Traumatic brain injury (TBI), a leading cause of death and disability in the United States, causes potentially preventable damage in part through the dysregulation of neural calcium levels. This dysregulation likely affects the activity of the calcium-sensitive phosphatase, calcineurin, with serious implications for neural function. To test this possibility, the present study characterized the role of calcineurin in a rat model of brain trauma, the lateral fluid percussion injury model. Golgi-Cox histochemistry revealed an acute post-TBI loss and delayed overgrowth of dendritic spines on principal cortical cells. The spine loss appeared to require calcineurin activity, since administering a calcineurin inhibitor, FK506, 1 hour after TBI prevented the spine loss. Additional experiments showed how calcineurin activity might be related to the spine loss. Specifically, Western blots and enzyme activity assays revealed an acute increase in the cortical activity of calcineurin and its downstream effector, the actin-depolymerizing protein, cofilin. The cofilin activation was blocked by the same FK506 treatment that prevented spine loss, suggesting a relationship between cofilin activation and spine loss. To investigate long-term consequences of calcineurin activation after TBI, rats were administered FK506 (Tacrolimus) 1 hour after TBI and then monitored for spontaneous seizure activity months later. Acute post-TBI treatment with FK506 reduced the frequency of late non-convulsive seizures but did not prevent late convulsive seizures, cortical atrophy, or thalamic damage. The results of the present study implicate calcineurin in the acute dendritic remodeling and late non-convulsive seizures that occur after TBI. Importantly, these findings reveal calcineurin as a potential therapeutic target in the treatment of TBI and its sequalae.

The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death. In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival. The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.

The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death. In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival. The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.

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In dieser Dissertation versuche ich, die zentralen Nitrogen-heterocyclischen Kerne, die Seitenketten und deren Position zu variieren. Als synthetische Strategie wurden Palladium-katalysierte Kupplungsreaktionen verwendet, um Seitenketten und Aryl-Substituenten einzuführen. Halogensubstituierte Diarylheterocyclen sind wichtige Intermediate in der Synthese der allgemeine Strukture. Die Einführung der gewünschten Seitenketten durch Carbon-Carbon und Carbon-Nitrogen-Bindungsknüpfung wurde durch Sonogashira-Kupplung, Heck-Kupplung und Buchwald-Hartwig-Aminierung erzielt. Mit der Sonogashira-Reaktion kann eine funktionalisierte Alkynylgruppe in die heterocyclischen Kerne effektiv und bequem eingeführt werden. Eine anschliessende katalytische Hydrierung der Alkynylgruppe führt zu funktionalisierten Alkyl substituierten Diarylheterocyclen. In der vorliegenden Arbeit wurden mehr als 180 Substanzen synthetisiert. Unter ihnen sind ungefähr 130 neue Substanzen. 86 von ihnen passen in die allgemeine Strukture. / In the present thesis, I tried to vary the central nitrogen-heterocyclic cores, the functionalised side chains and its position of attachment. As a synthetic strategy, palladium-catalyzed coupling reactions were used to introduce side chains and aryl substituents into the central heterocycle. In this way the utility of such reactions to heterocyclic systems, which were neglected so far, could be figured out. Halogen substituted diaryl heterocycles are important intermediates in the synthesis of general structures. The introduction of the desired side chains by Carbon-Carbon bond formation reactions was achieved by Sonogashira coupling and Heck coupling. Buchwald-Hartwig amination and nucleophilic substitution were used to establish side chains which are connected to the core heterocycle by heteroatom-Carbon bonds. Sonogashira reaction turned out to be the most effective and convenient method to introduce functionalized alkynyl group into the heterocyclic cores. In the present work, more than 180 compounds were synthesized. Among them, about 130 compounds are new products. 86 of them fit into the general structure.