Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

Für das Überleben von Bacillus subtilis ist eine verlässliche Überwachung der Integrität der Zellhülle essentiell, um diese zu schützen und bei Schäden adäquat zu reagieren. Neben den ECF � Faktoren spielen Zwei-Komponenten-Systeme (2KS) in der Zellhüllstressantwort von B. subtilis eine zentrale Rolle. Eines dieser Systeme, das LiaRS- 2KS reagiert auf eine große Anzahl verschiedener Zellwand-Antibiotika sowie andere zellhüllstress-auslösende Substanzen. Die zelluläre Funktion und Rolle des Lia-Systems konnte bisher nicht genau definiert werden. In der hier vorliegenden Dissertation wurde das Lia-System erstmals hinsichtlich seiner funktionalen Rolle in B. subtilis untersucht. Im ersten Teil der Ergebnisse wurde eine detaillierte Analyse der LiaR-vermittelten Zellhüllstressantwort in B. subtilisvorgenommen. Transkriptom-Studien dienten zur Identifizierung des LiaR-Regulons. Hierbei wurde die Genexpression des Wildtyps mit zwei Mutanten, die den „ON“ (�liaF) und „OFF“ (�liaR) Zustand des Lia-Systems repräsentierten, verglichen. Von den dabei identifizierten drei potentiellen LiaR-Zielloci (liaIH, yhcYZ-ydhA, ydhE) konnten durch anschließende Folgeuntersuchungen nur die Gene liaI und liaH als in vivo relevante Zielgene für LiaR verifiziert werden. Umfangreiche phänotypische Analysen zeigten, dass �liaIH-Mutanten nur schwach sensitiv auf einige Antibiotika sowie oxidativen Stress reagierten. Ebenso vermittelt eine Überexpression von LiaH in einer �liaF-Mutante keine Resistenz gegenüber stressauslösenden Substanzen. LiaH gehört zur Familie der Phagenschock-Proteine. Weitere Mitglieder dieser Familie sind PspA aus Escherichia coli und Vipp1 aus Arabidopsis thaliana, die große oligomere Ringstrukturen bilden. Die strukturelle Untersuchung von LiaH ergab, dass auch dieses Protein große Ringe bildet (>1MDa). Der zweite Ergebnisteil befasst sich mit der Untersuchung der Stimuluswahrnehmung der Zellhüllstress-detektierenden Systeme in B. subtilis. Die Zellhüllstressantwort auf das Antibiotikum Bacitracin wurde hierbei mittels �-Galaktosidase-Assay sowie Western Blot- Analyse erforscht. Das Bce-System reagiert dabei am stärksten und spezifischsten auf Bacitracin-Stress. Es wurde ebenfalls festgestellt, dass der ABC-Transporter BceAB essentiell für die Stimuluswahrnehmung ist und dass das Bce-System an sich eine Resistenzdeterminante in B. subtilis darstellt. Das Lia-System hingegen wird erst bei höheren Bacitracin-Konzentrationen induziert. Zusammengefasst deuten diese Ergebnisse darauf hin, dass das Bce-System Bacitracin direkt wahrnimmt (drug sensing) und das LiaSystem in indirekter Weise auf Zellhüllstress ausgelöst durch Bacitracin reagiert (damage sensing). Im dritten Teil der Ergebnisse wurdendie zelluläre Lokalisation von LiaI, LiaH und LiaG sowie die Beziehung der Proteine untereinander mittels Fluoreszenz-Mikroskopie und biochemische Ansätze untersucht. Die Membranproteine LiaI und LiaG sind unter Stressbedingungen in der Zellmembran lokalisiert. LiaH, ein cytoplasmatisches Protein verändert unter Stressbedingungen seine Lokalisation vom Cytoplasma an die Membran. Die Funktion von LiaH scheint sich also an der Zellmembran zu vollziehen, wobei LiaI als Interaktionspartner identifiziert wurde. Da in einer �liaI-Mutante LiaH unter Stressbedingungenebenfallsnoch an die Zellmembran assoziert ist, wurde nach weiteren Interaktionspartnern von LiaH gesucht. Eine umfangreiche bacterial-two-hybrid-Analyse ergab, dass sowohl LiaH als auch LiaI und LiaG in ein Interaktionsnetzwerk eingebettet sind, in welchem das bisher uncharakterisierte Protein YvlB eine Schlüsselrolle spielt.Die ebenso in dieses Netzwerk involvierten Proteine YjoB, DnaK und HtpG üben als Proteasen/Chaperone Funktionen in der Faltung und Degradierung von Proteinen aus. Ein Zusammenspiel des Lia-Systems und des Schlüsselproteins YvlB mit den Proteasen/Chaperonen als Reaktion auf Zellhüllstress ist denkbar. Die Phagenschock-Homologe PspA in Streptomyces lividans und E. coli üben einen erheblichen Einfluss auf die Proteinsekretion sowie die elektronenmotorische Kraft der Zelle aus. Daher wurde im letzten Teil der Ergebnisse die Rolle von LiaH in der Proteinsekretion sowie im Energiestoffwechsel näher analysiert. Ein Einfluß des Lia- Systems in der Aufrechterhaltung der elektronenmotorischen Kraft der Zelle konnte nicht bestätigt werden. Durch die Analyse des Sekretoms in B. subtilis konnte gezeigt werden, dass das extrazelluläre Proteom einer �PliaI-liaIH-Mutante im Vergleich zum Wildtyp signifikante Veränderungen in der Komposition aufwies.So wurde im Sekretom der �PliaIliaIH- Mutante vor allem das Zellwand-assoziierte Protein WapAidentifiziert, welches im Wildtyp oder in einer �liaF-Mutante nicht auftrat. Das Lia-System beeinflußt somit auch die Proteinsekretion von B. subtilis, wobei die molekularen Mechanismen noch unbekannt sind.

DNA methylation plays a crucial role in the epigenetic control of gene expression during mammalian development and differentiation. Whereas the de novo DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, establish DNA methylation patterns during development; Dnmt1 stably maintains DNA methylation patterns during replication. DNA methylation patterns change dynamically during development and lineage specification, yet very little is known about how DNA methylation affects gene expression profiles upon differentiation. Therefore, we determined genome-wide expression profiles during differentiation of severely hypomethylated embryonic stem cells (ESCs) lacking either the maintenance enzyme Dnmt1 (dnmt1-/- ESCs) or all three major Dnmts (dnmt1-/-; dnmt3a-/-, dnmt3b-/- or TKO ESCs), resulting in complete loss of DNA methylation, and assayed their potential to transit in and out of the ESC state. Our results clearly demonstrate that upon initial differentiation to embryoid bodies (EBs), wild type, dnmt1-/- and TKO cells are able to activate differentiation processes. However, transcription profiles of dnmt1-/- and TKO EBs progressively diverge with prolonged EB culture, with dnmt1-/- EBs being more similar to wild type EBs, indicating a higher differentiation potential of dnmt1-/- EBs compared to TKO EBs. Remarkably though, after dissociation of late EBs and further cultivation under pluripotency promoting conditions, both dnmt1-/- and TKO but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation is dispensable for the initial activation of differentiation programs, it seems to be crucial for permanently restricting the developmental fate during differentiation. Based on the essential role of Uhrf1 in maintenance DNA methylation, we investigated the structurally highly similar second member of the Uhrf protein family, Uhrf2, whose function in maintenance methylation or other biological processes is completely unknown. Expression analysis of uhrf1 and uhrf2 in various cell lines and tissues revealed a time- and developmental switch in transcript levels of both genes with uhrf1 being highly expressed in undifferentiated, proliferating cells and uhrf2 being predominately expressed in differentiated, non-dividing cells. These opposite expression patterns together with no detectable effect on DNA methylation levels upon knock down of uhrf2 suggests that Uhrf2 is rather involved in maintaining DNA methylation patterns in differentiated cells and points to non-redundant functions of both proteins. The discovery of the “6th base” of the genome, 5-hydroxymethylcytosine (5hmC), resulting from the oxidation of 5mC by the family of Tet dioxygenases (Tet1-3), once again ignited the debate about how DNA methylation marks can be modified and removed. To gain insights into the biological function of this newly identified modification, we developed a sensitive enzymatic assay for quantification of global 5hmC levels in genomic DNA. Similar to 5mC levels, we found that also 5hmC levels dynamically change during differentiation of ESCs to EBs, which correlates with the differential expression of tet1-3. Furthermore, we characterized a novel endonuclease, PvuRts1I that selectively cleaves 5hmC containing DNA and show first data on its application as a tool to map and analyze 5hmC patterns in mammalian genomes. Finally, we investigated designer transcription activator-like effector (dTALEs) proteins targeting the oct4 locus. Our results show that the epigenetic state of the target locus interferes with the ability of dTALEs to activate transcriptionally silent genes, which however can be overcome using dTALEs in combination with low doses of epigenetic inhibitors. In conclusion, this work gives further insights into the biological roles of methylation mark writers (Dnmts), readers (Uhrfs) and modifiers (Tets) and advances our understanding on the function of DNA methylation in the epigenetic control of gene expression during development and cellular differentiation.

Stress is part of everyday life. And while acute and short periods of stress can help to overcome challenges, exposure to chronic - and especially uncontrollable - stress can lead to maladaption of the organism, which can ultimately increase the risk of disease. However, vulnerability to stress and vulnerability to risk increases are strongly dependent on the individual. The molecular underpinnings of this vulnerability and resilience are still largely unknown. Therefore, the present thesis aims at identifying novel molecules involved in modulating individual stress vulnerability in the brain of male mice. In a first step, we investigated long-term gene expression changes in the hippocampus of male mice that underwent chronic social stress. Adolescent male CD1 mice were subjected to 7 weeks of chronic social stress and were investigated after 5 weeks of recovery via an unbiased whole-genome approach utilising microarray technology. Here, we did not find strong differences caused by the stress exposure, possibly because not all animals were affected by the stress exposure. Nevertheless, we identified Iffo1 as a gene that seems to be affected by at least acute stress and might also be involved in the long-term effect of chronic stress exposure. In the next step, we classified the animals from the same paradigm into stress-vulnerable and stress-resilient individuals based on their corticosterone levels after recovery. Animals which still showed elevated levels of corticosterone 5 weeks after stress were defined as vulnerable, while animals in which levels returned to baseline comparable to controls were termed stress resilient. With an additional whole-genome experiment, we were able to show distinct patterns of gene expression between the groups, including genes like Arc, Gria1 and Gria2. In addition, we also investigated differences in peripheral lymphocytes, which showed regulation in genes like Hsp90b1 or SLA. When we compared the expression profiles between brain and peripheral blood, we showed that stress-vulnerable and stress-resilient animals show different patterns of correlations. In the final part of the thesis, we decreased the expression of Arc, one of the genes we found overrepresented in vulnerable individuals, in the hippocampal formation of male mice via AAV-mediated shRNA knockdown. As we performed the modulation of Arc before the stress exposure, we were able to investigate the causal influence of Arc expression on stress exposure. Animals that were subjected to 3 weeks of chronic social defeat, showed an increase in anxiety-related behaviour, impairment in spatial memory, an increase in social behaviour and did not differ in depression-like behaviour. Concomitant with the behavioural alterations, stressed animals showed alterations in multiple physiological parameters, like increased adrenal glands or corticosterone response. Intriguingly, we were able to prevent most of the behavioural, but not the physiological, changes with the Arc knockdown. This strongly suggests that Arc is at least partly causally involved in the molecular machinery that underlies stress vulnerability. As Arc is a downstream molecule in multiple pathways already connected to stress vulnerability or stress in general, it might be that Arc actually is one of the major molecular factors that translate the effects of these pathways.

Only a small fraction of the estimated species diversity on Earth already has been discovered, and expected high extinction rates force biologists to rapid surveys. Molecular barcoding techniques meet such goals, but taken alone they can hardly connect genetic discoveries with the large morphology-based body of taxonomic knowledge that accumulated during centuries. Also, the study of organismic evolution requires reliable information on phenotypes. Morphological and biological knowledge on formally described species can be, however, very heterogeneous regarding both quality and quantity. Especially problematic are meiofaunal taxa – biodiversity generally is poorly explored, and species are small, hard to collect, externally quite uniform and difficult to distinguish by means of traditional taxonomic techniques. Old species descriptions often are fragmentary and information may or may not be reliable. Novel microanatomical imaging techniques raised the hope to combine the rapid examinations with the obligatory accuracy and desired comprehensiveness of structural information obtainable. Among the most successful interstitial gastropod taxa are the Acochlidia, combining extremely high morphological and biological diversity with modest species diversity. The state of research at the beginning of my PhD thesis considered the Acochlidia as poorly known, enigmatic and morphologically and biologically aberrant Opisthobranchia, comprising only 27 valid species. Most of the acochlidian species are marine mesopsammic and distributed along the coasts of the world’s oceans, but some species succeeded to invade freshwater systems on tropical islands. Uniquely among the otherwise hermaphroditic euthyneurans, some acochlidians have separate sexes. Previous sampling efforts were biased to European waters and a few other places that had been visited by experts. Original descriptions of the acochlidian species were often limited to the external morphology, the structure of calcareous spicules and the examination of the radula by light microscopy; furthermore, some anatomical data were traditionally obtained from gross-morphological dissection or from paraffin-based histology. Inner acochlidian classification was controversial and neither morphology-based nor molecular phylogenetic studies resolved the origin of this traditional “order” among euthyneuran heterobranch gastropods. In a case study for Mollusca, and for the first time for heterobranch gastropods, I comparatively explored the microanatomy of a representative sampling of known acochlidian taxonomic diversity applying computer-aided 3D reconstructions with Amira based on serial semi-thin histological sections. My dissertation aimed (1) to revise the morphology and taxonomy of acochlidians, including the most dubiously and incompletely described species, (2) to generate detailed microanatomical data sets for comparative purposes, (3) to reconstruct global acochlidian phylogeny and major traits of their evolution, and (4) to explore the power and the limits of modern microanatomy against traditional taxonomy and molecular approaches, and to develop integrative approaches. Original type material was traced in museums and institutions according to the literature and loaned for re-examination whenever possible. Most of the acochlidian species were re-collected at the type localities. Seven acochlidian species covering seven of eight families were re-examined in full detail; other species were studied to the level necessary and possible considering time constraints; additionally five species were introduced new to science. The microanatomical part of my dissertation clearly demonstrates that traditional acochlidian taxonomy did not provide sufficiently detailed and reliable anatomical information. In contrast, computer-based 3D reconstructions with the software Amira are an efficient, powerful tool for microanatomy, providing a wealth of new data on all major organ systems of the Acochlidia. Transforming specimens into serial histological sections is “invasive”, but generates vouchers that carry testable information. Semithin-sectioning (1-2 µm) and staining as applied herein provide resolution adequate to trace relevant organs, ducts and tissues; limits of this method refer to quantitative detection of fine nerves. The process of preparing complete 3D models is time consuming, but greatly supports accurateness of finding and identifying structures and includes several steps of internal quality control. 3D models, especially when interactive, are attractive and instructive, comprise verifiable high-quality data, and revealed considerable amounts of erroneous data within original species descriptions. Former outliers – i.e. apparently aberrant and enigmatic species - fit well into the pattern of known acochlidian species after the correction of the original data. 3D modeling from serial sections as applied herein is discussed as the best currently available method for exploring complex soft part microanatomy in small invertebrate specimens. Using the verified and supplemented morphological data, more than 100 morphological characters were defined and coded for all 27 acochlidian species considered valid at that time, and 11 euthyneuran outgroups. A cladistic analysis with PAUP recovered monophyletic Acochlidia originating from an unresolved basal opisthobranch level. The Acochlidia split into the Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia (‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina (Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))). The formerly enigmatic Ganitidae, resembling sacoglossan opisthobranchs by having dagger-like rhachidian radular teeth, were recovered as highly derived microhedylids. This topology is largely well-supported, robust to modifications of outgroup taxon sampling, and in principles was supported by a recent multi-locus molecular analysis. In addition, molecular analyses revealed the formerly enigmatic, amphibious Aitengidae also clustering within hedylopsacean Acochlidia. Although my phylogenetic hypothesis is not considered definitive, the paraphyly of some of the traditionally recognized family level taxa induced a preliminary reclassification of the Acochlidia. Rarely among invertebrates, morphology-based and molecular acochlidian topologies are compatible, and thus may closely reflect natural relationships. Major traits of the acochlidian evolutionary history were reconstructed tracing character state changes on the tree. The previous hypothesis of a general regression of morphological complexity in the Acochlidia applies only for microhedylacean species. Within Microhedylacea, we confirmed a tendency towards successive reductions, particularly in the reproductive system. Species are aphallic, sperm transfer occurs by spermatophores and dermal fertilisation and the secondary gonochorism evolved once in the ancestor of the Microhedylidae. In contrast, already basal hedylopsacean species show a complex excretory system adapted to a freshwater influenced environment. An evolutionary trait from a simple unarmed copulatory system towards complex hypodermal injection systems was recognised culminating in a large, trap-like spiny rapto-penis of several limnic Acochlidiidae. In spite of a high level of convergence involved, precise microanatomical data sets on a vast (yet incomplete) ingroup sampling thus allowed reconstructing a novel, plausible and detailed hypothesis on acochlidian phylogeny and evolution. This approach may have considerable potential also in other groups with similarly small and rare members that are elusive to molecular studies. Limits of morphology-based phylogeny concern any subgroups with just limited information available, old and possibly rapid diversifications, such as the origin of Acochlidia among Euthyneura, and relatively recent subgroups with little phenotypical differences fixed. We show that traditional taxonomy fails to differentiate some genetically clearly distinct lineages. In Pseudunela, sophisticated microanatomy alone cannot reliably delimitate all of the evolutionary lineages, but may reveal diagnosable differences among pseudocryptic species once they have been delimited by molecular analyses. Integrative taxonomy combining modern microanatomical data on acochlidians with molecular analyses thus is superior to individual approaches. With all key species revised in microanatomical and testable detail, and many additional species compared to such standard, now the Acochlidia probably range among the best described heterobranch groups. There is, however, still a critical gap of knowledge regarding biological observations and ontogenetic stages. Future work also should focus on resolving the exact origin of Acochlidia among Panpulmonata and on generating comparative anatomical data from potential sister groups. In spite of the urgency for speed facing the biodiversity crisis, my dissertation showed the essential need for revisory work on acochlidians, and this may be true also for other poorly known micromolluscs. Integrative 3D microanatomical and molecular approaches as exemplified herein are efficient, and thus suitable to explore the diversity and evolution of neglected micromolluscs within overall reasonable time scales.

An aberrant regulation of the hypothalamic-pituitary-adrenal (HPA) axis is closely associated with the pathophysiology of affective disorders such as major depression (MD). Accordingly, patients suffering from MD frequently show profound neuroendocrine alterations with hyper- or hypo-cortisolism as a result of a dysregulated stress hormone system. Focussing on this key endophenotype of MD, the ‘stress reactivity’ (SR) mouse model was recently established, consisting of three independent mouse lines, the high (HR), intermediate (IR) and low (LR) stress reactivity line, selectively bred for differences in their corticosterone (CORT) secretion in response to a psychological stressor. Previous studies revealed distinct differences between HR, IR and LR animals regarding sleep architecture, activity rhythms, emotional behaviour, cognition as well as neuroendocrine functions, resembling several endophenotypes observed in depressed patients. In the series of studies presented in this work, we aimed to investigate whether the differences between HR, IR and LR mice were restricted to the peripheral phenomenon of adrenal CORT secretion, or whether these endophenotypes were brought about by an aberrant regulation of upstream control centres of the HPA axis. To this end, we performed experiments investigating all functional levels of HPA axis control, i.e. the adrenals, the pituitary and brain centres known to be involved in the neuroendocrine stress response. Moreover, we assessed the expression of corticosteroid-binding globulin (CBG), which contributes to the transport and delivery of CORT to its target tissues. Finally, we studied HPA axis regulatory mechanisms by means of the combined dexamethasone/corticotropin-releasing hormone (Dex/CRH) test. At the level of the adrenal, we found that pharmacological inhibition of the biosynthesis and secretion of CORT using metyrapone had a significant impact on the stress-coping behaviour of HR, IR and LR animals as determined in the forced swim test (FST). As another peripheral factor influencing the secretion of CORT, we assessed the adrenal sensitivity of the animals to adrenocorticotropic hormone (ACTH) in vivo. After a Dex-mediated inhibition of endogenous ACTH release from the anterior pituitary, LR animals showed a markedly reduced CORT surge compared to HR mice in response to a stimulation of the adrenals with two doses of exogenous ACTH, indicating an enhanced adrenal sensitivity in HR mice and a blunted responsiveness to ACTH in LR mice. In addition, we found significant differences in plasma CBG levels between the three mouse lines (HR>IR>LR), concomitant with differences in free plasma CORT both, basal and in response to 15 min restraint stress (HR>IR>LR). Since only free CORT is biologically active, these results indicate that CBG might play a role in the endophenotypes of the SR mouse lines. At the pituitary level, we detected significantly altered ACTH protein levels (HR>IR≥LR) and proopiomelanocortin mRNA expression (HR>IR>LR), suggesting a differential activation of the anterior pituitary between the three lines, which is in line with the observed differences in stress reactivity. In the brain, we assessed the neuronal activation induced by an acute stressor in regions known to be involved in HPA axis function such as the prefrontal cortex, the basolateral amygdala, the hippocampus and the paraventricular nucleus of the hypothalamus (PVN) using c-fos in-situ hybridisation. Acute stress exposure markedly increased neuronal activation in all investigated brain areas. However, significant differences in the neuronal excitation between the three lines were only detected in the PVN (HR>IR>LR), indicating an altered activation of the animals’ HPA system orchestrated by this nucleus. Finally, we assessed HPA axis regulatory mechanisms by means of the Dex/CRH test. Our results revealed considerable similarities to clinical studies, with HR mice showing signs of Dex non-suppression in addition to an overshooting CORT surge after CRH stimulation, mirroring the HPA axis hyper-active state of patients suffering from the psychotic or melancholic subtype of MD. In contrast, LR animals presented a strong Dex-induced CORT suppression and a blunted response to the CRH stimulation, resembling the situation observed in atypically depressed patients. Strikingly, chronic fluoxetine (Flx) treatment enhanced the negative feedback regulation of the HPA axis in all three lines of the SR mouse model. In particular, the Dex-mediated CORT suppression in HR mice was restored by Flx. Furthermore, Flx affected the stress-coping behaviour of the animals in the FST. Flx induced a reduction in active coping, indicating an attenuation of the hyper-aroused state, particularly in HR mice. Taken together, the series of studies presented here demonstrated that the SR mouse model shows functional alterations on all levels of the HPA axis - peripheral, central and regarding the regulation – similar to the endophenotypes of MD patients, thus revealing a high level of face and construct validity of the model. Hence, the SR mouse model can serve as a valuable tool in the discovery and validation of new drug targets and improve already existing treatments of MD, particularly those targeting the HPA system.

Anxiety disorders are among the most common psychiatric diseases and contribute to the development of other psychiatric conditions, such as major depression, leading to a high impairment of daily life quality. Although it is obvious that the physiological architecture of neuronal networks and its modifications are essential for the ability of the brain to process incoming information and to control highly organized behaviour, the mechanisms underlying anxiety disorders still remain poorly understood. We focused our attention on two brain structures, which are strongly involved in emotional responses of mammals, namely the hippocampus and the amygdala. Both structures belong to the limbic system and play fundamental roles in information processing. Recent findings indicate that alterations in neuronal network properties of these two brain areas critically contribute to the development of such disorders. To potentially uncover changes in neuronal network features associated with abnormal anxiety, we performed experiments in a well-established animal model of extremes in trait anxiety, the high vs. low anxiety-related behaviour (HAB/LAB) mice. HAB mice exposed to an enriched environment (HAB E.E.) and stressed LAB mice (LAB Str.) were also used in the present study. HAB E.E. animals showed decreased anxiety compared to standard HABs, whereas LAB Str. animals displayed an increase in anxiety levels compared to standard LABs. Anxiety levels were measured by means of the elevated plus maze. For our investigations, we employed classical electrophysiological techniques and high-speed voltage-sensitive dye imaging (VSDI) in acute hippocampal (dorsal & ventral) and amygdalar brain slices. Field potential recordings revealed that HAB animals exhibit weaker long-term potentiation at CA3-CA1 synapses (CA1 LTP) in the dorsal hippocampus and an increased LTP in the ventral hippocampus compared to LAB and control CD1 mice. These observations could support the idea of an exacerbated activation of the “emotional” (ventral) hippocampus concomitantly with a decreased activity in the “cognitive” (dorsal) hippocampus, findings that have also been made in patients suffering from anxiety disorders. To examine whether neuronal activity propagation through the amygdala differs between HAB, HAB E.E., LAB and LAB Str. mice, we used a quantitative VSDI approach. Our results demonstrate that HAB animals exhibit stronger neuronal activity propagation through the amygdala compared to LAB mice. This indicates that differences in anxiety levels may correlate with the effectiveness of neuronal activity flow through the amygdalar network. Our study also provides strong evidence that environmentally induced shifts in trait anxiety are associated with changes in intrinsic amygdalar network properties. To summarize, HAB animals showed increased “excitability” in the ventral hippocampus and in the amygdalar network, both structures known to be involved in the control of emotional states and in the stress response in mammals. In addition, the differences in amygdalar network activity were rescued by environmental conditions (enriched environment). Dysregulation of these structures could lead to the “pathologic anxiety-like” behaviour, which can be observed in HAB animals.

Aspergillus fumigatus is a saprophytic mold that naturally inhabits the soil. Asexual reproduction yields hardy conidia that circulate in the air and are inhaled daily by humans. The fungus seems not to have evolved distinct mechanisms of pathogenicity, but is capable of responding to many stressful environmental cues present in its naturally harsh niche. The robust conidia present no problem to a fully functioning immune system, but if the innate immune system is compromised, the conidia can become activated and differentiate within the lung tissue to form invasive and disseminating hyphae. The resulting disease is called aspergillosis and is difficult to detect and to treat. To date, scientists have yet to find the factor(s) missing during immunosuppression that allow a healthy patient to easily dispose of A. fumigatus. We explored two possibilities: the production of neutrophil extracellular traps (NETs) and the release of IFN-γ by natural killer (NK) cells. We report here that NETs alone cannot kill the fungus, but do inhibit polar growth. Elongation of hyphal tips is abrogated due to zinc starvation, likely a consequence of the zinc-chelating, NETs-associated protein calprotectin. NK cells alone are also incapable of fungicidal activity, but their release of IFN-γ upon contact with A. fumigatus abrogates hyphal growth by a yet unknown mechanism. In vitro studies of the innate immune response, though helpful, are far from representative of the in vivo response. Neither NETs nor IFN-γ alone can manage Aspergillus infection, but in combination, these and other immune assaults certainly can. The difficulty lies in identifying the precise combination of immune cells and cytokine milieu that in a healthy individual prevent infection. Additionally, we explored mechanisms by which the fungus responds to stress, namely the HOG MAPK pathway, historically involved in osmotic stress response. In filamentous fungi, certain stress signals are sensed by a cytoplasmic hybrid histidine kinase sensor and then passed through the HOG system via phosphorylation. We identified the putative hybrid sensor kinase in A. fumigatus, and generated a corresponding knockout mutant. The ΔtcsC mutant was indeed sensitive to osmotic stress, and resistant to the phenolpyrrole fungicide fludioxonil. In the wild type the addition of either osmotic stress or fludioxonil resulted in SakA phosphorylation and translocation to the nucleus. SakA, the Hog1 homolog in A. fumigatus, is located at the end of the HOG pathway, confirming the role of TcsC as the cytoplasmic sensor upstream of SakA. In hypoxia, on farnesol, and in high concentrations of divalent cations the ΔtcsC mutant exhibited a striking “fluffy” phenotype characterized by the production of tremendous aerial hyphae and little or no differentiation, i.e., no conidiation. Though the ΔtcsC mutant showed no change in virulence compared to wild type, components of the TcsC signalling pathway remain promising targets for antifungal agents.

Besides inducing transcription by activating nuclear receptors, corticosterone (CORT) acts rapidly to alter cellular activity through multiple signaling cascades. Pharmacology-based experiments presented here show that nanomolar doses of CORT activate several pathways in primary hippocampal cultures within 20 minutes, a time-frame that excludes genomic mechanisms mediated by classical glucocorticoid (GR) and mineralocorticoid (MR) nuclear receptors. Moreover, none of these effects were subject to inhibition by either a series of structurally-unrelated antagonists of the classical GR and MR or by inhibitors of translation. Accordingly, a major aim of this work was to identify mechanisms proximal to the cell membrane that could potentially mediate these rapid actions of CORT. Initial time-course studies showed that 10 nM CORT rapidly triggers protein tyrosine kinase (PTK) activation that can be blocked by the small molecule inhibitor, PP2. It was also found that Src kinase is rapidly activated by CORT through tyrosine phosphorylation at Y416 and dephosphorylation at Y527. These events were subject to regulation by another key PTK member, Pyk2 (proline-rich tyrosine kinase 2). A series of experiments designed to study the potential mechanisms that regulate Pyk2, revealed that CORT increases the phosphorylation status of at least three tyrosine sites within Pyk2, namely, Y402, Y579/580 and Y881. Further, pharmacological probing uncovered requisite roles for other signaling pathways, such as the classical PLC-PKC pathway and the “novel” PKA and PKB pathways. Interestingly, further investigations revealed that the rapid CORT-induced activation of Pyk2/Src occurs in a G-protein dependent manner. This, together with the observation that membrane-impermeable BSA-conjugated CORT (CORT-BSA) produces similar responses profile to that obtained with CORT, led to pilot experiments to probe whether GPR30, a recently-described G protein-coupled receptor that appears to transduce signals from other steroid ligands, might be the putative receptor for CORT. Support for this view was provided by the finding that the rapid actions of CORT and CORT-BSA on Pyk2/Src activation could be blocked by a novel inhibitor of GPR30. Nevertheless, further studies will be needed to establish the role of GPR30 more firmly. Other studies sought to identify downstream targets of Pyk2/Src. Results show that Pyk2/Src regulates activation of c-Abl (another PTK) and RhoA, both of which are regulators of a number of cellular processes, including actin cytoskeleton remodeling. Furthermore, it was found that CORT triggers phosphorylation of the NR2B subunit of NMDAR, increases surface expression of NMDAR and activates downstream MAP kinases; all of these events depend on Src, one of whose direct substrates is the NR2B subunit. In a summary, the evidence presented in this dissertation suggests that, by acting via a G protein-coupled receptor, rather than through classical nuclear receptor mechanisms, CORT rapidly activates a series of intracellular signaling cascades that lie proximal to the neuronal plasma membrane; Pyk2/Src are early kinases involved and beyond these divergent pathways come into play, ultimately influencing functions ranging from rearrangements of the actin cytoskeleton, spine structure, scaffold protein clustering and function, to synaptic plasticity and transcriptional regulation.