Kar Wing To. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 200-223). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.v / 撮要 --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / Contents --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1 / Chapter 1.1.1 --- Astrocytes --- p.1 / Chapter 1.1.2 --- Oligodendrocytes --- p.5 / Chapter 1.1.3 --- Microglial --- p.6 / Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6 / Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9 / Chapter 1.4 --- Astrocytes and Immune Response --- p.10 / Chapter 1.5 --- Cytokines --- p.10 / Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12 / Chapter 1.5.2 --- Cytokines and brain injury --- p.13 / Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13 / Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14 / Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16 / Chapter 1.5.4.2 --- Binding to TNF-α --- p.17 / Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17 / Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19 / Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23 / Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26 / Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27 / Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28 / Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29 / Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30 / Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30 / Chapter 1.6 --- Ion Channels and Astrocytes --- p.31 / Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33 / Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33 / Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34 / Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34 / Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36 / Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36 / Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38 / Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39 / Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39 / Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41 / Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43 / Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45 / Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46 / Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49 / Chapter 1.7.1 --- PKC and Brain Injury --- p.50 / Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51 / Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52 / Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54 / Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54 / Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55 / Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56 / Chapter 1.10 --- The Aim of This Project --- p.58 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.61 / Chapter 2.1.1 --- Sources of the Chemicals --- p.61 / Chapter 2.1.2 --- Materials Preparation --- p.65 / Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65 / Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65 / Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65 / Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66 / Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66 / Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67 / Chapter 2.1.2.5 --- Antibodies --- p.67 / Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67 / Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67 / Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68 / Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68 / Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68 / Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69 / Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69 / Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70 / Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71 / Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72 / Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74 / Chapter 2.2 --- Methods --- p.74 / Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74 / Chapter 2.2.2 --- Cell Preparation for Assays --- p.75 / Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76 / Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76 / Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77 / Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77 / Chapter 2.2.3.4 --- Protein Assay --- p.78 / Chapter 2.2.3.5 --- Data Analysis --- p.79 / Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79 / Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79 / Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79 / Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80 / Chapter 2.2.4.1 --- Confocal Microscopy --- p.80 / Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81 / Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82 / Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83 / Chapter 2.2.5.1 --- RNA Preparation --- p.83 / Chapter 2.2.5.1.1 --- RNA Extraction --- p.83 / Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84 / Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84 / Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85 / Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85 / Chapter 2.2.5.5 --- Quantification of Band Density --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87 / Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88 / Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92 / Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92 / Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95 / Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96 / Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102 / Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102 / Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104 / Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107 / Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109 / Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109 / Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114 / Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114 / Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117 / Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117 / Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119 / Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121 / Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121 / Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126 / Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128 / Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130 / Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130 / Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135 / Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138 / Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138 / Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141 / Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143 / Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143 / Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146 / Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149 / Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149 / Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153 / Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157 / Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159 / Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161 / Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165 / Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167 / Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167 / Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172 / Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172 / Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175 / Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177 / Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177 / Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179 / Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179 / Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS / Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184 / Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186 / Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187 / Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187 / Chapter 4.3.2 --- CaSR Expression --- p.190 / Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192 / Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193 / Chapter 4.4 --- Conclusion --- p.194 / REFERENCES --- p.200

Le glufosinate d’ammonium (GLA) est un herbicide organophosphoré couramment utilisé en agriculture. De nombreux cas d’ingestions intentionnelles ont mis en évidence sa neurotoxicité. Cependant, ses effets sur le neurodéveloppement ne sont peu étudiés. En effet, le cerveau est une cible importante du GLA en raison de son homologie de structure avec le glutamate, principal neurotransmetteur excitateur du système nerveux central. Des résultats précédents du laboratoire ont permis de montrer qu’une exposition périnatale à de faibles doses de GLA induisait des perturbations de la neurogenèse et de la migration des neuroblastes au niveau de la zone sous ventriculaire vers les bulbes olfactifs. Ces modifications sont associées à l’apparition de troubles du spectre autistique dans la descendance. Ma thèse s’inscrit dans la continuité de ses travaux en abordant les aspects cellulaires et moléculaires mis en jeux lors d’une exposition précoce au GLA. Etant donné que dans la vie de tous les jours, nous sommes continuellement exposés aux pesticides mais également à leurs métabolites, j’ai étudié en parallèle les effets du principal métabolite du GLA, l’acide 4-méthylphosphinyl-2-oxo-butanoïque (PPO).Le premier travail de ma thèse a été de développer un protocole in vitro de culture primaire de cellules souches neurales issues de la zone sous-ventriculaire de souris pour l’analyse des effets neurotoxiques du GLA et du PPO. Les résultats de la première étude de ma thèse montrent une induction d’un stress oxydatif lié impliquant le système glutamatergique et associé à une perturbation de l’homéostasie calcique. Etant donné que les cellules souches neurales sont sensibles aux effets d’un stress oxydatif, dans une seconde étude, j’ai étudié l’impact de ces effets sur les mécanismes de différenciation cellulaire des cellules souches neurales. Mes résultats indiquent un effet significatif d’une exposition au GLA et au PPO sur la formation et le maintien de la niche neurogénique sous-ventriculaire in vitro. Le GLA et le PPO interfèrent avec la formation de l’épithélium épendymaire et induisent une perturbation dans la différenciation neurogliale des cellules souches neurales, sans influencer leur capacité de croissance ou de prolifération.L’ensemble des données de cette thèse mettent l’accent sur l’intérêt d’étudier les mécanismes cellulaires et moléculaires liés à la neurotoxicité des substances actives des pesticides, des métabolites de ces mêmes pesticides, mais également des mélanges substances actives-métabolites auxquels nous sommes continuellement exposés dans notre environnement. / The glufosinate-ammonium (GLA) is an organophosphorus herbicide commonly used in agriculture. Many cases of intentional ingestions have highlighted its neurotoxicity. However, its effects on neurodevelopment are not well studied. Indeed, the brain is an important target of GLA due to its structural homology with glutamate, the main excitatory neurotransmitter of the central nervous system. Our previous data are shown that a perinatal exposure to low doses of GLA induces disturbances in neurogenesis and in neuroblasts migration from the subventricular zone to the olfactory bulbs. These changes are associated with the development of autism spectrum disorders in the offspring. My thesis is in the continuity of his work and addresses the cellular and molecular aspects involved in early exposure to GLA. Since we are continuously exposed to pesticides, but also to their metabolites, I studied in parallel the effects of the main metabolite of GLA, the 4 methylphosphinyl-2-oxo-butanoic acid (PPO).The first work of my thesis was to develop an in vitro protocol for the primary culture of neural stem cells from the subventricular zone of mice, for the analysis of the neurotoxic effects of GLA and PPO. The results of the first study of my thesis showed an induction of related oxidative stress involving the glutamatergic system, and associated with a disruption of calcium homeostasis. Since neural stem cells are sensitive to the effects of oxidative stress, in a second study, I studied the impact of these effects on the cellular differentiation mechanisms of neural stem cells. My results indicated a significant effect of exposure to GLA and PPO on the formation and maintenance of the subventricular neurogenic niche in vitro. GLA and PPO interfere with the formation of ependyma and induce a disruption in the neuroglial differentiation of neural stem cells, without influencing their growth or proliferation capacity.All these data highlight on the interest of studying the cellular and molecular mechanisms linked to the neurotoxicity of the active substances of pesticides, the metabolites of these same pesticides, but also the mixtures of active substances and metabolites to which we are continuously exposed in our environment.

La poly(ADP-ribosyl)ation est une modification post-traductionnelle de protéines catalysée par les Poly(ADP-ribose) polymérases (PARPs, 17 membres). Depuis 2011, le laboratoire décortique les propriétés biologiques de PARP3 qui est désormais bien décrite pour son rôle dans la réparation des cassures double-brin de l’ADN, la mitose et la transition épithélio-mésenchymateuse. Ces recherches combinées aux données de la littérature semblent indiquer que les fonctions de PARP3 sont très étendues et participent aussi à des processus physiologiques. Ainsi, mes travaux de thèse révèlent une nouvelle fonction clé de PARP3 dans la différenciation des cellules souches neurales et musculaires. Nous avons observé une forte augmentation de l’expression de PARP3 au cours de la neurogénèse, la gliogenèse et la myogénèse. En l’absence de PARP3, la différenciation des cellules souches neurales (NSPC) en astrocytes et neurones est perturbée et les souris PARP3KO présentent une incapacité à régénérer le tissu cérébral au niveau du striatum après ischémie hypoxique. Concernant les cellules musculaires, la disruption de PARP3 (Crispr/Cas9) empêche toute différenciation des myoblastes C2C12 en myotubes et conduit à une désoganisation du cytosquelette, une dégénérescence mitochondriale et une répression de gènes de l’identité. La réexpression de PARP3 catalytiquement active restaure la capacité de différenciation des C2C12. Enfin, nous avons identifié de nouvelles protéines cibles de PARP3 qui permettent de suspecter un rôle dans l’autophagie et le métabolisme énergétique au cours de la différenciation cellulaire. L’ensemble de ces résultats nous ont permis de découvrir que PARP3 a un rôle central dans la différenciation cellulaire et d’ouvrir de solides pistes de recherche afin d’identifier les mécanismes mis en jeu. / Poly (ADP-ribosyl)ation is a post-translational modification of proteins catalysed by Poly (ADP-ribose) polymerases (PARPs, 17 members). Since 2011, the laboratory has been dissecting the biological properties of PARP3 which is now well described for its role in the repair of DNA double-strand breaks, in mitosis and in epithelial-mesenchymal transition. This investigation combined with data from the literature suggests that PARP3 functions are very wide and could participate in physiological processes. Thus, my thesis work reveals a new key function of PARP3 in neural and muscular stem cell differentiation. We observed a strong increase in PARP3 expression during neurogenesis, gliogenesis and myogenesis. In the absence of PARP3, the differentiation of neural stem cells (NSPCs) into astrocytes and neurons is impaired and PARP3KO mice display an inability to regenerate brain tissue in the region of the striatum after hypoxic ischemia. Regarding muscle cells, PARP3 disruption (Crispr/Cas9) prevents C2C12 myoblast differentiation into myotubes and leads to cytoskeleton disorganisation, mitochondrial degeneration, and repression of identity genes. The reexpression of a catalytically active PARP3 restores the C2C12 differentiation capacity. Finally, we have identified new PARP3 target proteins that suggest a role in autophagy and energetic metabolism during cell differentiation.Together, these results reveal that PARP3 has a central role in cell differentiation and opens solid lines of research to identify the mechanisms involved.

Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.

Pluripotent stem cells (PSCs) are specialized cells, which have remarkable ability to maintain in an undifferentiated state and are capable of undergoing differentiation to three germ-layer lineage cell types, under differentiation-enabling conditions. PSCs include embryonic stem (ES)-cells, embryonal carcinoma (EC)-cells and embryonic germ (EG)-cells. ES-cells are derived from the inner cell mass (ICM) of day 3.5 blastocysts (mouse). On the other hand, EC- and EG-cells have different source of origin and exhibit some differences in terms of their differentiation abilities and culture requirements. These PSCs act as an ideal in-vitro model system to study early mammalian development and cell differentiation and, they could potentially be used for experimental cell-based therapy for a number of diseases. However, one of the problems encountered is the immune rejection of transplanted cells. For this, immune-matched induced pluripotent stem (iPS)-cells have been derived from somatic cells, by forced expression of a few stemness genes. Although, human PSCs lines are being experimented, their cell-therapeutic potential is still far from being thoroughly tested due to lack of our understanding regarding lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked PSCs and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse: GU-3 line (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. Recently, we have derived a ‘GS-2’ ES-cell line from the GU-3 mouse line (Singh et al., 2012). Additionally, we envisaged the need for developing an iPS-cell line from the GU-3 mouse and then use them for studying cell differentiation. Thus, aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked iPS-cell line from a genetically non-permissive FVB/N mouse strain, characterize the established iPS-cell line and achieve differentiation of various cell types from EGFP-expressing iPS-cell line; (2) to study differentiation phenomenon, in particular to cardiac lineage, using select-cardiogenesis modulators and (3) to assess the gene-expression profiles and signaling system associated with cardiomyocyte differentiation of PSCs. This thesis is divided into four chapters with the 1st chapter being a review of literature followed by three data chapters. In the chapter I of the thesis, a comprehensive up-to¬date review of literature is provided pertaining to PSCs, their classification, derivation strategies especially for reprogramming of somatic cells for iPSC generation, their differentiation potential and characterization, particularly to cardiac lineage. Various molecular regulators involved in cardiac differentiation of PSCs with emphasis on epigenetic regulation involving DNA methylation and signaling pathways involved are described in detail. Subsequently, various approaches used for enhanced cardiac differentiation of PSCs and the therapeutic potential of PSC-derived differentiated cell types to treat disease(s) are discussed. Chapter-II describes the successful establishment of a permanent iPS-cell line (named ‘N9’ iPS-cell line) from the non-permissive FVB/N EGFP-transgenic GU-3 ‘green’ mouse. This chapter provides results pertaining to detailed derivation strategy and characterization of the ‘N9’ iPS-cell line which includes colony morphology, expansion (proliferation) efficiency, alkaline phosphatase staining, pluripotent markers’ expression analysis by qPCR and immunostaining approaches and karyotyping analysis. Further, in order to thoroughly assess the differentiation competence of the ‘N9’ iPS¬cell line, assessment of in-vitro and in-vivo differentiation potential of the ‘N9’ iPS-cell line by embryoid body (EB) formation and teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives were performed, followed by the generation of chimeric blastocysts by aggregation method. This established N9 iPS-cell line could potentially offer a suitable model system to study cardiac differentiation along with other established PSC lines such as the GS-2 and D3 ES-cell lines and the P19 EC-cell line. Following the establishment of the system to study cardiac differentiation of PSC lines, efforts were made to understand the biology of cardiac differentiation of PSCs (wild¬type and EGFP-transgenic PSC lines and P19 EC-cell line) using small molecules as modulators. Data pertaining to this is described in Chapter-III. The possible involvement of epigenetic regulation of cardiogenesis for example, DNA methylation changes in cardiogenesis-associated genes is studied using 5-aza cytidine as one of the chromatin modifiers. In order to understand the cardiac differentiation phenomenon, as a consequence of using 5-aza cytidine in cell culture, it was important to investigate its ability to induce/mediate cardiac differentiation. This involved an assessment by quantitating the cardiac beating phenotype and correlating this with enhanced cardiac-gene expression profiles. Further, DNA methylation regulation of cardiogenesis¬associated genes is described using various DNA methylation analysis techniques. Moreover, the possible involvement of other signaling members in mediating the cardiac differentiation is also studied using the P19 EC-cells. Results pertaining to the above findings are described in detail in the Chapter-III. Chapter-IV is focused on various efforts made towards investigating the ability of ascorbic acid to enhance cardiac differentiation of mouse ES-cells (GS-2 and D3 lines). Ascorbic acid has been implicated to be influencing cardiogenesis and it is reported to enhance differentiation of various cell types under certain culture conditions. Results pertaining to enhancement of cardiac differentiation of PSCs using ascorbic acid are presented in this chapter. This included assessment by quantitating cardiac beating phenotype and its correlation with enhanced cardiogenesis-associated gene expression profiles. Besides, estimation on the sorted cardiomyocyte population, derived from PSCs was also made using mature-cardiac marker. The possible underlying signaling mechanism involved was also studied in detail, using specific inhibitors for pERK (U0126), integrin signaling (pFAK; PP2) and collagen synthesis (DHP), in order to ascertain their involvement in ascorbic acid-mediated cardiac differentiation of mouse ES-cells. Subsequent to the three data chapters (II-IV), separate sections are provided for ‘Summary and Conclusion’ and for ‘Bibliography’, cited in the thesis. The overall scope of the study has been to understand the basic biology of cardiac differentiation from PSCs (EC-cells, iPS-cells and transgenic and wild-type ES-cells) and to assess, by using certain small molecules, whether PSCs could be coaxed to enhance the differentiation to a particular cell type (cardiac). The data contained in this thesis addresses the above theme.

Healthy bone is bombarded with many different mechanical strain derived signals during normal daily activities. One of these signals is present as a direct connective tissue strain on the cells. However, there is also the presence of an electrically charged streaming potential during this straining. The electrical potential is created from the movement of charged fluid through the small bone porosities. To date, little focus has been applied to elucidating the possible synergistic effects of these two stimulants. The aim of this project was to evaluate the effects of mechanical strain and indirect electrical stimulation upon the development of bone forming osteoblast cells and any possible synergistic effects of the two stimulants. This aim was achieved by using a novel device, designed and developed with the capability of creating a cell substrate surface strain along with an exogenous electrical stimulant individually or at the same time. Proliferation and differentiation were determined as a measure of cellular development. The indirect electrical stimulation was achieved through the use of a pulsed electromagnetic field (PEMF) while the mechanical strain was produced from dynamic stretching of a deformable cell substrate. Strain and strain rate were modelled from recent studies proposing that relatively high frequency, low strain osteogenic mechanical stimulants are more indicative of what healthy bone would be experiencing during normal activities. The PEMF signal mimicked a clinically available bone growth stimulator signal. Results showed a PEMF stimulus on monolayers of SaOS-2 and MG-63 osteoblast-like cells leads to a depression in proliferation. A concomitant increase in alkaline phosphatase production was also observed for the SaOS-2 cultures, but not for the MG-63 cell line. It was hypothesised that this was due to the MG-63's lack of phenotypic maturity compared to the SaOS-2 cells. Mechanical strain of the cell substrate alone, at a relatively high frequency (5Hz) but small strain, did not significantly effect either cell proliferation or differentiation for the MG-63 cells. However, when the electrical and mechanical stimulants were combined a significant increase in cellular differentiation occurred with MG-63 cultures, revealing a possible synergistic effect of these two stimulants on the development of bone cells.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

The Tec family kinases Itk and Rlk have been previously shown to have an important role in signaling downstream of the T cell receptor [TCR]. Almost all of the work done in the past on these two kinases looked at their role in conventional αβ T cells, specifically CD4+ T cells. These studies demonstrated functions for Itk [primarily] and Rlk in T cell development, activation, and differentiation. However, despite the wealth of knowledge on conventional CD4+ T cells, prior to the work presented here little to no studies addressed the role of Tec family kinases on CD8+ or innate T cell development. My studies show a clear role for Itk [and in some cases Rlk] in innate T cell development; whether it be deprecating, in the case of innate CD8+ T cells or some subsets of γδ T cells, or beneficial, in the case of NKT cells. I show that Itk has a crucial role in conventional CD8+ T cell development, as absence of Itk [or Itk and Rlk] causes strongly reduced numbers of conventional CD8+ T cells and a vigorous enhancement of an innate-like CD8+ T cell population. In NKT cells, my work demonstrates that Itk [and to a lesser extent Rlk] is required for terminal maturation, survival, and cytokine secretion. Finally, on γδ T cells Itk is important in maintaining the Th1 cytokine secretion profile usually associated with these cells, and regulating the development of CD4+ or NK1.1+ γδ T cells. Taken together, this work clearly illustrates an important role for Tec family kinases in innate T cell development and function.

The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K. The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways.