt mit einem OT-I Lymphozyt die passiven elektrischen Parameter der Neuronenmembran gemessen. In diesen Messungen konnte gezeigt werden, dass nach unmittelbarem Kontakt eines Neurons mit einem OT-I Lymphozyt der neuronale Membranwiderstand reduziert wird bzw. die Leitfähigkeit der Zellmembran erhöht wird. Diese Änderung der neuronalen Membran-Leitfähigkeit findet in einem Zeitraum von 10 min nach dem Zell-Zell-Kontakt statt. Auch hier konnte gezeigt werden, dass dieser Einfluss von OT-I Lymphozyten auf Neurone strikt Antigen-abhängig ist. Zur Untersuchung des Mechanismus der OT-I T-Lymphozyten auf Neurone wurde das Augenmerk auf verschiedene T-Zell-induzierte Apoptosewegegelegt. Es konnte gezeigt werden, dass durch Blockieren der Fas/FasL-Interaktion mittels eines Antikörpers kein Unterschied, weder in der neuronalen Apoptoserate nach Co-Kultivierung, noch eine Änderung der passiven neuronalen Membran-Leitfähigkeit auftritt. Weiterhin wurde die Rolle der von T-Zellen sezernierten Granula Perforin und Granzym B untersucht. Um den Einfluss dieser Granula aufzuklären, wurden OT-I Lymphozyten verwendet, die entweder defizient für Perforin oder Granzym B waren. In diesem experimentellen Ansatz wurde gezeigt, dass ausschließlich Perforin für die Erniedrigung des passiven neuronalen Membran-Widerstandes verantwortlich ist.
Diese Erhöhung der neuronalen Membranleitfähigkeit führte aber nicht direkt zum neuronalen Zelltod. Vielmehr wurde durch die einhergehende Depolarisation des Neurons die elektrische Aktivität der Zelle vermindert, sodass es zu einem sogenannten „electrical silencing“ kommt. Dieser Umstand konnte auch in der Betrachtung der spontanen Netzwerkaktivität von Neuronenkulturen gezeigt werden. Hierfür wurden hoch dichte Neuronenkulturen auf MEA-Chips kultiviert. Mit Hilfe dieser MEA konnten die Summenfeldpotentiale der Neuronenkulturen detektiert werden. Hierbei wurde beobachtet, dass nach Beladung der Neuronen mit dem spezifischen OT-I-Antigen und OT-I Zellen eine Verringerung der spontanen Netzwerkaktivität einhergeht. Auch in diesem Effekt konnte eine Antigen-Spezifität nachgewiesen werden.
Da der Prozess der zellulären Apoptose mit einem Anstieg der intrazellulären Ca2+-Konzentration einhergeht, und Perforin als Ca2+-durchlässiger unselektiver Porenbildner fungiert, wurden zur Überprüfung der Hypothese calcium imaging-Experimente durchgeführt. Analog zu den elektrophysiologischen Messungen wurde gezeigt, dass nach direktem Zell-Zell-Kontakt zwischen Neuron und OT-I Lymphozyt eine Erhöhung der intrazellulären Ca2+-Konzentration zu messen ist. Dass diese Änderung des neuronalen Ca2+-Einstroms durch Perforin-abhängige Membranporen hervorgerufen wird, konnte durch die Verwendung von Perforin-defizienten OT-I Lymphozyten bewiesen werden. Unter Verwendung von Perforin-defizienten OT-I Lymphozyten wurde keine Änderung der neuronalen Ca2+-Konzentration ermittelt. Weiterhin wurde in diesem experimentellen Ansatz gezeigt, dass auch der OT-I-vermittelte neuronale Ca2+-Anstieg strikt Antigen-abhängig ist.Zusammengefasst konnte in dieser Arbeit gezeigt werden, dass MHC-I/Antigen-vermittelte CD8+ Lymphozyten-Interaktion mit einem Neuron zu „electrical silencing“ des Neurons führt. Dieser Prozess ist klar Perforin-abhängig, führt jedoch nicht zum unmittelbaren Zelltod des Neurons.
This thesis explores the influence of social and environmental cues on the nest building behavior of leaf-cutting ants. Especially, the investigations are aimed at evaluating the mechanisms of nest building and how the nest environment can spatially guide building responses that lead to an adaptive nest architecture. The emergence of nest chambers in the nest of the leaf-cutting ant Acromyrmex lundi were evaluated. Rather than excavating nest chambers in advance, at places where workers encounter suitable environmental conditions for brood and fungus rearing, these items have to be present at a site. When presented in the laboratory with a choice between two otherwise identical digging sites, offering suitable environmental conditions, but one containing brood, the workers displayed a higher excavation activity at the site where they encountered the putative content of a chamber. The shape of the excavated cavity was also more round and chamber-like. It is concluded that leaf-cutting ants respond to social cues during nest building. Excavation is a costly process and colonies have to spend a part of their energy stores on nest building, so that regulatory responses for the control of nest excavation are expected to occur. Worker density at the beginning of the digging process influenced digging activity while the presence of in-nest stores did not. Stored brood and fungus did however influence the architecture of the excavated nest, leading to the excavation of larger chambers and smaller tunnels. While self-organized mechanisms appear to be involved in the nest building process, the social cues of the ants’ environment during building clearly influence the nest architecture and lead to an adjustment of the nest size to the current space needs of the colony. Workers secondarily regulated nest size by the opportunistic refilling of unused space with excavated soil pellets. As the ants should provide suitable conditions for brood and fungus rearing, they should show a behavioral response to CO2 concentrations, as the gas is known to hinder fungus respiration. Workers of A. lundi did indeed avoid high CO2-levels for fungus rearing but actually preferred CO2-values in the range encountered close to the soil surface, where this species excavates their nests. However, different CO2-levels did not affect their excavation behavior. While fungus chambers make up part of a leaf-cutting ant nest, most leaf-cutting ants of the genus Atta also spent part of the colony’s energy on excavating large, voluminous chambers for waste disposal, rather than scattering the material aboveground. It is expected that leaf-cutting ants also show environmental preferences for waste management. In experiments Atta laevigata workers preferred deposition in a warm and dry environment and showed no preference for specific CO2-levels. The continued accumulation of waste particles in a waste chamber seems to be based on the use of volatiles. These originate from the waste itself, and seem to be used as an orientation cue by workers relocating the material. The ensuing large accumulation of waste at one site should result in the emergence of more voluminous chambers for waste disposal.