Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cells

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

Effect of multiple sources of disturbance on rocky benthic assemblages in some localities of Salento, Apulia

The first part of my work consisted in samplings conduced in nine different localities of the salento peninsula and Apulia (Italy): Costa Merlata (BR), Punta Penne (BR), Santa Cesarea terme (LE), Santa Caterina (LE), Torre Inserraglio (LE), Torre Guaceto (BR), Porto Cesareo (LE), Otranto (LE), Isole Tremiti (FG). I collected data of species percentage covering from the infralittoral rocky zone, using squares of 50x50 cm. We considered 3 sites for location and 10 replicates for each site, which has been taken randomly. Then I took other data about the same places, collected in some years, and I combined them together, to do a spatial analysis. So I started from a data set of 1896 samples but I decided not to consider time as a factor because I have reason to think that in this period of time anthropogenic stressors and their effects (if present), didn’t change considerably. The response variable I’ve analysed is the covering percentage of an amount of 243 species (subsequently merged into 32 functional groups), including seaweeds, invertebrates, sediment and rock. 2 After the sampling, I have been spent a period of two months at the Hopkins Marine Station of Stanford University, in Monterey (California,USA), at Fiorenza Micheli's laboratory. I've been carried out statistical analysis on my data set, using the software PRIMER 6. My explorative analysis starts with a nMDS in PRIMER 6, considering the original data matrix without, for the moment, the effect of stressors. What comes out is a good separation between localities and it confirms the result of ANOSIM analysis conduced on the original data matrix. What is possible to ensure is that there is not a separation led by a geographic pattern, but there should be something else that leads the differences. Is clear the presence of at least three groups: one composed by Porto cesareo, Torre Guaceto and Isole tremiti (the only marine protected areas considered in this work); another one by Otranto, and the last one by the rest of little, impacted localities. Inside the localities that include MPA(Marine Protected Areas), is also possible to observe a sort of grouping between protected and controlled areas. What comes out from SIMPER analysis is that the most of the species involved in leading differences between populations are not rare species, like: Cystoseira spp., Mytilus sp. and ECR. Moreover I assigned discrete values (0,1,2) of each stressor to all the sites I considered, in relation to the intensity with which the anthropogenic factor affect the localities. 3 Then I tried to estabilish if there were some significant interactions between stressors: by using Spearman rank correlation and Spearman tables of significance, and taking into account 17 grades of freedom, the outcome shows some significant stressors interactions. Then I built a nMDS considering the stressors as response variable. The result was positive: localities are well separeted by stressors. Consequently I related the matrix with 'localities and species' with the 'localities and stressors' one. Stressors combination explains with a good significance level the variability inside my populations. I tried with all the possible data transformations (none, square root, fourth root, log (X+1), P/A), but the fourth root seemed to be the best one, with the highest level of significativity, meaning that also rare species can influence the result. The challenge will be to characterize better which kind of stressors (including also natural ones), act on the ecosystem; and give them a quantitative and more accurate values, trying to understand how they interact (in an additive or non-additive way).

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Tactile Perception - Perception of tactile distance on a single skin surface changes with stimulus orientation: a neuronal network modelling study

L’interazione che abbiamo con l’ambiente che ci circonda dipende sia da diverse tipologie di stimoli esterni che percepiamo (tattili, visivi, acustici, ecc.) sia dalla loro elaborazione per opera del nostro sistema nervoso. A volte però, l’integrazione e l’elaborazione di tali input possono causare effetti d’illusione. Ciò si presenta, ad esempio, nella percezione tattile. Infatti, la percezione di distanze tattili varia al variare della regione corporea considerata. Il concetto che distanze sulla cute siano frequentemente erroneamente percepite, è stato scoperto circa un secolo fa da Weber. In particolare, una determinata distanza fisica, è percepita maggiore su parti del corpo che presentano una più alta densità di meccanocettori rispetto a distanze applicate su parti del corpo con inferiore densità. Oltre a questa illusione, un importante fenomeno osservato in vivo è rappresentato dal fatto che la percezione della distanza tattile dipende dall’orientazione degli stimoli applicati sulla cute. In sostanza, la distanza percepita su una regione cutanea varia al variare dell’orientazione degli stimoli applicati. Recentemente, Longo e Haggard (Longo & Haggard, J.Exp.Psychol. Hum Percept Perform 37: 720-726, 2011), allo scopo di investigare come sia rappresentato il nostro corpo all’interno del nostro cervello, hanno messo a confronto distanze tattili a diverse orientazioni sulla mano deducendo che la distanza fra due stimoli puntuali è percepita maggiore se applicata trasversalmente sulla mano anziché longitudinalmente. Tale illusione è nota con il nome di Illusione Tattile Orientazione-Dipendente e diversi risultati riportati in letteratura dimostrano che tale illusione dipende dalla distanza che intercorre fra i due stimoli puntuali sulla cute. Infatti, Green riporta in un suo articolo (Green, Percpept Pshycophys 31, 315-323, 1982) il fatto che maggiore sia la distanza applicata e maggiore risulterà l’effetto illusivo che si presenta. L’illusione di Weber e l’illusione tattile orientazione-dipendente sono spiegate in letteratura considerando differenze riguardanti la densità di recettori, gli effetti di magnificazione corticale a livello della corteccia primaria somatosensoriale (regioni della corteccia somatosensoriale, di dimensioni differenti, sono adibite a diverse regioni corporee) e differenze nella dimensione e forma dei campi recettivi. Tuttavia tali effetti di illusione risultano molto meno rilevanti rispetto a quelli che ci si aspetta semplicemente considerando i meccanismi fisiologici, elencati in precedenza, che li causano. Ciò suggerisce che l’informazione tattile elaborata a livello della corteccia primaria somatosensoriale, riceva successivi step di elaborazione in aree corticali di più alto livello. Esse agiscono allo scopo di ridurre il divario fra distanza percepita trasversalmente e distanza percepita longitudinalmente, rendendole più simili tra loro. Tale processo assume il nome di “Rescaling Process”. I meccanismi neurali che operano nel cervello allo scopo di garantire Rescaling Process restano ancora largamente sconosciuti. Perciò, lo scopo del mio progetto di tesi è stato quello di realizzare un modello di rete neurale che simulasse gli aspetti riguardanti la percezione tattile, l’illusione orientazione-dipendente e il processo di rescaling avanzando possibili ipotesi circa i meccanismi neurali che concorrono alla loro realizzazione. Il modello computazionale si compone di due diversi layers neurali che processano l’informazione tattile. Uno di questi rappresenta un’area corticale di più basso livello (chiamata Area1) nella quale una prima e distorta rappresentazione tattile è realizzata. Per questo, tale layer potrebbe rappresentare un’area della corteccia primaria somatosensoriale, dove la rappresentazione della distanza tattile è significativamente distorta a causa dell’anisotropia dei campi recettivi e della magnificazione corticale. Il secondo layer (chiamato Area2) rappresenta un’area di più alto livello che riceve le informazioni tattili dal primo e ne riduce la loro distorsione mediante Rescaling Process. Questo layer potrebbe rappresentare aree corticali superiori (ad esempio la corteccia parietale o quella temporale) adibite anch’esse alla percezione di distanze tattili ed implicate nel Rescaling Process. Nel modello, i neuroni in Area1 ricevono informazioni dagli stimoli esterni (applicati sulla cute) inviando quindi informazioni ai neuroni in Area2 mediante sinapsi Feed-forward eccitatorie. Di fatto, neuroni appartenenti ad uno stesso layer comunicano fra loro attraverso sinapsi laterali aventi una forma a cappello Messicano. E’ importante affermare che la rete neurale implementata è principalmente un modello concettuale che non si preme di fornire un’accurata riproduzione delle strutture fisiologiche ed anatomiche. Per questo occorre considerare un livello astratto di implementazione senza specificare un’esatta corrispondenza tra layers nel modello e regioni anatomiche presenti nel cervello. Tuttavia, i meccanismi inclusi nel modello sono biologicamente plausibili. Dunque la rete neurale può essere utile per una migliore comprensione dei molteplici meccanismi agenti nel nostro cervello, allo scopo di elaborare diversi input tattili. Infatti, il modello è in grado di riprodurre diversi risultati riportati negli articoli di Green e Longo & Haggard.

Synaptic connectivity in micropatterned networks of neuronal cells

The presented thesis describes the formation of functional neuronal networks on an underlying micropattern. Small circuits of interconnected neurons defined by the geometry of the patterned substrate could be observed and were utilised as a model system of reduced complexity for the behaviour of neuronal network formation and activity. The first set of experiments was conducted to investigate aspects of the substrate preparation. Micropatterned substrates were created by microcontact printing of physiological proteins onto polystyrene culture dishes. The substrates displayed a high contrast between the repellant background and the cell attracting pattern, such that neurons seeded onto these surfaces aligned with the stamped structure. Both the patterning process and the cell culture were optimised, yielding highly compliant low-density networks of living neuronal cells. In the second step, cellular physiology of the cells grown on these substrates was investigated by patch-clamp measurements and compared to cells cultivated under control conditions. It could be shown that the growth on a patterned substrate did not result in an impairment of cellular integrity nor that it had an impact on synapse formation or synaptic efficacy. Due to the extremely low-density cell culture that was applied, cellular connectivity through chemical synapses could be observed at the single cell level. Having established that single cells were not negatively affected by the growth on patterned substrates, aspects of network formation were investigated. The formation of physical contact between two cells was analysed through microinjection studies and related to the rate at which functional synaptic contacts formed between two neighbouring cells. Surprisingly, the rate of synapse formation between physically contacting cells was shown to be unaltered in spite of the drastic reduction of potential interaction partners on the micropattern. Additional features of network formation were investigated and found consistent with results reported by other groups: A different rate of synapse formation by excitatory and inhibitory neurons could be reproduced as well as a different rate of frequency-dependent depression at excitatory and inhibitory synapses. Furthermore, regarding simple feedback loops, a significant enrichment of reciprocal connectivity between mixed pairs of excitatory and inhibitory neurons relative to uniform pairs could be demonstrated. This phenomenon has also been described by others in unpatterned cultures [Muller, 1997] and may therefore be a feature underlying neuronal network formation in general. Based on these findings, it can be assumed that inherent features of neuronal behaviour and cellular recognition mechanisms were found in the cultured networks and appear to be undisturbed by patterned growth. At the same time, it was possible to reduce the complexity of the forming networks dramatically in a cell culture on a patterned surface. Thus, features of network architecture and synaptic connectivity could be investigated on the single cell level under highly defined conditions.

Impact of forest fragmentation and disturbance on the endangered tropical tree Prunus africana (Rosaceae)

Anthropogene Fragmentierung und Störung von Wäldern beeinflussen ökologische Prozesse. Darüber hinaus werden genetische Drift und Inzucht verstärkt und die Fitness von Populationen beeinträchtigt. Um die Einflüsse von Fragmentierung und Störung auf die Biodiversität und Prozesse in tropischen Wäldern zu ermitteln, habe ich im „Kakamega Forest“, West-Kenia, die Baumart Prunus africana genauer untersucht. Dabei lag der Fokus auf (i) der Frugivorengemeinschaft und Samenausbreitung, (ii) der Kleinsäugergemeinschaft im Kontext der Samenprädation und (iii) der genetische Populationsstruktur von Keimlingen und adulten Bäumen. Der Vergleich von Keimlingen mit adulten Bäumen ermöglicht es, Veränderungen im Genfluss zwischen Generationen festzustellen. Die Ergebnisse zeigten, dass im untersuchten Waldgebiet insgesamt 49 frugivore Arten (Affen und Vögel) vorkommen. Dabei lag die Gesamtartenzahl im zusammenhängenden Wald höher als in den isoliert liegenden Fragmenten. An den Früchten von P. africana konnten insgesamt 36 Arten fressend beobachtet werden. Hier jedoch wurden in Fragmenten eine leicht erhöhte Frugivorenzahl sowie marginal signifikant erhöhte Samenausbreitungsraten nachgewiesen. Der Vergleich von stark gestörten mit weniger gestörten Flächen zeigte eine höhere Gesamtartenzahl sowie eine signifikant höhere Frugivorenzahl in P. africana in stark gestörten Flächen. Entsprechend war die Samenausbreitungsrate in stark gestörten Flächen marginal signifikant erhöht. Diese Ergebnisse deuten darauf hin, dass die quantitative Samenausbreitung in fragmentierten und gestörten Flächen etwas erhöht ist und somit eine gewisse Artenredundanz besteht, die den Verlust einzelner Arten ausgleichen könnte. Prunus africana Samen, die auf dem Boden lagen, wurden hauptsächlich von einer Nagerart (Praomys cf. jacksonii) erbeutet. Dabei war in gestörten Waldbereichen eine tendenziell höhere Prädatoraktivität zu beobachten als in weniger gestörten. Zudem waren einzelne Samen im Gegensatz zu Samengruppen in gestörten Flächen signifikant höherem Prädationsdruck ausgesetzt. Diese Ergebnisse zeigen, dass Fragmentierung sowie anthropogene Störungen auf unterschiedliche Prozesse im Lebenszyklus eines tropischen Baumes gegensätzliche Effekte haben können. Eine Extrapolation von einem auf einen anderen Prozess kann somit nicht erfolgen. Die genetische Differenzierung der adulten Baumpopulationen war gering (FST = 0.026). Der Großteil ihrer Variation (~ 97 %) lag innerhalb der Populationen, was intensiven Genfluss in der Vergangenheit widerspiegelt. Die genetische Differenzierung der Keimlinge war etwas erhöht (FST = 0.086) und ~ 91 % ihrer Variation lag innerhalb der Populationen. Im Gegensatz zu den adulten Bäumen konnte ich für Keimlinge ein „Isolation-by-distance“-Muster feststellen. Somit sind erste Hinweise auf begrenzten Genfluss im Keimlingsstadium infolge von Fragmentierung gegeben. Obwohl die Momentaufnahmen im Freiland keine Abnahme in der Frugivorenzahl und Samenausbreitung von P. africana als Folge von Fragmentierung beobachten ließen, weisen die Ergebnisse der genetischen Studie auf einen bereits reduzierten Genaustausch zwischen den Populationen hin. Somit lässt sich feststellen, dass die Faktoren Fragmentierung und Störung genetische Diversität, ökologische Prozesse und Artendiversität in Wäldern jeweils auf unterschiedliche Weise beeinflussen. Um Konsequenzen derartiger Einflüsse folgerichtig abschätzen zu können, sind Studien auf unterschiedlichen Diversitätsebenen unabdingbar.

Neuronal differentiation and epithelial integrity: the role of Drosophila Short stop

The nervous system is the most complex organ in animals and the ordered interconnection of neurons is an essential prerequisite for normal behaviour. Neuronal connectivity requires controlled neuronal growth and differentiation. Neuronal growth essentially depends on the actin and microtubule cytoskeleton, and it has become increasingly clear, that crosslinking of these cytoskeletal fractions is a crucial regulatory process. The Drosophila Spectraplakin family member Short stop (Shot) is such a crosslinker and is crucial for several aspects of neuronal growth. Shot comprises various domains: An actin binding domain, a plakin-like domain, a rod domain, calcium responsive EF-hand motifs, a microtubule binding Gas2 domain, a GSR motif and a C-terminal EB1aff domain. Amongst other phenotypes, shot mutant animals exhibit severely reduced dendrites and neuromuscular junctions, the subcellular compartmentalisation of the transmembrane protein Fasciclin2 is affected, but it is also crucially required in other tissues, for example for the integrity of tendon cells, specialised epidermal cells which anchor muscles to the body wall. Despite these striking phenotypes, Shot function is little understood, and especially we do not understand how it can carry out functions as diverse as those described above. To bridge this gap, I capitalised on the genetic possibilities of the model system Drosophila melanogaster and carried out a structure-function analysis in different neurodevelopmental contexts and in tendon cells. To this end, I used targeted gene expression of existing and newly generated Shot deletion constructs in Drosophila embryos and larvae, analyses of different shot mutant alleles, and transfection of Shot constructs into S2 cells or cultured fibroblasts. My analyses reveal that a part of the Shot C-terminus is not essential in the nervous system but in tendon cells where it stabilises microtubules. The precise molecular mechanism underlying this activity is not yet elucidated but, based on the findings presented here, I have developed three alternative testable hypothesis. Thus, either binding of the microtubule plus-end tracking molecule EB1 through an EB1aff domain, microtubulebundling through a GSR rich motif or a combination of both may explain a context-specific requirement of the Shot C-terminus for tendon cell integrity. Furthermore, I find that the calcium binding EF-hand motif in Shot is exclusively required for a subset of neuronal functions of Shot but not in the epidermal tendon cells. These findings pave the way for complementary studies studying the impact of [Ca2+] on Shot function. Besides these differential requirements of Shot domains I find, that most Shot domains are required in the nervous system and tendon cells alike. Thus the microtubule Gas2 domain shows no context specific requirements and is equally essential in all analysed cellular contexts. Furthermore, I could demonstrate a partial requirement of the large spectrin-repeat rod domain of Shot in neuronal and epidermal contexts. I demonstrate that this domain is partially required in processes involving growth and/or tissue stability but dispensable for cellular processes where no mechanical stress resistance is required. In addition, I demonstrate that the CH1 domain a part of the N-terminal actin binding domain of Shot is only partially required for all analysed contexts. Thus, I conclude that Shot domains are functioning different in various cellular environments. In addition my study lays the base for future projects, such as the elucidation of Shot function in growth cones. Given the high degree of conservation between Shot and its mammalian orthologues MACF1/ACF7 and BPAG1, I believe that the findings presented in this study will contribute to the general understanding of spectraplakins across species borders.

Frugivores, seed dispersal and tree regeneration along a human disturbance gradient in East African tropical rainforests

Samenausbreitung und Regeneration von Bäumen sind wichtig für den langfristigen Bestand von Baum- und Frugivorengemeinschaften in tropischen Regenwäldern. Zunehmende Rohdung und Degradation gefährden den Ablauf dieser mutualistischen Prozesse in diesem Ökosystem. Um den Einfluss von kleinräumiger menschlicher Störung auf die Frugivorengemeinschaft und die zentralen Ökosystemprozesse Samenausbreitung und Regeneration zu erforschen, habe ich 1) die Frugivorengemeinschaft und die Samenausbreitungsrate von Celtis durandii (Ulmaceae) und 2) den Zusammenhang zwischen Baumarten mit fleischigen Früchten, Frugivoren und der Etablierung von Keimlingen dieser Baumarten in unterschiedlich stark gestörten Flächen dreier ostafrikanischer tropischer Regenwälder untersucht. Insgesamt konnte ich 40 frugivore Vogel- und Primatenarten in den drei untersuchten Waldgebieten nachweisen. Auf gering gestörten Flächen wurden mehr Frugivore als auf stark gestörten Flächen aufgenommen. Auch die Beobachtungen an C. durandii ergaben mehr frugivore Besucher in Bäumen auf gering gestörten als auf stark gestörten Flächen. Dies führte zu einer marginal signifikant höheren Samenausbreitungsrate auf den gering gestörten Flächen. Diese Ergebnisse waren auf regionaler Ebene in allen drei untersuchten Wäldern konsistent. Dies zeigt, dass kleinräumige Störung einen umfassenderen negativen Einfluss auf Frugivore und ihre Funktion als Samenausbreiter hat als zuvor angenommen. Bei der Vegetationserfassung nahm ich 131 verschiedene Baumarten mit fleischigen Früchten in den drei Regenwäldern auf. Kleinräumige menschliche Störung erhöhte den Artenreichtum dieser Baumarten marginal signifikant, hatte jedoch keinen direkten Einfluss auf die Frugivorendichte und den Artenreichtum von Keimlingen dieser Baumarten. Der Artenreichtum von Baumarten mit fleischigen Früchten zeigte einen marginal signifikant positiven Einfluss auf die Frugivorendichte, allerdings nicht auf die Keimlinge. Allerdings führte die Dichte der Frugivoren zu signifikant erhöhtem Artenreichtum der Keimlinge. Folglich scheint kleinräumige Störung die Keimlingsetablierung indirekt durch erhöhten Baumartenreichtum und erhöhte Frugivorendichte zu beeinflussen. Die Frugivorendichte hatte einen größeren Einfluss auf die Waldregeneration als kleinräumige Störung und Baumartenreichtum. Demnach scheint kleinräumige menschliche Störung sowohl positive als auch negative Effekte auf Samenausbreitung und Regeneration zu haben. Somit sind weitere Studien notwendig, die den Einfluss von kleinräumiger menschlicher Störung auf Mutualismen tropischer Regenwälder aufklären.

Modulation der Genexpression und des neuronalen Zelltods durch das Amyloid-Vorläufer-Protein (APP)

Das Amyloid-Vorläufer-Protein (APP) spielt eine zentrale Rolle in der Entstehung und Entwicklung von Morbus Alzheimer. Hierbei ist die proteolytische Prozessierung von APP von entscheidender Bedeutung. Das Verhältnis von neurotoxischen und neuroprotektiven Spaltprodukten, die über den amyloidogenen und nicht-amyloidogenen Weg der APP-Prozessierung gebildeten werden, ist für das Überleben von Neuronen und deren Resistenz gegen zytotoxische Stress-Stimuli von hoher Relevanz. Störungen der Calcium-Homöostase sind ein bekanntes Phänomen bei Morbus Alzheimer. Im ersten Teil der vorliegenden Arbeit wurde die Rolle von überexprimiertem APP in der Regulation des neuronalen Zelltods nach Calcium Freisetzung untersucht. Die Calcium Freisetzung aus dem endoplasmatischen Retikulum wurde durch die Inhibition der sarko- und endoplasmatischen Calcium-ATPasen (SERCA) ausgelöst. Dies führt zur Induktion der sogenannten „unfolded protein response“ (UPR) und zu einer Aktivierung von Effektor-Caspasen. Für APP-überexprimierende PC12 Zellen konnte bereits zuvor eine im Vergleich zur Kontrolle nach der durch Calcium Freisetzung-induzierten Apoptose eine erhöhte intrazelluläre Calcium Konzentration nachgewiesen werden. Über die Messung der Aktivierung von Effektor-Caspasen konnte zudem ein gesteigerter Zelltod in den APP-überexprimierenden Zellen gemessen werden. Zudem konnte gezeigt werden, dass der pro-apoptotische Transkriptionsfaktor CHOP, nicht aber die klassischen UPR-Zielgene spezifisch hochreguliert wurden. Die APP-modulierte gesteigerte Induktion von Apoptose nach Calcium Freisezung konnte durch Komplexierung der intrazellulären Calcium Ionen und durch Knockdown von CHOP im Vergleich zur Kontrolle gänzlich unterdrückt werden. Ferner bewirkte die Inhibition der Speicher-aktivierten Calcium-Kanälen (SOCC) eine signifikante Unterdrückung der beobachteten erhöhten intrazellulären Calcium Konzentration und der gesteigerten Apoptose in den APP-überexprimierenden PC12 Zellen. In diesem Teil der Arbeit konnte eindeutig gezeigt werden, dass APP in der Lage ist den durch Calcium-Freisetzung-induzierten Zelltod zu potenzieren. Diese Modulation durch APP verläuft in einer UPR-unabhängigen Reaktion über die Aktivierung von SOCC’s, einer erhöhten Aufnahme von extrazellulärem Calcium und durch erhöhte Induktion des pro-apoptotischen Transkriptionsfaktors CHOP. Im zweiten Teil dieser Arbeit wurde die sAPPα-vermittelte Neuroprotektion untersucht. Dabei handelt es sich um die N-terminale Ektodomäne von APP, die über die Aktivität der α-Sekretase prozessiert wird und anschließend extrazellulär abgegeben wird. Ziel dieser Versuchsreihe war die neuroprotektive physiologische Funktion von APP im Hinblick auf den Schutz von neuronalen Zellen vor diversen für Morbus Alzheimer relevanten Stress-Stimuli bzw. Apoptose-Stimuli zu untersuchen. Durch die Analyse der Effektor-Caspasen konnte gezeigt werden, dass sAPPα in der Lage ist PC12 Zellen potent vor oxidativem Stress, DNA-Schäden, Hypoxie, proteasomalem Stress und Calcium-Freisetzung zu schützen. Außerdem konnte gezeigt werden, dass sAPPα in der Lage ist den pro-apoptotischen Stress-induzierten JNK/Akt-Signalweg zu inhibieren. Eine Beteiligung des anti-apoptotischen PI3K/Akt-Signalwegs bei der sAPPα-vermittelten Protektion konnte über die Inhibition der PI3-Kinase ebenfalls demonstriert werden, die eine Aufhebung der sAPPα-vermittelten Neuroprotektion bewirkte. Diese Daten zeigen neue molekulare Mechanismen auf, die dem sAPPα-vermittelten Schutz vor pathophysiologisch relevanten Stress-Stimuli in neuronalen Zellen zugrunde liegen. Im letzten Teil der Arbeit wurden verschieden Gruppen von pharmakologischen Substanzen im Hinblick auf ihre neuroprotektive Wirkung untersucht und mit ihren Effekten auf den APP-Metabolismus korreliert. Die Untersuchungen ergaben, dass Galantamin, ein schwacher Acetycholinesterase Inhibitor und allosterisch potenzierender Ligand von nikotinischen Acetylcholin-Rezeptoren in der Lage war, naive, und mit noch höherer Effizienz APP-überexprimierende Zelllinien vor dem Stress-induzierten Zelltod zu schützen. Zudem bewirkte Galantamin in APP-überexprimierenden HEK293 Zellen eine rasche Erhöhung der sAPPα Sekretion, so dass hier von einer Rezeptor-vermittelten Modulation des APP Metabolismus ausgegangen werden kann. Omega-3 Fettsäuren wirken sich positiv auf die Membranfluidität von Zellen aus und es konnte bereits gezeigt werden, dass die Bildung des toxischen Aβ Peptids hierdurch vermindert wird. In Analogie zu Galantamin schützte die Omega-3 Fettsäure Docosahexaensäure (DHA) neuronale Zellen vor dem Stress-induzierten Zelltod, wobei der Schutz in APP-überexprimierenden Zellen besonders effizient war. Diese Daten legen nahe, dass die Aktivierung des antiamyloidogenen Wegs der APP-Prozessierung ein viel versprechender Ansatz für die Entwicklung neuer Therapien gegen Morbus Alzheimer sein könnte.

The role of MYCN-mediated transcriptional repression in neuronal physiopathology

MYC is a transcription factor that can activate transcription of several targets by direct binding to their promoters at specific DNA sequences (E-box). Recent findings have also shown that it can exert its biological role by repressing transcription of other set of genes. C-MYC can mediate repression on its target genes through interaction with factors bound to promoter regions but not through direct recognition of typical E-Boxes. In this thesis, we investigated whether MYCN can also repress gene transcription and how this is mechanistically achieved. Moreover, expression of TRKA, P75NTR and ABCC3 is attenuated in aggressive MYCN-amplified tumors, suggesting a causal link between elevated MYCN activity and transcriptional repression of these three genes. We found that MYCN is physically associated with gene promoters in vivo in proximity of the transcriptional start sites and this association requires interactions with SP1 and/or MIZ-1. Furthermore, we show that this interaction could interfere with SP1 and MIZ-1 activation functions by recruiting co-repressors such as DNMT3a or HDACs. Studies in vitro suggest that MYCN interacts through distinct domains with SP1, MIZ-1 and HDAC1 supporting the idea that MYCN may form different complexes by interacting with different proteins. Re-expression of endogenous TRKA and P75NTR with exposure to the TSA sensitizes neuroblastoma to NGF-mediated apoptosis, whereas ectopic expression of ABCC3 decreases cell motility without interfering with growth. Finally, using shRNA whole genome library, we dissected the P75NTR repression trying to identify novel factors inside and/or outside MYCN complex for future therapeutic approaches. Overall, our results support a model in which MYCN can repress gene transcription by direct interaction with SP1 and/or MIZ-1, and provide further lines of evidence on the importance of transcriptional repression induced by Myc in tumor biology.

Stimulus-induced gamma activity in the electrocorticogram of freely moving telemetric implanted rats: the neuronal signature of novelty detection

During this thesis a new telemetric recording system has been developed allowing ECoG/EEG recordings in freely behaving rodents (Lapray et al., 2008; Lapray et al., in press). This unit has been shown to not generate any discomfort in the implanted animals and to allow recordings in a wide range of environments. In the second part of this work the developed technique has been used to investigate what cortical activity was related to the process of novelty detection in rats’ barrel cortex. We showed that the detection of a novel object is accompanied in the barrel cortex by a transient burst of activity in the γ frequency range (40-47 Hz) around 200 ms after the whiskers contact with the object (Lapray et al., accepted). This activity was associated to a decrease in the lower range of γ frequencies (30-37 Hz). This network activity may represent the optimal oscillatory pattern for the propagation and storage of new information in memory related structures. The frequency as well as the timing of appearance correspond well with other studies concerning novelty detection related burst of activity in other sensory systems (Barcelo et al., 2006; Haenschel et al., 2000; Ranganath & Rainer, 2003). Here, the burst of activity is well suited to induce plastic and long-lasting modifications in neuronal circuits (Harris et al., 2003). The debate is still open whether synchronised activity in the brain is a part of information processing or an epiphenomenon (Shadlen & Movshon, 1999; Singer, 1999). The present work provides further evidence that neuronal network activity in the γ frequency range plays an important role in the neocortical processing of sensory stimuli and in higher cognitive functions.

LPS-induced modifications in spontaneous network activity causes neuronal apoptosis in neonatal cerebral cortex

During the perinatal period the developing brain is most vulnerable to inflammation. Prenatal infection or exposure to inflammatory factors can have a profound impact on fetal neurodevelopment with long-term neurological deficits, such as cognitive impairment, learning deficits, perinatal brain damage and cerebral palsy. Inflammation in the brain is characterized by activation of resident immune cells, especially microglia and astrocytes whose activation is associated with a variety of neurodegenerative disorders like Alzheimer´s disease and Multiple sclerosis. These cell types express, release and respond to pro-inflammatory mediators such as cytokines, which are critically involved in the immune response to infection. It has been demonstrated recently that cytokines also directly influence neuronal function. Glial cells are capable of releaseing the pro-inflammatory cytokines MIP-2, which is involved in cell death, and tumor necrosis factor alpha (TNFalpha), which enhances excitatory synaptic function by increasing the surface expression of AMPA receptors. Thus constitutively released TNFalpha homeostatically regulates the balance between neuronal excitation and inhibition in an activity-dependent manner. Since TNFalpha is also involved in neuronal cell death, the interplay between neuronal activity MIP-2 and TNFalpha may control the process of cell death and cell survival in developing neuronal networks. An increasing body of evidence suggests that neuronal activity is important in the regulation of neuronal survival during early development, e.g. programmed cell death (apoptosis) is augmented when neuronal activity is blocked. In our study we were interested on the impact of inflammation on neuronal activity and cell survival during early cortical development. To address this question, we investigated the impact of inflammation on neuronal activity and cell survival during early cortical development in vivo and in vitro. Inflammation was experimentally induced by application of the endotoxin lipopolysaccharide (LPS), which initiates a rapid and well-characterized immune response. I studied the consequences of inflammation on spontaneous neuronal network activity and cell death by combining electrophysiological recordings with multi-electrode arrays and quantitative analyses of apoptosis. In addition, I used a cytokine array and antibodies directed against specific cytokines allowing the identification of the pro-inflammatory factors, which are critically involved in these processes. In this study I demonstrated a direct link between inflammation-induced modifications in neuronal network activity and the control of cell survival in a developing neuronal network for the first time. Our in vivo and in vitro recordings showed a fast LPS-induced reduction in occurrence of spontaneous oscillatory activity. It is indicated that LPS-induced inflammation causes fast release of proinflammatory factors which modify neuronal network activity. My experiments with specific antibodies demonstrate that TNFalpha and to a lesser extent MIP-2 seem to be the key mediators causing activity-dependent neuronal cell death in developing brain. These data may be of important clinical relevance, since spontaneous synchronized activity is also a hallmark of the developing human brain and inflammation-induced alterations in this early network activity may have a critical impact on the survival of immature neurons.

Membrane properties and synaptic integration of identified neuronal populations in the developing rodent neocortex

In my PhD work I concentrated on three elementary questions that are essential to understand the interactions between the different neuronal cell populations in the developing neocortex. The questions regarded the identity of Cajal-Retzius (CR) cells, the ubiquitous expression of glycine receptors in all major cell populations of the immature neocortex, and the role of taurine in the modulation of immature neocortical network activity.rnTo unravel whether CR cells of different ontogenetic origin have divergent functions I investigated the electrophysiological properties of YFP+ (derived from the septum and borders of the pallium) and YFP− CR cells (derived from other neocortical origins). This study demonstrated that the passive and active electrophysiological properties as well as features of GABAergic PSCs and glutamatergic currents are similar between both CR cell populations. These findings suggest that CR cells of different origins most probably support similar functions within the neuronal networks of the early postnatal cerebral cortex.rnTo elucidate whether glycine receptors are expressed in all major cell populations of the developing neocortex I analyzed the functional expression of glycine receptors on subplate (SP) cells. Activation of glycine receptors by glycine, -alanine and taurine elicited membrane responses that could be blocked by the selective glycinergic antagonist strychnine. Pharmacological experiments suggest that SP cells express functional heteromeric glycine receptors that do not contain 1 subunits. The activation of glycine receptors by glycine and taurine induced a membrane depolarization, which mediated excitatory effects. Considering the key role of SP cells in immature cortical networks and the development of thalamocortical connections, this glycinergic excitation may influence the properties of early cortical networks and the formation of cortical circuits.rnIn the third part of my project I demonstrated that tonic taurine application induced a massive increase in the frequency of PSCs. Based on their reversal potential and their pharmacological properties these taurine-induced PSCs are exclusively transmitted via GABAA receptors to the pyramidal neurons, while both GABAA and glycine receptors were implicated in the generation of the presynaptic activity. Accordingly, whole-cell and cell-attached recordings from genetically labeled interneurons revealed the expression of glycine and GABAA receptors, which mediated an excitatory action on these cells. These findings suggest that low taurine concentrations can tonically activate exclusively GABAergic networks. The activity level maintained by this GABAergic activity in the immature nervous system may contribute to network properties and can facilitate the activity dependent formation of adequate synaptic projections.rnIn summary, the results of my studies complemented the knowledge about neuronal interactions in the immature neocortex and improve our understanding of cellular processes that guide neuronal development and thus shape the brain.rn

Resonance properties of different neuronal populations in the immature mouse neocortex

Subthreshold resonance is a characteristic membrane property of different neuronal classes, is critically involved in the generation of network oscillations, and tunes the integration of synaptic inputs to particular frequency ranges. In order to investigate whether resonance properties of distinct neuronal populations in the immature neocortex contribute to these network oscillations, I performed whole-cell patch-clamp recordings from visually identified neurons in tangential and coronal neocortical slices from postnatal day (P) P0-P7 C57Bl/6 and P6-P13 GAD67-GFP knock-in mice. Subthreshold resonance was analyzed by sinusoidal current injection of varying frequency. All Cajal-Retzius cells showed subthreshold resonance with an average frequency of 2.6 ± 0.1 Hz (n=60), which was massively reduced by ZD7288, a blocker of hyperpolarization-activated cation currents. About 65.6% (n=61) of the supragranular pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.1 Hz (n=40). Application of 1 mM Ni2+ suppressed subthreshold resonance, suggesting that low-threshold Ca2+ currents contribute to resonance in these neurons. About 63.6% (n=77) of the layer V pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.2 Hz (n=49), which was abolished by ZD7288. Only 44.1% (n=59) of the subplate neurons showed subthreshold resonance with an average frequency of 1.3 ± 0.2 Hz (n=26) and a small resonance strength. Finally, 50% of the investigated GABAergic interneurons showed subthreshold resonance with an average frequency of 2.0 ± 0.2 Hz (n=42). Membrane hyperpolarization to –86 mV attenuated the frequency and strength of subthreshold resonance. Subthreshold resonance was virtually abolished in the presence of 1 mM Ni2+, suggesting that t-type Ca2+ currents are critically involved in the generation of resonance, while ZD7288 had no effect. Application of 0.4 µM TTX suppressed subthreshold resonance at depolarized, but not hyperpolarized membrane potential, suggesting that persistent Na+ current contribute to the amplification of membrane resonance. rnIn summary, these results demonstrate that all investigated neuronal subpopulations reveal resonance behavior, with either hyperpolarization-activated cation or low-threshold Ca2+ currents contributing to the subthreshold resonance. GABAergic interneurons also express subthreshold resonance at low frequencies, with t-type Ca2+ and persistent Na+ currents underlying the generation of membrane resonance. The membrane resonance of immature neurons may contribute to the generation of slow oscillatory activity pattern in the immature neocortex and enhance the temporal precision of synaptic integration in developing cortical neurons.rn

NLGA based Active Control in Aerospace: fault tolerability, disturbance rejection, and parameter estimation

A new control scheme has been presented in this thesis. Based on the NonLinear Geometric Approach, the proposed Active Control System represents a new way to see the reconfigurable controllers for aerospace applications. The presence of the Diagnosis module (providing the estimation of generic signals which, based on the case, can be faults, disturbances or system parameters), mean feature of the depicted Active Control System, is a characteristic shared by three well known control systems: the Active Fault Tolerant Controls, the Indirect Adaptive Controls and the Active Disturbance Rejection Controls. The standard NonLinear Geometric Approach (NLGA) has been accurately investigated and than improved to extend its applicability to more complex models. The standard NLGA procedure has been modified to take account of feasible and estimable sets of unknown signals. Furthermore the application of the Singular Perturbations approximation has led to the solution of Detection and Isolation problems in scenarios too complex to be solved by the standard NLGA. Also the estimation process has been improved, where multiple redundant measuremtent are available, by the introduction of a new algorithm, here called "Least Squares - Sliding Mode". It guarantees optimality, in the sense of the least squares, and finite estimation time, in the sense of the sliding mode. The Active Control System concept has been formalized in two controller: a nonlinear backstepping controller and a nonlinear composite controller. Particularly interesting is the integration, in the controller design, of the estimations coming from the Diagnosis module. Stability proofs are provided for both the control schemes. Finally, different applications in aerospace have been provided to show the applicability and the effectiveness of the proposed NLGA-based Active Control System.