FE65 es una proteína adaptadora que actualmente se ha relacionado con la formación del péptido β-amiloide y la hiperfosforilación de tau, procesos que se ven incrementados en pacientes con Alzheimer, probablemente como consecuencia de un aumento en la actividad transcripcional de FE65. Una de las principales interacciones que se asocian con la activación de ésta proteína es la unión de su dominio PTB2 con el dominio citoplasmático de la proteína precursora del amiloide (AICD), interacción que se ve estabilizada por la proteína adaptadora 14-3-3γ. En esta tesis se llevó a cabo la construcción de un modelo por homología de PTB2 y un segmento de AICD, correspondiente a la secuencia de interacción con FE65. A partir de éstos, mediante técnicas de docking se determinó el modo conformacional de interacción más probable para el complejo, cuya interfaz de interacción fue estudiada mediante un análisis de geometrías de interacción, principalmente de formación de puentes de hidrógeno y también mediante el estudio del cambio en el área de superficie accesible a solvente entre los monómeros y el complejo. Una vez determinados los aminoácidos que interaccionan en la interfaz se realizó un estudio de la contribución de éstos en la estabilidad del complejo mediante una técnica conocida como Alanine Scanning. Se logró determinar la importancia de cada uno de estos aminoácidos en la formación del complejo, presentando así las bases necesarias para el futuro diseño de ligandos inhibidores de la interacción como estrategia farmacoterapéutica para combatir la patología del Alzheimer. A su vez se presenta un estudio preliminar de otro complejo involucrado en la interacción de FE65 y AICD, aquel formado entre 14-3-3γ con AICD, pudiendo determinar dos posibles sitios de unión. Por otra parte se realizó un breve estudio de un potencial sitio de unión de PTB2 a fosfoinositidos de membrana, interacción que se ha señalado como probable, pero cuyo posible papel en los procesos moleculares de FE65 no ha sido determinado aun.

Amyloid plaques, a neuropathological hallmark of Alzheimer’s disease (AD), are largely composed of amyloid beta (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretase. The endosome is increasingly recognized as an important crossroads for APP and the secretases, with major implications for APP processing and amyloidogenesis. Amongst various posttranslational modifications affecting APP, ubiquitination of cytodomain lysines may represent a key signal controlling endosomal sorting. Here, we show that substitution of APP COOH-terminal lysines with arginines disrupts APP ubiquitination, though the pool of ubiquitinated APP is small or transient. Nonetheless, this small deficiency in ubiquitination can have a significant impact on APP, such that the number of lysines mutated trends toward an increase in APP metabolism. An APP mutant lacking all COOH-terminal lysines undergoes the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40, without change in Aβ42. This phenotype is abolished by artificial ubiquitination of APP using rapalog-mediated proximity inducers. Lack of APP COOH-terminal lysines does not affect APP endocytosis, but leads to a redistribution of APP from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with subsequent decrease in APP COOH-terminal fragment (CTF) content of secreted exosomes, but minimal effects on APP lysosomal degradation. Both the secreted and intracellular increase in Aβ40 is abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared to presenilin 1 (PSEN1). In a separate set of studies, we found that a familial AD mutant, L723P, which occurs immediately next to a string of three lysines in the juxtamembrane region, behaves more similarly to other FAD-causing mutations. APP L723P exhibits a selective increase in Aβ42, and a delay in degradation, but no change in exosomal content, despite some missorting to the endosomal limiting membrane. Our findings demonstrate that ubiquitin can act as a signal for endosomal sorting at five lysines in the APP cytodomain, disruption of which prevents sequestration of APP in ILVs and results in the processing of a larger pool of APP-CTF by PSEN2 on the endosomal membrane.

In the brain, axons are wrapped by myelin sheaths which ensure fast saltatory conduction of impulses and provide metabolic support. In multiple sclerosis (MS), the myelin sheaths are lost which leaves the axon denuded. This not only results in slower conduction of action potentials, but if prolonged, can also lead to axon death due to the loss of metabolic support. This neurodegeneration is the main cause of permanent disability in multiple sclerosis patients. The axon death and disability which stem from it could be prevented by restoring the myelin wrap before axon damage has occurred. This remyelination process is carried out by oligodendrocyte precursor cells which are present throughout life. To remyelinate, OPCs migrate to the area of damage and differentiate into myelinating oligodendrocytes which ensheathe axons with new myelin. In multiple sclerosis, this process occurs but is insufficient to overcome the damage. Therefore, central to the therapeutic efforts in multiple sclerosis is the aim to improve endogenous remyelination. Enhancing recruitment of oligodendrocyte precursor cells (OPCs) to the areas of damage is a clinically unexplored target. To investigate the therapeutic potential of OPC recruitment modulators, I have looked at 2 different targets involved in migration NDST1/HS and Sema3A/NP1. The first target, heparan sulfate (HS) is a proteoglycan which is important to OPC migration, investigated by Pascale Durbec's group in France. In a demyelinating mouse model, its key synthesising enzyme, NDST1, is upregulated by oligodendroglia in a belt around the lesion to aid OPC recruitment. Loss of NDST1 in oligodendrocytes was found to impair remyelination and reduce OPC migration in mice. In collaboration with them, I investigated the relevance of this molecule in post-mortem MS human tissue. I found that in human as well as mouse, NDST1 was primarily expressed by oligodendroglia. The protein level and the proportion of oligodendroglia expressing NDST1 was increased in MS compared to control indicating NDST1 upregulation as a disease response in human. We also found that low numbers of NDST1+ oligodendroglia correlate with bigger sizes of lesions and chronic lesion types that fail to repair, highlighting its importance in repair. Moreover, high numbers of NDST1+ cells in a patient correlated with increased remyelination potential. This indicates that in human, intra-patient variation in NDST1 level may explain differences in potential for endogenous repair. Secondly, I looked at Sema3A, a chemorepulsive molecule which is upregulated in demyelinated injury rodent models aswell as multiple sclerosis lesions, particularly in OPC-depopulated chronic active lesions. Research has consistently found that the level of Sema3A negatively correlates to remyelination because Sema3A hinders OPC migration. This has highlighted Sema3A as a potential target to improve OPC recruitment in MS however the size and shape of the molecule make it hard to design therapeutics against it. Therefore, I looked at its druggable receptor, Neuropilin 1 (NP1), to see whether inhibition of NP1 had the same positive effect on OPC recruitment and remyelination as lowering the level of Sema3A. NP1 is a tyrosine kinase receptor for both Sema3A and vascular endothelial growth factor (VEGF) and is found in many cell types. To check if NP1 inhibition is beneficial, I assessed remyelination in a mouse where the Sema3A binding site of NP1 has been mutated to prevent Sema3A binding and exerting its effect. This is a proxy for a (currently unavailable) ideal NP1 inhibitor of the Sema3A site only. Contrary to my expectations, OPC recruitment and remyelination in the mutant mice were not improved. However, the NP1 mutation resulted in an altered immune response. To exclude the possibility that no improvement in the OPC recruitment and remyelination of those mice was seen because it was negated by the altered immune response, I explored a cell specific mutant mouse in which NP1 was deleted in oligodendroglia only. In this mutant as well, I did not see improvement of OPC recruitment and remyelination. I therefore propose that Neuropilin 1 is not imperative for Sema3As action in remyelination and is not suitable as a therapeutic target in multiple sclerosis. Loss of the whole NP1, but not loss of the Sema3A site also resulted in biggermyelinated and unmyelinated axons as well as a different myelin thickness post remyelination. This showed that VEGF and the VEGF site on NP1 in oligodendroglia have a previously unknown but important role in determining axon size and myelin thickness which should be further investigated. To further elucidate those results in a simple system, I looked at how Sema3A, NP1-Sema3A inhibitors, VEGF and NP1-VEGF inhibitor affect OPC behaviour. I confirmed Sema3As chemorepulsive effect but also showed that at different concentrations it can improve proliferation and survival of OPCs. Inhibiting the Sema3A site and the VEGF site of NP1 by specific blocking antibodies also affects OPC proliferation and maturation. This suggested that NP1s ligands are involved in more than just OPC migration. In summary, this work supports the relevance of the mouse findings that NDST1 is upregulated in demyelination and important for repair for human illustrating that it might be a suitable therapeutic target to investigate. However, despite the importance of Sema3A in MS models, its only reported receptor, NP1, is not essential for Sema3As action. Therefore, it is an unsuitable therapeutic target. The fact that NP1 is an inappropriate drug target for MS is further demonstrated by the involvement of its ligands in multiple OPC behaviours both in positive and negative aspects.

Criminological Aspects of Drug-Related Crime ABSTRACT The thesis deals with the topic of criminological aspects of drug-related crime. Considering the breadth of the issue, it focuses only on the so-called primary drug-related crime, i.e. criminal activity consisting of violation of laws and regulations on the handling of narcotic drugs and psychotropic substances. The aim of the thesis is particularly to describe the situation and development of the primary drug-related crime in the territory of the Czech Republic and to summarize possible development trends in the following years, as well as to map the development of national and international legislation and assess its possible effects on the drug problem. Finally, based on empirical research, the thesis aims to characterize drug-related crime committed on the territory of the capital city of Prague for which the perpetrators were finally convicted in 2017. The first chapter of the thesis defines the terms relevant to the topic, especially with respect to applicable legislation. The second chapter outlines the development of the issues of drug abuse and distribution in the territory of the Czech Republic and the corresponding evolution of legislation, while the third chapter is dedicated to the current, particularly criminal legislation on the handling...

Separation methods involving a mixture of metals typically include upfront processing that leads to one final product. To lower the waste and ultimately environmental burden, the potential to synthesize multiple functional products from a mixed metal precursor solution is explored. The initial precursor solutions contained varying ratios of gold and copper, gold and nickel, and finally a ternary solution of gold, copper, and nickel. The amount of gold was kept constant, while the amount of copper and/or nickel was sequentially increased. Two separate synthesis processes were tested, the traditional Turkevich method involving trisodium citrate and another chemical reduction method involving sodium borohydride. The particle size and chemical composition of the synthesized particles were characterized using TEM, DLS and ICP-OES. It was determined that gold nanoparticles still formed at a ratio of 1 to 2 gold to copper while using trisodium citrate or sodium borohydride as the reducing agent. The same limiting ratio was observed for the gold to nickel reactions in the presence of either trisodium citrate or sodium borohydride. The ternary mixture limit for gold nanoparticle formation was a molar ratio of 1 to 1 to 1 gold to copper to nickel. The repeatability and stability results for the optimized binary reaction conditions indicate that using sodium borohydride as the reducing agent consistently produces more stable particle suspensions. Quantifying the environmental impact using green chemistry metrics indicate the Turkevich reactions for the optimized reactions have the lower environmental factors.

Neonatal hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles. This disease is associated with significant morbidity and mortality and is often fatal if left untreated. Hydrocephalus was first described over 2500 years ago by Hippocrates, the father of medicine, and remains poorly understood today. Current therapies still rely on invasive procedures developed over 60 years ago that are associated with high failure and complication rates. Thus, the identification of molecular mechanisms and the development of non-invasive medical treatments for neonatal hydrocephalus are high priorities for the medical and scientific communities. The prevailing doctrine in the field is that hydrocephalus is strictly a "plumbing problem" caused by impaired cerebrospinal fluid (CSF) flow. Recently, animal models with impaired cilia have provided insight into the mechanisms involved in communicating (non-obstructive) hydrocephalus. However, as a result of a poor understanding of hydrocephalus, no animal studies to date have identified an effective non-invasive treatment. The goal of this thesis project is to investigate the molecular mechanisms underlying this disease and to identify a non-invasive, highly effective treatment strategy. In Chapter 2, we utilize a novel animal model with idiopathic hydrocephalus, mimicking the human ciliopathy Bardet-Biedl Syndrome (BBS), to examine the role of cilia in hydrocephalus. We find that these mice develop communicating hydrocephalus prior to the development of ependymal "motile" cilia, suggesting that this phenotype develops as a result of dysfunctional "primary" cilia. Primary cilia are non-motile and play a role in cellular signaling. These results challenge the current dogma that dysfunctional motile cilia underlies neonatal hydrocephalus and implicate a novel role for primary cilia and cellular signaling in this disease. Chapter 3 focuses on identifying the link between primary cilia and neonatal hydrocephalus. In this chapter, we report that disrupting the molecular machinery within primary cilia leads to faulty PDGFRα signaling and the loss of a particular class of neural progenitor cells called oligodendrocyte precursor cells (OPCs). We find that the loss of OPCs leads to neonatal hydrocephalus. Importantly, we identify the molecular mechanism underlying both the loss of OPCs and the pathogenesis of neonatal hydrocephalus. Chapter 4 explores the therapeutic potential of targeting the defective cellular signaling pathways to treat neonatal hydrocephalus. By targeting the faulty signaling, we restore normal development of oligodendrocyte precursor cells, and curtail the development of hydrocephalus. This work challenges the predominant view of hydrocephalus being strictly a "plumbing problem" treatable solely by surgical diversion of CSF. Here, we propose that hydrocephalus is a neurodevelopmental disorder that can be ameliorated by non-invasive means. Importantly, we introduce novel molecular targets and a non-invasive treatment strategy for this devastating disorder. To our knowledge, we are the first to successfully treat neonatal hydrocephalus in any model organism by targeting neural progenitor cells.

Alzheimer�s disease (AD) is a neurodegenerative disorder, charaeterised by progressive loss of memory. It is important to understand what factors initiate the onset of AD so that effective therapeutic treatments can be developed to target the precise mechanisms that initiate this disease. Currently, synaptic dysfunction is widely believed to be the first significant alteration preceding the onset of AD, and is thought to be initiated by an intracellular accumulation of amyloid-β (Aβ), or a free radical-induced increase of oxidative stress. As Aβ levels rise during the onset of AD, a concomitant reduction of secreted amyloid precursor protein-α (sAPPα) is observed, as the two proteins exist in equilibrium. Intriguingly, the neuroprotective and neurotrophic properties of sAPPα indicate that it is intimately involved in the physiological pathways of the major hypotheses for the cause of AD, and may also be involved in the mechanisms that underlie learning and memory. Therefore, it is possible that during the onset of AD, the decrease of sAPPα may contribute to synaptic dysfunction by disrupting the mechanisms of synaptic plasticity. Long-term potentiation (LTP) is the leading experimental model for investigating the neural substrate of memory formation, and describes the molecular mechanisms that underlie an increase in the strength of synaptic transmission. The role sAPPα may play in the induction and maintenance of LTP has not previously been addressed in vivo. Therefore, the aim of this thesis was to investigate whether sAPPα affects the induction of LTP in the hippocampus of the anaesthetised rat. The present findings are the first to suggest that sAPPα may modulate the induction of LTP in vivo. Decreasing the function of endogenous sAPPα (with sAPPα-binding antibodies and a pharmacological inhibition of α-secretase) significantly reduced the magnitude of LTP induced in the dentate gyrus. Therefore, the reduction of sAPPα during AD is likely to have a detrimental impact on the mechanisms of synaptic plasticity, and by extension, learning and memory. The present investigation has also found that the application of recombinant, purified sAPPα to the rat hippocampus has an �inverted U-shaped� dose-response effect on the magnitude of LTP. Low concentrations of sAPPα significantly enhanced LTP, supporting previous findings that exogenous sAPPα can facilitate in vitro LTP and enhance memory performance in animals. On the other hand, comparatively high concentrations of sAPPα significantly decreased the magnitude of LTP. This observation is also consistent with previous findings, in which high concentrations of sAPPα have been shown to be less synaptogenic and memory enhancing than lower doses. These results are the first to suggest that sAPPα modulates in vivo synaptic plasticity, and have important implications for the development of strategies to treat AD.

Adult neural precursor cells (NPCs) are a heterogeneous population of mitotically active, self-renewing multipotent cells of both adult and developing CNS. They can be expanded in vitro in the presence of mitogens. The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). A stereological count demonstrates that mice with significant cell loss exhibit highest survival of grafted NPCs and migration to the vicinity of PCs as compared to wt and younger grafted animals. These animals showed improved motor skills as compared to sham animals. Confocal analysis and profiling shows that many of implanted cells present in the cerebellar cortex have formed gap junctions with host PCs and express connexin43. Grafted cells did not adopt characteristics of PCs, but stereological and morphometric analysis of the cerebellar cortex revealed that grafted animals had more surviving PCs and a better preserved morphology of these cells than the control groups. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. In this study, evidence for a neuroprotective effect came, in addition to motor behavior improvement, from stereological and electrophysiological analyses and suggest that timing of stem cell delivery is important to determine its therapeutic effect. In a brain stem cell niche, NSCs reside in a complex cellular and extracellular microenvironment comprising their own progeny, ependymal cells, numerous blood vessels and various extracellular matrix molecules. Recently, it was reported that blood vessel ECs-NSCs crosstalk plays an important role in tissue homeostasis. Bloodstream offers a natural delivery vehicle especially in case of diffuse neurodegenerative diseases which require widespread distribution of exogenous cells. As NSCs are confronted with blood-brain barrier endothelial cells (BBB-ECs) before they can enter into brain parenchyma, we investigated their interaction using primary cultures in an in vitro BBB model. We isolated human fetal neural precursor cells (hfNPCs) from aborted fetal brain tissues and expanded in vitro. We showed that in an in vitro model, human BBB endothelium induces the rapid differentiation of hfNPCs and allows them to cross the endothelial monolayer, with the differentiated progeny remaining in close contact with endothelial cells. These results are not reproduced when using a non-BBB endothelium and are partly dependent on the cytokine MCP1. Our data suggest that, in the presence of attractive signals released by a damaged brain, intravascularly administered NPCs can move across an intact BBB endothelium and differentiate in its vicinity. Overall, our findings have implications for the development of cellular therapies for cerebellar degenerative diseases and understanding of the brain stem cell niche.

The present study evaluated olfactory and cognitive abilities of the Tg6799 (also called 5xFAD) strain of Alzheimer’s disease (AD) model mice of two different age groups (2-3 and 8-10 months of age), and one group of healthy control mice (9-10 months). Employment of an operant conditioning paradigm using an automated olfactometer, an olfactory habituation/dishabituation test and a spatial learning test with non olfactory cues resulted in data showing that the 5xFAD mice develop olfactory impairments already at 2-3 months of age. The impairments consisted in a robust impairment in olfactory sensitivity, decreased responsiveness to novel odors and an inability to discriminate between enantiomeric odor molecules in the 5xFAD mice compared to control mice. Spatial learning deficits were also detected at this age, suggesting that cognitive functions were also affected. No differences in magnitude of the olfactory or spatial learning impairments could be detected between the age groups of model mice tested. Histological examination of development and presence of amyloid β (Aβ) plaques in the brains showed that plaques develop mainly between the ages of 3 and 8 months. This indicates that soluble Aβ rather than the formation of plaques might be responsible for the olfactory impairment and spatial learning impairments found. By 10 months of age plaque load of the 5xFAD mice was massive. The results of the present study clearly show that the 5xFAD strain might be suitable for research on human AD with regard to the early onset of olfactory impairments.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.