Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities.

L'objectif de cette thèse est d'étudier les propriétés de variétés toriques particulières. La thèse est divisée en trois parties, les deux premières étant fortement liées. Dans la première partie, nous étudions des variétés algébriques associées aux processus de Markov sur les arbres. A chaque processus de Markov sur un arbre on peut associer une variété algébrique. Motivé par la biologie, nous nous concentrons sur les processus de Markov dé finis par une action de groupe. Nous étudions les conditions pour que la variété obtenue soit torique. Nous donnons un résultat où les variétés obtenues sont normales, ainsi que des exemples où elles ne le sont pas. L'une des principales méthodes que nous utilisons est la généralisation des notions de prises et de réseaux introduites dans [BW07] à des groupes abéliens arbitraires. Dans notre contexte, les réseaux forment un groupe qui agit sur la variété. Par ailleurs, l'espace ambiant de lavariété est la représentation régulière de ce groupe. Le principal problème ouvert que nous essayons de résoudre dans cette partie est une conjecture de Sturmfels et Sullivant [SS05, Conjecture 2] indiquant que le schéma a fine associé au modèle 3-Kimura estdé fini par un idéal engendré en degré 4. Notre meilleur résultat dit que le schéma projectif associé peut être dé fini par un idéal engendré en degré 4. Avec Maria Donten -Bury, nous proposons une méthode pour engendrer l'idéal associé à la variété pour tous les modèles. Nous montrons que notre méthode fonctionne pour de nombreux modèles ainsi que pour les arbres si et seulement si la conjecture de Sturmfels et Sullivant est vraie. Nous présentons quelques applications, par exemple au problème d'identi abilité en biologie. La deuxième partie concerne les variétés algébriques associées aux graphes trivalents pour le modèle de Jukes-Cantor binaire. Il s'agit d'un travail en commun avec Weronika Buczyńska, Jarosław Buczyński et Kaie Kubjas. La variété associée á un graphe peut être représentéevpar un semi-groupe gradué. Nous étudions les liens entre les propriétés du graphe et le semigroupe. Le théorème principal borne le degré en lequel le semi-groupe est engendré par le premier nombre de Betti du graphe, plus un. Dans la dernière partie, nous étudions la structure de la catégorie dérivée des faisceaux cohérents des variétés toriques lisses. Dans un travail commun avec Michał Lasoń [LM11], nous construisons une collection fortement exceptionnelle complète de fi brés en droites pour une grande classe de variétés toriques complètes lisses dont le nombre de Picard est égal á trois. De nombreuses questions concernant le type de collections auxquelles on peut s'attendre sur les variétés toriques de certains types sont encore ouvertes. A ce titre, nous prouvons que Pn éclaté en deux points ne possède pas de collection fortement exceptionnelle complète de fibrés en droites pour n assez grand. Ceci fournit une collection infi nie de contre-exemples à la conjecture de King. Le premier contre-exemple est dû à Hille et Perling [HP06]. Récemment, des contre-exemples ont également été trouvés par E mov [E ] dans le cadre des variétés de Fano. Nous allons travailler sur le corps des nombres complexes C. Toutes les variétés considérées sont des variétés algébriques dans le sens de [Har77]. / The aim of this thesis is to investigate the properties of special toric varieties. The thesis is divided into three parts. The first two of them are strongly related to each other.In the fi rst, main part we study algebraic varieties associated to Markov processes on trees. To each Markov process on a tree one can associate an algebraic variety. Motivated by biology, we focus on Markov processes de fined by a group action. We investigate underwhich conditions the obtained variety is toric. We provide conditions ensuring that the obtained varieties are normal, as well as give examples when they are not. One of the main tools we use is the generalization of the notions of sockets and networks introduced in [BW07] to arbitrary abelian groups. In our setting the networks form a group, that acts on the variety. Moreover the ambient space of the variety is the regular representation of this group. The main open problem that we address in this part is a conjecture of Sturmfels and Sullivant [SS05, Conjecture 2] stating that the afi ne scheme associated to the 3-Kimura model is de fined by an ideal generated in degree 4. Our strongest result states that the associated projective scheme can be generated in degree 4. Together with Maria Donten -Bury we also propose a method for generating the ideal defi ning the variety for any model. We prove that our method works for many models and trees if and only if the conjecture of Sturmfels and Sullivant holds. We present some applications, for example to theidenti ability problem in biology. The second part concerns algebraic varieties associated to trivalent graphs for the binary Jukes-Cantor model. It is a joint work with Weronika Buczyńska, Jarosław Buczyński and Kaie Kubjas. In case of the graph, the associated variety can be represented by a graded semigroup. We investigate the connections between properties of the graph and the semigroup. The main theorem bounds the degree in which the semigroup is generated by the first Betti number of the graph plus one. Due to connections with the first part much of the terminology that we use is either a specialization or generalization of previous de finitions. From the one hand, as we are working with graphs with possible loops the notions of leaves, nodes and valency are more subtile than for trees. From the other hand, as we are dealing only with the binary Jukes-Cantor model, sockets and networks have got a very special form. In the last part we study the structure of the derived category of coherent sheaves for smooth toric varieties. As a result of a joint work with Michał Lasoń [LM11] we construct a full, strongly exceptional collection of line bundles for a large class of smooth, complete toric varieties with Picard number three. Many questions concerning what kind of collections should be expected on toric varieties of certain types are still open. As a contribution we prove that Pn blown up in two points does not have a full, strongly exceptional collection of line bundles for n large enough. This provides an in finite collection of counterexamples to King's conjecture. The first such counterexample is due to Hille andPerling [HP06]. Recently also counterexamples in the Fano case were found by E mov [E ].

Glycoengineering is a technology that could improve protein therapeutics. While protein glycosylation in general enhances solubility and stability, and reduces aggregation, immunogenicity and proteolysis, specific kinds of glycosylation may also be critical. For example, capping of glycans with N-acetylneuraminic acid (Neu5Ac) maximises circulatory half-life in humans. Moreover, some glycans directly participate in molecular recognition and other aspects of glycoprotein function. Glycoproteins produced by non-human mammalian cells carry glycans capped by N-glycolyl-neuraminic acid rather than Neu5Ac. Yet production in human cell lines is costly and slow, requires specialist facilities, produces low yields and is subject to additional regulations. Hence there is a case for glycoengineering alternative expression systems capable of rapid, low-cost, high-yield glycoprotein production. This report focuses on the glycoengineering of Pichia pastoris, a yeast, to produce recombinant human glycoprotein factor H (FH) bearing human-like glycans. FH is a potent down-regulator of the complement system. Mutations and SNPs in FH result in autoimmune diseases such as atypical haemolytic ureamic syndrome and age-related macular degeneration (AMD). Recombinant FH is an enticing therapeutic candidate for treating AMD, but high doses are required since FH is abundant (200-300 mg l-1) in normal human serum. Human FH (155 kDa), with eight sites of N-linked glycosylation and 40 disulphides, is a challenging target for recombinant production. Yet FH was previously expressed to 10s of milligrams in P. pastoris. In this study, methods were established to confirm that human plasma-derived (h)FH carries predominantly N-linked diantennary disialylated complex-type glycans, with monosialylated diantennary structures and triantennary structures in fucosylated and non-fucosylated forms, contributing to glycan heterogeneity. Functional comparison of native hFH, enzymatically desialylated (DeSia-) hFH and deglycosylated recombinant P. pastoris-produced (DeGly-r)FH showed that DeSia-hFH had the lowest affinity for complement protein C3b, its key target. Moreover, DeSia-hFH binds C3d, an opsonic C3b-breakdown product, whereas native hFH does not. DeSia-hFH had an improved ability to accelerate decay of the C3 convertase (an enzyme that cleaves C3 to C3b) compared to native hFH, but neither was as good as DeGly-rFH in this respect. In contrast, DeGly-rFH had reduced cofactor activity (for factor I-mediated degradation of C3b) compared to native hFH whereas DeSiahFH did not have reduced cofactor activity. These data suggest that sialylation of FH glycans may play a role in stabilising a conformation of circulating FH that is not fully effective, consistent with specificity for self-surfaces and resistance to bacterial hijack. Aiming eventually to produce human-like glycosylated FH in glycoengineered P. pastoris, the SuperMan 5 strain served as a starting point. While conventional strains of P. pastoris put hypermannosylated N-linked glycans on proteins, glycans on SuperMan 5-produced FH were shown to contain just five mannose (Man) residues. In further glycoengineering, and following unsuccessful efforts to use inABLE technology for this purpose, commercially available (GlycoSwitch) vectors were used to introduce genes encoding the glycosyltransferase enzymes N-acetylglucosamine (GlcNAc) transferase I (GnTI) and galactose (Gal) transferase. These catalysed the formation of a hybrid-type glycan containing an N-acetyllactosamine (Gal-β(1,4)-GlcNAc (LacNAc)) antennae on a five-mannose glycan. Then two more GlycoSwitch plasmids, containing genes encoding α-Mannosidase II (ManII) and GnTII, were introduced into P. pastoris to catalyse the formation of a second LacNAc antennae. MALDI-TOF analysis found the glycosylation of this strain to be heterogeneous, containing the humanised diantennary digalactosyl glycan as well as other endogenous yeast glycans. This strain was designated SuperGal. Large-scale expression of rFH with terminally galactosylated complex-type glycans (Gal-rFH) in SuperGal yielded 100s of milligrams of purified Gal-rFH. Yeast-type glycans were enzymatically removed from rFH and the remaining complex-type humanised glycans were sialylated with a recombinant bacterial α(2,6)-sialyltransferase from Photobacterium sp. expressed in E.coli. Purified sialylated (Sia-) and non-sialylated (Gal-) rFH expressed in SuperGal were functionally characterised in vitro using SPR-based assays. In C3b-binding assays Sia-rFH had lower affinity compared to Gal-rFH. Both bound with lower affinity than DeGly-rFH. A similar pattern of binding affinity was seen for C3d. In C3 convertase decay-acceleration assays, all rFH glycoforms performed equally well and had greater activity than hFH. Conversely, Sia-and Gal-rFH were shown to perform equally as well as hFH in CA assays, while all three versions outperformed DeGly-rFH. However, in vivo complement activity assay carried out in a FH-knockout mouse model showed that humanisation of the glycosylation of rFH did not significantly improve activity compared to DeGly-rFH. In addition, analysis of the circulatory half-life of rFH showed that humanisation did not improve half-life. Further engineering steps will be required to increase the complex-type glycan site occupancy on rFH with a view to improving circulatory half-life and efficacy. However, this study represents a significant step forward in developing a therapeutically useful source of rFH.

Methylphenidate (trade name: Ritalin) produced a more robust conditioned place male as compared to female juvenile rats. This effect was blocked by a D1 antagonist (SCH 23390), which resulted in a conditioned place aversion in male as compared to female rats. Effects on Brain-derived neurotrophic factor (BDNF) will be reported.

La présence synchrone de métastases hépatiques (MH) et carcinose péritonéale (CP)d'origine colorectale (CRC) est associée à une survie globale médiocre et est traditionnellement considérée comme une contre-indication à l’approche chirurgicale curative. Cependant, suite aux résultats encourageants après traitement chirurgicale, quelques séries ont rapporté une survie prolongée atteignant 3 ans chez des patients sélectionnés, ce qui suggère qu’un traitement chirurgicale curative est possible. À ce jour, en cas de chirurgie majeure hépatique et péritonéale associée, aucune stratégie thérapeutique n'a été établie, Nous avons postulé que la régénération hépatique après une résection hépatique pourrait favoriser la croissance de la CP. Nous avons construit un modèle animal immunnocompetent de CP limitée. L'objectif de l’étude était d'analyser les effets de l’hépatectomie majeure et de la régénération hépatique dans notre modèle murin de PC et le processus d'angiogenèse associé. En outre, nous avons analysée une cohorte prospective multicentrique de patients ayant eu une résection hépatique et une chirurgie cytoréductive avec HIPEC synchrone. L'objectif de cette étude était d'évaluer les outcomes et identifier les facteurs pronostiques afin d'optimiser la sélection des candidats au traitement chirurgical et de déterminer la stratégie chirurgicale optimale. / The synchronous presence of liver metastases (LM) and peritoneal carcinomatosis (PC)from colorectal cancer (CRC) is associated with poor outcome and is traditionally considered acontraindication to any surgical approach. However, few series reported a prolonged survival aftersurgical management, reaching 3 years in selected patients thus suggesting that a curative surgicalmanagement may be possible. To date, no standard management pathway has been established,especially if a major liver and peritoneal surgery has to be performed. We postulated that liverregeneration after liver resection could promote PC growth. We constructed an immunocompetentanimal model of limited PC. The objective of our study was to analyze the effects of major LR andliver regeneration after hepatectomy on peritoneal carcinomatosis growth and the associatedangiogenesis process. Furthermore, we have analyzed a prospective international cohort of patientsundergoing synchronous liver resection and cytoreductive surgery with HIPEC. The aim of this studywas to describe the outcomes, to identify variables potentially related to poor outcome, in order toestablish future guideline for the management of these patients, to optimize the selection of candidatesfor surgical treatment and determine the best surgical strategy.

Initial studies to determine the thermodynamic solubility product (Ksp) of gypsum and CaCO3 were conducted. The influence of different electrolyte salts at different concentrations upon the solubility of gypsum and CaCO3 was then tested. Analytical data was utilized in conjunction with a computer to calculate the activity of CaCO3 and gypsum, the ion pair concentration and the solubility product. Indifferent salts increased the solubility of gypsum and CaCO3, and salts with a common ion decreased the solubility of gypsum and CaCO3. Lithium was found to be the dominant monovalent cation present in these marine derived soils. In most cases the lithium concentration was greater than the sodium plus potassium (Na++K+) concentrations. All soils were found to be high in calcium and sulfate and the 1:1 soil water suspensions were saturated with respect to the constituent mineral gypsum. Salt release from Mancos shale is controlled by the parabolic diffusion law. Two diffusion controlled reactions occur: (a) a fast surface reaction and (b) a slow mineral weathering reaction. The fast reaction, accounting for 80-90 percent of the total salt production is due to the dissolution of salt from the surface of the mineral particles and to the dissolution of the fine (<.10 mm) mineral fraction. This reaction occurred in less than 2 minutes. The slow reaction accounting for 10-20 percent of the salt production is due to the dissolution of the larger more resistant mineral fraction, and proceeds for several days. Chemical equilibrium was reached in less than 72 hours for the small natural occurring size fraction (<.10 mm), while 7 to 9 days was required for equilibrium in the larger (>.25 to >1.0 mm) size fractions, respectively. Soil columns were leached with deionized water to allow the calculation of potential to produce 3.15 tons of salt per acre inch, and the salt accounted for 1.89 percent of the soil's total mass.

Scientific surveys provide sufficient evidence that anxiety disorders are one of the most common psy-chiatric disorders in the world. The lifetime prevalence rate of anxiety disorder is 28.8% (Kessler, et al., 2005). The most widely studied anxiety disorders are as follows panic disorder (PD), post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), social phobia (or social anxiety disorder), specific phobias, and generalized anxiety disorder (GAD). (NIMH Article, 2009). Classical conditioning is the stable paradigm used from the last one century to understand the neurobi-ology of fear learning. Neurobiological mechanism of fear learning is well documented with the condi-tioning studies. In the therapy of anxiety disorders, exposure based therapies are known to be the most effective approaches. Flooding is a form of exposure therapy in which a participant is exposed to the fear situation and kept in that situation until their fear dissipates. The exposure therapy is based on the phenomena of extinction; this means that a conditioned response diminishes if the conditioned stimulus (CS) is repeatedly presented without an unconditioned stimulus (UCS). One problem with extinction as well as with exposure-based therapy is the problem of fear return (for e.g. renewal, spontaneous recov-ery and reinstatement) after successful extinction. Therefore, extinction does not delete the fear memory trace. It has been well documented that memory processes can be modulated or disrupted using several sci-entific paradigms such as behavioral (for e.g. exposure therapy), pharmacological (for e.g. drug manipu-lation), non-invasive stimulation (for e.g. non-invasive stimulation such as electroconvulsive shock (ECS), transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), etc. However, modulation of memory processes after reactivation or via non-invasive stimulation is still not clear, which is the focus of the current study. In addition, study of genetic variant suggests that genetic differences play a vital role in the psychiatric disorder especially in fear learning. Hence, it is also one of the concerns of the current dissertation to investigate the interaction between gene and reconsolidation of memory. With respect to fear-conditioning, there are three findings in the current dissertation, which are as fol-lows: (i) In the first study we investigated that non-invasive weak electrical stimulation interferes with the consolidation process and disrupts the fear consolidation to attain stable form. This might offer an effective treatment in the pathological memories, for e.g. PTSD, PD, etc. (ii) In the second study we demonstrated whether a brief single presentation of the CS will inhibit the fear recovery. Like earlier studies we also found that reactivation followed by reconsolidation douses fear return. Attenuation of fear recovery was observed in the reminder group compared to the no-reminder group. (iii) Finally, in our third study we found a statistically significant role of brain derived neurotrophic factor (BDNF) polymorphism in reconsolidation. Results of the third study affirm the involvement of BDNF variants (Met vs. Val) in the modulation of conditioned fear memory after its reactivation. In summary, we were able to show in the current thesis modulation of associative learning and recon-solidation via transcranial direct current stimulation and genetic polymorphism. / Mit einer lebenslangen Prävalenz von etwa 28% (Kessler Rc, 2005) stellen Angststörungen eine der häufigsten psychischen Störungen weltweit dar. Zu den am besten untersuchten Angststörungen gehö-ren Panikstörungen (PD), posttraumatische Belastungsstörungen (PTSD), Zwangsstörungen (OCD), soziale Phobien (oder soziale Angststörungen), spezifische Phobien und generalisierte Angststörungen (GAD) (NIMH Artikel, 2009). Die klassische Konditionierung ist das seit dem letzten Jahrhundert gültige Paradigma zur Erforschung der neurobiologischen Mechanismen des Angstlernens. Bei der Behandlung von Angststörungen haben sich Konfrontationstherapien als äußerst wirksam herausgestellt. Reizüberflutung (Flooding) ist bei-spielsweise eine Form der Konfrontationstherapie, bei der der Teilnehmer einer furchteinflößenden Situation ausgesetzt und in ihr gehalten wird, bis seine Furcht vergeht. Die Konfrontationstherapie ba-siert auf dem Phänomen der Extinktion, also dem Rückgang eines konditionierten Verhaltens nach wie-derholter Präsentation eines konditionierten Stimulus (CS) ohne einen unkonditionierten Stimulus (UCS). Ein Problem der Extinktion und der Konfrontationstherapien ist, dass das Furchtgefühl nach einer erfolgreichen Extinktion zurückkehren kann, was darauf hinweist, dass eine Extinktion nicht die Spuren des Angstgedächtnisses löscht. Vieles deutet darauf, dass der Erinnerungsprozess mittels verschiedenener wissenschaftlicher Para-digmen moduliert oder unterbrochen werden kann. Hierzu gehören etwa behavioristische (z.B. Kon-frontationstherapie), pharmakologische oder nicht-invasive Interventionen (z.B. Elektrokonvulsions-therapie (ECS), transkranielle Magnetstimulation (TMS) oder transkranielle Gleichstromstimulation (tDCS)). Da die Modulation von Erinnerungsprozessen nach einer Reaktivierung oder durch eine nicht-invasive Stimulation derzeit noch unzureichend erforscht ist, wurde der Schwerpunkt der vorliegenden Studie auf diese Thematik gelegt. Ein weiteres Ziel ist es, die Wechselwirkung bestimmter Gene mit der Rekonsolidierung des Gedächtnisses zu untersuchen, also Prozesse, denen eine entscheidende Rolle für Angststörungen im Allgemeinen und Furcht-Lernen im Speziellen zugeschrieben wird. Die vorliegende Dissertation umfasst drei zentrale Ergebnisse zur konditionierten Angst: (i.) In der ers-ten Studie wurde herausgefunden, dass eine nicht-invasive, schwache Stimulation den Konsolidie-rungsprozess beeinflusst und verhindert, dass die Angstkonsolidierung eine stabile Form erreicht. Dies könnte eine neue Möglichkeit darstellen, pathologische Gedächtnisinhalte, die z.B. bei Störungen wie PTSD oder PD vorkommen, effektiv zu behandeln. (ii.) Die zweite Studie untersuchte, ob eine kurze, einfache Präsentation des CS das Wiederaufkommen von Angst hemmen kann. Ähnlich wie in früheren Studien beschrieben, fanden auch wir, dass eine Reaktivierung gefolgt von einer Rekonsolidierung die Rückkehr der Angst unterbindet. Insbesondere wurde in der Gruppe, deren Teilnehmer erneut kon-frontiert wurden (reminder), im Vergleich zur Kontroll-Gruppe (no-reminder) ein verringertes Wieder-aufkommen von Angst beobachtet. (iii.) Die dritte Studie zeigte, dass ein Polymorphismus im BDNF-Gen (Met vs Val) eine signifikante Rolle für die Rekonsolidierung und die Modulation des konditionierten Angstgedächtnisses nach seiner Reaktivierung spielt.

Neurotrophine beeinflussen durch die Modulation von Prozessen wie Zellproliferation, -migration, Apoptose und Synapsenbildung entscheidend die neuronale Plastizität. Sie gelten deshalb als Kandidatengene neuronaler Entwicklungsstörungen wie Autismus-Spektrum-Störungen (ASS). Die vorgelegte Arbeit zielt auf die weitere Klärung der Rolle von Brain Derived Neurotrophic Factor (BDNF) bei der Ätiopathophysiologie der ASS durch Expressionsanalysen im Blut als potenziellem Surrogat zentralnervöser Prozesse. In gut charakterisierten ASS-Stichproben und - neben gesunden Kontrollprobanden - einer klinischen Kontrollgruppe von Patienten mit Aufmerksamkeitsdefizit-/ Hyperaktivitätsstörung (ADHS) wurde die BDNF-mRNA-Expression in Vollblut sowie BDNF-Proteinserumkonzentrationen untersucht. Zusätzlich wurden mögliche Einflussfaktoren auf die BDNF-Werte wie Alter, IQ, autismusspezifische Symptomatik, Komorbidität und Medikation analysiert. In einer ersten Stichprobe (ASS-Patienten versus gesunde Kontrollen) wurden signifikant erniedrigte BDNF-Serumkonzentrationen in der Patientengruppe mittels Enzyme-Linked-Immunosorbent-Assay gemessen (p = 0,040). In einer zweiten unabhängigen Stichprobe (Patienten mit ASS, Patienten mit ADHS und gesunde Kontrollen) wurde auf mRNA-Ebene mittels quantitativer Real-Time-Polymerasekettenreaktion ebenfalls ein signifikanter Gruppenunterschied ermittelt mit erniedrigter BDNF-Expression in der ASS-Gruppe im Vergleich zu gesunder Kontrollgruppe (p = 0,011), sowie einem Trend zu erniedrigten BDNF-Werten bei ADHS-Patienten im Vergleich zu gesunden Probanden (p = 0,097). Des Weiteren wurde eine signifikante negative Korrelation zwischen Alter und BDNF-mRNA-Expression bei Patienten mit ASS sowie eine positive Korrelation von Alter und BDNF-Serumkonzentrationen bei gesunden Kontrollen gemessen. Auch korrelierten die BDNF-Werte im Serum mit der Ausprägung des autistischen Phänotyps. In einer Subgruppe der ADHS-Patienten wurde kein Einfluss von Psychostimulanzien auf die BDNF-mRNA-Expression gemessen. Der Einbezug größerer Stichproben sowie die systematische Erfassung weiterer potenzieller Einflussfaktoren auf die BDNF-Expression (wie pubertärer Entwicklungsstand bzw. Geschlechtshormonkonzentrationen) könnten in zukünftigen Studien zu einer weiteren Klärung der pathophysiologischen Rolle von BDNF bei Kindern und Jugendlichen mit ASS beitragen. / Neurotrophins impact on neuronal plasticity by modulating processes such as cell proliferation, cell migration, apoptosis and synaptic plasticity. Therefore, they are regarded as candidate genes for neurodevelopmental disorders such as autism spectrum disorders (ASD). The following work aims at further clarifying the role of brain derived neurotrophic factor (BDNF) in the pathophysiology of ASD by expression analyses in blood as a potential surrogate for BDNF effects observed in the central nervous system. BDNF mRNA expression in whole blood and BDNF serum concentrations were analyzed in well characterized samples of ASD patients, healthy controls, and a clinical control group of patients with attention deficit hyperactivity disorder (ADHD). In addition, potential modulating factors such as age, IQ, autistic phenotype, comorbidity and medication were further investigated. In a first project (ASD patients vs. healthy controls) significantly lower BDNF serum concentrations in the ASD group were observed via enzyme-linked immunosorbent assay (p = 0.040). In a second independent sample and project (patients with ASS, patients with ADHD, and healthy controls), BDNF mRNA expression was analyzed using quantitative real time polymerase chain reaction. Also in this sample, a significant group difference was found with lower BDNF expression in the ASD group compared to the health controls (p = 0.011). Moreover, a trend of decreased BDNF mRNA levels was observed for patients with ADHD in comparison to the normally developing controls (p = 0.097). Furthermore, with regard to potential influencing factors, we found a significant negative correlation between age and BDNF mRNA expression in patients with ASD, as well as a positive correlation between age and BDNF serum concentrations in healthy controls. A positive correlation was moreover detected between the serum BDNF concentrations and autistic phenotype. Testing a sub-group of ADHD patients, no significant influence of stimulants was observed on BDNF mRNA expression. In future studies, bigger sample sizes as well as a systematic assessment of other factors that potentially influence BDNF expression (like pubertal developmental status or concentration of sex hormones) could further clarify the pathophysiological role of BDNF in children and adolescents with ASD.

Neurotrophine beeinflussen durch die Modulation von Prozessen wie Zellproliferation, -migration, Apoptose und Synapsenbildung entscheidend die neuronale Plastizität. Sie gelten deshalb als Kandidatengene neuronaler Entwicklungsstörungen wie Autismus-Spektrum-Störungen (ASS). Die vorgelegte Arbeit zielt auf die weitere Klärung der Rolle von Brain Derived Neurotrophic Factor (BDNF) bei der Ätiopathophysiologie der ASS durch Expressionsanalysen im Blut als potenziellem Surrogat zentralnervöser Prozesse. In gut charakterisierten ASS-Stichproben und - neben gesunden Kontrollprobanden - einer klinischen Kontrollgruppe von Patienten mit Aufmerksamkeitsdefizit-/ Hyperaktivitätsstörung (ADHS) wurde die BDNF-mRNA-Expression in Vollblut sowie BDNF-Proteinserumkonzentrationen untersucht. Zusätzlich wurden mögliche Einflussfaktoren auf die BDNF-Werte wie Alter, IQ, autismusspezifische Symptomatik, Komorbidität und Medikation analysiert. In einer ersten Stichprobe (ASS-Patienten versus gesunde Kontrollen) wurden signifikant erniedrigte BDNF-Serumkonzentrationen in der Patientengruppe mittels Enzyme-Linked-Immunosorbent-Assay gemessen (p = 0,040). In einer zweiten unabhängigen Stichprobe (Patienten mit ASS, Patienten mit ADHS und gesunde Kontrollen) wurde auf mRNA-Ebene mittels quantitativer Real-Time-Polymerasekettenreaktion ebenfalls ein signifikanter Gruppenunterschied ermittelt mit erniedrigter BDNF-Expression in der ASS-Gruppe im Vergleich zu gesunder Kontrollgruppe (p = 0,011), sowie einem Trend zu erniedrigten BDNF-Werten bei ADHS-Patienten im Vergleich zu gesunden Probanden (p = 0,097). Des Weiteren wurde eine signifikante negative Korrelation zwischen Alter und BDNF-mRNA-Expression bei Patienten mit ASS sowie eine positive Korrelation von Alter und BDNF-Serumkonzentrationen bei gesunden Kontrollen gemessen. Auch korrelierten die BDNF-Werte im Serum mit der Ausprägung des autistischen Phänotyps. In einer Subgruppe der ADHS-Patienten wurde kein Einfluss von Psychostimulanzien auf die BDNF-mRNA-Expression gemessen. Der Einbezug größerer Stichproben sowie die systematische Erfassung weiterer potenzieller Einflussfaktoren auf die BDNF-Expression (wie pubertärer Entwicklungsstand bzw. Geschlechtshormonkonzentrationen) könnten in zukünftigen Studien zu einer weiteren Klärung der pathophysiologischen Rolle von BDNF bei Kindern und Jugendlichen mit ASS beitragen. / Neurotrophins impact on neuronal plasticity by modulating processes such as cell proliferation, cell migration, apoptosis and synaptic plasticity. Therefore, they are regarded as candidate genes for neurodevelopmental disorders such as autism spectrum disorders (ASD). The following work aims at further clarifying the role of brain derived neurotrophic factor (BDNF) in the pathophysiology of ASD by expression analyses in blood as a potential surrogate for BDNF effects observed in the central nervous system. BDNF mRNA expression in whole blood and BDNF serum concentrations were analyzed in well characterized samples of ASD patients, healthy controls, and a clinical control group of patients with attention deficit hyperactivity disorder (ADHD). In addition, potential modulating factors such as age, IQ, autistic phenotype, comorbidity and medication were further investigated. In a first project (ASD patients vs. healthy controls) significantly lower BDNF serum concentrations in the ASD group were observed via enzyme-linked immunosorbent assay (p = 0.040). In a second independent sample and project (patients with ASS, patients with ADHD, and healthy controls), BDNF mRNA expression was analyzed using quantitative real time polymerase chain reaction. Also in this sample, a significant group difference was found with lower BDNF expression in the ASD group compared to the health controls (p = 0.011). Moreover, a trend of decreased BDNF mRNA levels was observed for patients with ADHD in comparison to the normally developing controls (p = 0.097). Furthermore, with regard to potential influencing factors, we found a significant negative correlation between age and BDNF mRNA expression in patients with ASD, as well as a positive correlation between age and BDNF serum concentrations in healthy controls. A positive correlation was moreover detected between the serum BDNF concentrations and autistic phenotype. Testing a sub-group of ADHD patients, no significant influence of stimulants was observed on BDNF mRNA expression. In future studies, bigger sample sizes as well as a systematic assessment of other factors that potentially influence BDNF expression (like pubertal developmental status or concentration of sex hormones) could further clarify the pathophysiological role of BDNF in children and adolescents with ASD.