Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Magdaléna Svobodová Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Assoc. Prof. Maria da Glória Correia da Silva Queiroz, Ph.D. Title of diploma thesis: Microglia control adenosine A2A-receptor mediated astrogliosis In the central nervous system, astrocytes and microglia are the main cells coordinating the inflammatory response. During inflammation, dying or temporarily damaged cells release ATP, as a danger-associated signal molecule, that contributes to the induction of astrogliosis and promotes clearance of the debris by immune cells such as microglia. Adenosine that results from ATP metabolism also stimulates astrogliosis. However, the effects of adenosine on astrogliosis may be more complex, since it also modulates microglia phenotype and microglia have been shown to prevent excessive astroglial proliferation mediated by nucleotides. In this context, ATP and adenosine are assumed as relevant signalling molecules in the control of astrogliosis and its modulation by microglia. However, it is still unknown whether and how microglia modulate adenosine-mediated astrogliosis. The present study aims to clarify the impact of microglia in the control of adenosine-induced astrogliosis. Two...

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The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

Purpose: The purpose of this study was to investigate the role of the vasculature and immune system in models of glaucoma. Vascular changes have been implicated in glaucoma. As well there is mounting evidence that the immune system plays a role in the disease. It is my hypothesis that the vasculature and immune system play a role in the retinal response to injury in models of glaucoma. Methods: Immunohistochemistry, in vivo retinal imaging (Bright field, fluorescent, optical coherence tomography), Slit2 injections and Evan’s blue labeling were used to investigate vascular and immune changes associated with retinal ganglion cell death after optic nerve crush up to 28 days after injury. Histology, immunohistochemistry, and intravascular labeling were utilized to investigate the role of the vascular degeneration and the systemic immune response to elevated intraocular pressure in 8-16 week old AP-2β Neural Crest Cell Knockout (AP-2β NCC KO) mice. Results: The vascular and immune responses to optic nerve crush were not found to play a significant role in the response to retinal ganglion cell death. Conversely the role of vascular degeneration and immune cell recruitment to the retinas of AP-2β NCC KO mice demonstrated that these factors played a significant role in the retinal response to injury. Conclusion: The vasculature and immune system play a varied role in the response to retinal injury and neurodegeneration depending on the model being studied. The vascular and immune changes were of minimal significance in acute optic nerve crush injury. On the other hand, the chronic injury associated with elevated intraocular pressure in AP-2β NCC KO mice was associated with significant vascular degeneration and systemic immune cell infiltration. / Thesis / Doctor of Philosophy (PhD)

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MicroRNAs (miRNAs) are short, non-coding RNA sequences that are generated from longer primary transcripts (pri-miRNA). These pri-miRNAs are processed by the endonuclease Drosha into a hairpin secondary structure (pre-miRNA), exported from the nucleus and cleaved by the enzyme Dicer to form a duplex RNA molecule. This miRNA:miRNA* duplex is subsequently further processed to form a single-stranded, mature miRNA. miRNAs have been extensively characterized and are known to play important roles in various physiologic and pathologic pathways. One hallmark of miRNAs function is their ability to modulate the downstream activities of protein-coding genes, as well as various other aspects of gene expression, by acting as post-transcriptional repressors of their messengerRNA (mRNA) targets. miR-184 is a highly conserved miRNA gene expressed in the Drosophila nervous system throughout development; and has been shown to target key regulators of differentiation, proliferation and apoptosis. Here we identify a novel role for miR-184 in regulating the development and maintenance of the Drosophila melanogaster post-embryonic nervous system. We present evidence which suggest miR-184 targets (i) paralytic (para), a voltage-gated sodium channel, shown to control neuronal excitability; and (ii) tramtrack69 (ttk69), a transcription factor known to regulate glial cell number and fate determination during embryonic development. In the absence of miR-184, homozygous loss-of-function mutant adult flies demonstrate hyperactive episodes in response to mechanical shock, indicative of increased susceptibility to seizures. Homozygous loss-of-function mutants also exhibit shortened lifespan, as well as reduced group longevity. Additionally, miR-184 deficient mutant larvae exhibit abnormal development of glia and glial progenitors; while expression of miR-184 exclusively in glia - reversed polarity- (repo) expressing cells - up-regulates development of glial cells. Phenotypes of the adult loss-of-function mutant are suppressed by genetic loss of para function; while larval phenotypes are rescued by reducing the genetic dosage of ttk69. These data imply that miR-184 functions to control post-embryonic gliogenesis, as well as in maintaining neuronal excitability and integrity of the Drosophila aging brain. / Biology

Recent studies examining cell-cell interactions during CNS development and following disease or trauma have highlighted our limited understanding of the in vivo functions of the myelinating cell of the CNS, the oligodendrocyte. With this in mind, our laboratory has developed techniques by which a profile of proteins derived from or regulated by oligodendrocytes can be elucidated. Specifically, we have demonstrated that oligodendrocytes can be selectively eliminated from one optic nerve of a rat by treating the animal with a unilateral exposure of X-irradiation at the time of birth. Consequently, this approach allowed us to experimentally create, within the same animal, one optic nerve devoid of oligodendrocytes and their progenitors (the X- irradiated side) and one optic nerve containing the normal oligodendrocyte population (the untreated side). Using this experimental animal model we hypothesized that uncharacterized proteins, derived from and regulated by oligodendrocytes, which are crucial for CNS development can be identified. Specifically, by comparing protein profiles found within the normal myelinating optic nerve versus the X-irradiated optic nerve, where oligodendrocytes are absent, potential oligodendrocyte-derived proteins can be quickly identified. Further verification that these proteins are indeed related to oligodendrocytes and/or the processes of myelination can be obtained by their reappearance in the 2-D gel protein profile of P28 X-irradiated nerves that, as we have shown previously, undergo a delayed myelination. We then employed mass spectrometric analysis to determine the identities of oligodendrocyte derivedregulated proteins. In this thesis, I will begin by describing our current knowledge of the proteins expressed by oligodendrocytes and their role(s) in oligodendrocyte function. This will be followed by a detailed description of the experimental model system we utilized in an attempt to elucidate the complete repertoire of oligodendrocyte-regulated proteins. We will then describe the results generated fiom our methodology and discuss the implications of our findings in relation to the functional cooperation between oligodendrocytes and other cells of the developing central nervous system. The results generated fiom this project should lead to a clearer understanding of the role of oligodendrocytes and'of the array of proteins whose expression patterns are associated with these cells during CNS development.

Les mécanismes développementaux qui spécifient le nombre et le phénotype laminaire des neurones du cortex cérébral jouent un rôle essentiel dans l’établissement de la cytoarchitecture corticale. Le nombre de neurones dans chaque couche d'une aire donnée est déterminé par le taux de production neuronale, qui dépend étroitement de l'équilibre entre les divisions prolifératives et différenciatives. Des observations clés suggèrent que la durée de la phase G1 (TG1) ferait partie intégrante d'un mécanisme cellulaire régulant le mode de division des précurseurs du cortex. Nous avons testé cette hypothèse par l'accélération expérimentale de la progression dans la phase G1 de précurseurs corticaux de souris in vivo, via la surexpression des cyclines E1 et D1. A E15, la réduction de TG1 promeut la rentrée dans le cycle cellulaire aux dépens de la différenciation neuronale, résultant en une modification de la cytoarchitecture du cortex adulte. Des données de modélisation confirment que les effets induits par la réduction de TG1 sont médiés par des changements du mode de division. Les effets de la surexpression des cyclines E1 et D2 à E13 sont plus modérés qu'à E15, indiquant des différences intrinsèques entre les précurseurs corticaux précoces et tardifs. La mesure des phases du cycle cellulaire des populations de précurseurs corticaux à l’aide de différentes techniques révèle un niveau important d’hétérogénéité et souligne la nécessité de prendre en compte la diversité des précurseurs co‐existant dans les zones germinales du télencéphale. / In the cerebral cortex, area‐specific differences in neuron number and phenotype are distinguishing features both within and across species. The developmental mechanisms that specify the number of neurons and their laminar fate are instrumental in specifying cortical cytoarchitecture. Neuron number in layers and areas correlate with changes in the rate of neuron production, largely determined by the balance between proliferative and differentiative divisions in cortical precursors. Key observations suggest a concerted regulation between the duration of the G1 phase (TG1) and mode of division and have led to the hypothesis that TG1 could be an integral part of a cellular mechanism regulating the mode of division of cortical precursors. To test this hypothesis we experimentally accelerated TG1 in mouse cortical precursors in vivo, via the forced expression of cyclinE1 and cyclinD1. At E15, TG1 reduction promoted cell‐cycle re‐entry at the expense of differentiation and led to cytoarchitectural modifications. Modeling confirms that the TG1‐induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. Forced expression of G1 cyclins was also applied to early cortical precursors. The effects of cyclinD1 and cyclinE1 up‐regulation at E13 were milder than those observed at E15, pointing to intrinsic differences between early and late cortical precursors. The used of various techniques to measure cell‐cycle kinetics in distinct precursor populations underlined the necessity of taking the full diversity of neural precursors co‐existing in the GZ of the telencephalon into account when performing cellcycle kinetics analysis.

A técnica de mobilização neural (NM) clinicamente é eficaz, porém ainda é pouco fundamentada. Neste trabalho, submetemos ratos Wistar no 14º dia após a lesão constritiva crônica (CCI) do nervo isquiático ao tratamento com NM, em 10 sessões, e avaliamos o comportamento doloroso utilizando testes comportamentais para hiperalgesia e alodinia. Ainda, observamos através de ensaios de Western blotting o envolvimento das células gliais e do fator neurotrófico derivado do cérebro (BDNF). No estudo comportamental, os animais com CCI mostraram diminuição no limiar nociceptivo, tratados com a NM apresentaram melhora no comportamento doloroso. Os ensaios de Western blotting mostraram que após a CCI houve aumento de OX-42, GFAP e BDNF, na medula, tálamo e mesencéfalo, também observado em analise de imuno-histoquímica e após a NM observamos diminuição desses mediadores através da primeira técnica mencionada. Sendo assim, sugerimos que a técnica de NM é eficaz como terapia analgésica, sendo possível observar o envolvimento das células gliais e do BDNF neste modelo experimental. / The technique of neural mobilization (NM) is clinically effective, although it is still poorly reasoned. In this study, Wistar rats on day 14th after chronic constrictive injury (CCI) of the sciatic nerve were submitted to treatment with NM in 10 sessions, and it was evaluated the painful behavior using tests for hyperalgesia and allodynia. Also, we observed through Western blotting assays the involvementof glial cells and brain-derived neurotrophic factor (BDNF). In the behavioral study, animals with CCI showed a decrease in nociceptive threshold, and those treated with NM showed an improvement in pain behavior. Western blotting assays showed an increase after CCI of OX-42, GFAP and BDNF levels in the spinal cord, thalamus and midbrain, also observed in immunohistochemical analysis, and after the NM we observed a decrease of these mediators through the first technique mentioned. Therefore, we suggest that the NM technique is an effective analgesic therapy, and it is possible to observe the involvement of glial cells and BDNF in this experimental model.