Doctor of Philosophy / Department of Biology / Stefan Rothenburg / Vaccinia virus is the prototypic poxvirus that has been widely used as a model for investigating poxvirus biology and genetics. Like several members of the Poxviridae family, vaccinia virus can infect several different species including mice, cows and humans. Because the entry of poxviruses into a host cell relies on ubiquitously expressed surface molecules, which are found in many species, the ability of poxviruses to infect and replicate in different host cells primarily depends on their ability to subvert the host’s innate immune response. One critical barrier to infection is overcoming the general shutdown of protein translation initiated by the cellular protein kinase PKR. PKR detects cytoplasmic double-stranded (ds) RNA generated during infection by the replicating virus, which activates it to phosphorylate the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2) and suppress general translation. Poxviruses are large viruses with dsDNA genomes that encode around 200 genes. Several of these genes are known as host range genes and are important for replication in different host species and many interact with components of the host immune response to promote viral replication. Two genes in vaccinia virus, called E3L and K3L, are known inhibitors of PKR and have previously been shown to be important for virus replication in cells from different species. The molecular explanation behind their host range function, however, is unknown. The main goal of the research presented in this thesis is to determine the molecular mechanisms for the host range function of vaccinia virus E3L and K3L, particularly in different hamster host cells. Along with an analysis of vaccinia virus host range genes, we have used genome-wide comparisons between host-restricted poxviruses in the Leporipoxvirus genus to parse out the potential genomic determinants of host range restriction in this clade of poxviruses. The overarching aim of this thesis work is to better understand the molecular mechanisms for host range in poxviruses.

Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNy-dependent autophagy, respectively. Indeed, sustained expression and activation of PKR reduced the intracellular survival of Mtb, an effect that could be enhanced by IFNy treatment. We further demonstrate that the enhanced anti-mycobacterial activity of THP-PKR macrophages is mediated by a mechanism dependent on selective autophagy as indicated by increased levels of LC3-II that colocalize with intracellular Mtb. Consistent with this mechanism, inhibition of autophagolysosome maturation with bafilomycin A1 abrogated the ability of THP-PKR macrophages to limit replication of Mtb, whereas pharmacological activation of autophagy enhanced the anti-mycobacterial effect of PKR overexpression. As such, PKR represents a novel and attractive host target for development of HDT for TB, and our data suggest value in the design of more specific and potent activators of PKR.

Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...

Die heterotetramere Proteinkinase CK2 nimmt aufgrund der großen Anzahl und Diversität ihrer Substrate, sowie aufgrund ihrer Eigenschaft Signalwege miteinander zu vernetzen eine Sonderstellung innerhalb der Kinasen ein. CK2 beeinflusst Proliferation, Differenzierung und Apoptose, Prozesse an denen auch Polyamine und der MAPK-Signalweg beteiligt sind. Eine vor kurzem durchgeführte Arbeit beschreibt die Bindung von CK2 an das Gerüstprotein KSR und die Verstärkung des MAPK-Signalwegs durch Phosphorylierung von Raf-Proteinen in Vertebraten. In dieser Arbeit konnte gezeigt werden, dass CK2 auch in Drosophila mit KSR interagiert und das einzige in Drosophila vorhandene Raf-Potein (DRaf) in vitro phosphoryliert. Im Gegensatz zur Phosphorylierung der humanen B-Raf und C-Raf Proteine an Serin 446 bzw. Serin 338 innerhalb der „negative charge regulatory region“ (N-Region), führten Kinasereaktionen und Massenspektrometrische Untersuchungen zur Identifizierung von Serin 11 als CK2 Phosphorylierungsstelle in DRaf, während ein zu Serin 446 in B-Raf äquivalentes Serin in der N-Region in Drosophila nicht durch CK2 phosphoryliert wird. Durch Überexpression von DRaf sowie von zwei DRaf-Varianten bei denen Serin 11 durch Alanin oder Aspartat substituiert wurde (DRafS11A und DRafS11D) konnte in Zellkulturexperimenten gezeigt werden, dass die Ladung an der Aminosäureposition 11 die Funktion von DRaf beeinflusst, wobei eine negative Ladung an dieser Stelle zur Phosphorylierung und Aktivierung der Effektorkinase Erk führt. Die Phosphorylierung durch CK2 ist unabhängig von regulatorischen Botenstoffen ("second messengers"), wird aber durch Bindung von Polyaminen moduliert. Intrazelluläre Polyamine entstammen zum grossen Teil dem zellulären Aminosäurekatabolismus und beeinflussen die Phosphorylierung von DRaf durch CK2 in vitro, wobei Spermin ein effizienter Inhibitor der Reaktion ist, während die Effekte von Putrescin und Spermidin gering sind. Auch in Drosophila Schneider S2 Zellen und in adulten weiblichen Fliegen hat Spermin einen inhibitorischen, CK2-abhängigen Effekt auf die Aktivierung von Erk. Ausserdem konnte gezeigt werden, dass Putrescin und Spermidin in der Lage sind die Aktivierung von Erk, im Vergleich zu Zellen die nur mit Spermin behandelt wurden, zu erhöhen. Das spricht dafür, dass die Phosphorylierung von DRaf und die davon abhängige Aktivierung von Erk durch CK2 von der Menge und Relation der verschiedenen Polyamine zueinander abhängt. Die Ergebnisse dieser Arbeit lassen den Schluss zu, dass der Polyaminmetabolismus über CK2 mit dem MAPK-Signalweg verknüpft ist. Nachdem Polyamine durch Aminosäurekatabolismus enstehen, kann auf diese Weise der MAPK-Signalweg in Abhängigkeit der Verfügbarkeit zellulärer Aminosäuren reguliert werden. Vorversuche zeigten eine Beeinflussung von Proliferation und Apoptose durch CK2 und Polyamine. Weitere Untersuchungen sind aber nötig um spezifische Einflüsse von Polyaminen und CK2 auf zelluläre Prozesse wie Proliferation, Differenzierung und Apoptose aufzudecken. / Because of its high number and diversity of substrates, as well as its ability to cross-link signalling pathways, the heterotetrameric protein kinase CK2 has an exceptional position within kinases. CK2 influences proliferation, differentiation and apoptosis, processes in which also polyamines and the MAPK-signalling pathway are involved. A recent publication delineates binding of CK2 to the scaffold protein KSR and the enhancement of the MAPK-signalling pathway by phosphorylation of Raf-proteins in vertebrates. In my thesis I could show that CK2 also interacts with KSR in Drosophila and phosphorylates the only existing Raf protein in Drosophila (DRaf) in vitro. In contrast to the phosphorylation of human B-Raf- and C-Raf-proteins on serine 446 respectively serine 338 within the "negative charge regulatory region" (N-region), kinase reactions and mass spectrometric analyses led to the identification of serine 11 as phosphorylation site in DRaf, whereas a serine in the N-region, which corresponds to serine 446 of B-Raf, is not phosphorylated by CK2 in Drosophila. In cell culture experiments overexpression of DRaf and two DRaf-variants, in which serine 11 was substituted by alanine or aspartate (DRafS11A and DRafS11D), revealed the charge at amino acid position 11 to affect the function of DRaf, with a negative charge leading to phosphorylation and activation of the effector kinase Erk. Phosphorylation by CK2 is independent of second messengers, whereas it is modified by binding of polyamines. Intracellular polyamines mainly derive from cellular amino acid catabolism and modulate the phosphorylation of DRaf by CK2 in vitro with spermine being an efficient inhibitor of the reaction, whereas the effects of putrescine and spermidine are minor. In Drosophila Schneider S2 cells and adult flies spermine inhibits the activation of Erk in a CK2-dependent way. Furthermore administration of putrescine and spermidine in combination with spermine leads to enhanced Erk activation in cells compared to cells that are treated with spermine. These results suggest that phosphorylation of DRaf and the subsequent activation of Erk by CK2 are dependent on the amount and relative concentrations of polyamines. Altogether the results of this work demonstrate a role for CK2 in linking polyamine metabolism to the MAPK-signalling pathway. Since polyamines derive from amino acid catabolism, the MAPK-signalling pathway can be regulated dependent on the availability of cellular amino acids. Preliminary experiments point to CK2- and polyamine-dependent effects on proliferation and apoptosis. Further investigations are necessary to reveal specific effects of polyamines and CK2 on cellular processes like proliferation, differentiation and apoptosis.

A insuficiência cardíaca é uma síndrome clínica de mau prognóstico caracterizada por disfunção cardíaca associada à intolerância aos esforços, retenção de fluído e redução da longevidade. Dentre as serina/treonina quinases associadas às alterações funcionais e estruturais cardíacas observadas na progressão da insuficiência cardíaca, a família das proteínas quinase C (PKC) composta por 12 diferentes isoformas parece modular a contratilidade miocárdica e o remodelamento cardíaco. No presente estudo, caracterizamos o fenótipo cardíaco e o perfil de ativação das diferentes isoformas de PKC na progressão da insuficiência cardíaca de etiologia isquêmica em ratos. Além disso, estudamos o efeito da inibição sustentada da isoforma PKCβII sobre a sobrevida, o remodelamento cardíaco e a função ventricular em modelo de insuficiência cardíaca de etiologia isquêmica. Conseguinte, identificamos possíveis substratos cardíacos da PKCβII envolvidos na progressão da insuficiência cardíaca. Para isso, avaliamos os efeitos agudo e crônico da inibição da PKCβII sobre o transiente de cálcio e a contratilidade de cardiomiócito isolados de ratos adultos com insuficiência cardíaca. Por fim, testamos as inibições específicas das PKCβII e PKCβI na progressão da hipertrofia cardíaca compensada para a insuficiência cardíaca em modelo animal de hipertensão arterial sustentada. Nossos resultados sugerem que a inibição sustentada da PKCβII reverte o quadro de insuficiência cardíaca, melhorando a função ventricular, o remodelamento cardíaco e a sobrevida dos diferentes modelos de insuficiência cardíaca estudados, constituindose em uma estratégia terapêutica celular promissora / Heart failure is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality worldwide. Protein kinase C isozymes emerge as important potential therapeutic targets in chronic cardiovascular disease. However, individual PKC isozymes play different roles in the pathogenesis of cardiac diseases. Here, we characterized the cardiac phenotype as well as the different PKC isozyme activation profile during myocardial-induced heart failure progression in rat. Furthermore, we evaluated the role of selective PKCβ II inhibition on survival, left ventricle remodeling and cardiac function in myocardial-induced heart failure. Moreover, we identified the cardiac PKCβII substrates related to heart failure. Finally, PKCβII and PKCβI specific inhibitors were chronically delivered to hypertensive-induced heart failure rats and the cardiac phenotype was evaluated. Our data suggest that 6-wks of PKCβII inhibition, but not PKCβI, improved animal survival by restoring cardiac function and promoting cardiac anti-remodeling effect in both myocardial infarctioninduced heart failure and hypertensive-induced heart failure rats. The improved cardiac function and anti-remodeling effect of PKCβII inhibition seems to be associated with increased contractility of cardiac myocytes, improved miofilaments/Ca2+ sensitivity and decreased cardiac inflammatory response. Altogether, the results provide evidence for beneficial effects of PKCβII specific intracellular inhibition on cardiac function and remodeling, which may be a promising cellular therapy for heart failure treatment

Apesar dos grandes avanços na produção in vitro (PIV) de embriões bovinos, a produção de blastocistos se mantém ainda aquém do observado in vivo, indicando que melhorais ainda são necessárias no processo de PIV. A competência para o desenvolvimento de oócitos tem sido relacionada, entre outros fatores, à interrupção do período de capacitação. Foi sugerido que o bloqueio meiótico poderia permitir ao oócito um tempo adicional para adquirir a competência. Embora ainda não se tenha melhorado o desenvolvimento embrionário com esse procedimento, o bloqueio meiótico com inibidores de cinases dependentes de ciclinas (CDK), como a butirolactona I (BLI), pode ser uma ferramenta útil na compreensão do desenvolvimento do oócito. O presente trabalho teve por objetivo averiguar o efeito da inibição de CDK com BLI, para bloquear temporariamente a retomada da meiose, sobre fatores que controlam o ciclo celular meiótico de oócitos bovinos. Oócitos bovinos obtidos de abatedouros foram bloqueados em vesícula germinativa (VG) in vitro com 10 µM de BLI por 24 h (BVG - bloqueado imaturo). Parte desses oócitos foi depois maturada in vitro por mais 24 h (BMII - bloqueado maturado). Como controles foram utilizados oócitos recém-aspirados dos folículos (VG - controle imaturo) ou maturados in vitro sem bloqueio meiótico prévio (MII - controle maturado). Os diferentes grupos de oócitos foram avaliados quanto a: 1) expressão de RNAm dos componentes do MPF (p34cdc2 e ciclina B1) e da MAPK; 2) expressão das proteínas do MPF (p34cdc2 e ciclina B1) e MAPK (p44 e p42); 3) atividade das proteínas MPF e MAPK durante a maturação in vitro de oócito submetidos ou não ao bloqueio da meiose pré-maturação e 4) a localização subcelular das proteínas p34cdc2, ciclina B1 e MAPK nos oócitos. Foi observado que: 1) a abundância relativa do RNAm de p34cdc2 e MAPK reduziu-se com a maturação, enquanto a ciclina B1 menteve-se estável; o bloqueio meiótico manteve o mesmo padrão de acordo com o estádio de maturação; 2) as proteínas de p34cdc2 e MAPK mantiveram-se estáveis durante a maturação, enquanto a ciclina B1 não foi detectada em oócitos imaturos e o foi nos maturados; o bloqueio meiótico também manteve a expressão das proteínas de acordo com o estádio de maturação; 3) as atividades de MPF e MAPK foram pouco afetadas pelo bloqueio meiótico; 4) as proteínas foram todas detectadas no citoplasma dos oócitos imaturos (bloqueados ou não) e maturados (bloqueados ou não), sendo apenas a p34cdc2 também localizada no núcleo de oócitos imaturos (bloqueados ou não). Diante dos resultados conclui-se que o bloqueio da meiose mantém o mesmo padrão de expressão, atividade e localização subcelular do MPF e da MAPK em oócitos imaturos e maturados, sugerindo que a tradução das mensagens, a ativação das cinases e a distribuição das proteínas foram controladas de acordo com o estádio da meiose, não sendo afetadas pelo bloqueio. / Although there have been great improvements in in vitro production (IVP) of bovine embryos, blastocyst production is still beyond that observed in vivo, indicating that more improvements are necessary in the IVP system. Developmental competence of oocytes has been related to, among other factors, the interruption in oocyte capacitation. Is has been suggested that meiosis block would allow the oocyte additional time to acquire competence. Although this procedure has not been successful in increasing embryo development, meiosis block with cyclin dependent kinase (CDK) inhibitors, such as butyrolactone I (BLI) can be a useful tool to study oocyte development. The present study aimed to assess the effects CDK inhibition with BLI, to temporarily block meiosis resumption, on factors involved in meiosis cell cycle control in bovine oocytes. Oocytes, obtained form slaughterhouse ovaries, were maintained in germinal vesicle (GV) arrest in vitro with 10 µM BLI for 24 h (BVG - blocked immature). A part of these oocytes was matured in vitro for another 24 h (BMII - blocked and matured). As controls, oocytes were also assessed soon after follicle aspiration (GV -immature control) or matured in vitro for 24 h without prior meiosis block (MII - matured control). The different groups of oocytes were assessed for: 1) mRNA expression for the MPF components (p34cdc2 and cyclin B1) and MAPK; 2) expression of MPF (p34cdc2 and cyclin B1) and MAPK (p44 and p42) proteins; 3) MPF and MAPK activities during maturation of oocytes submitted or not to prematuration meiosis block and 4) p34cdc2, cyclin B1 and MAPK subcellular localization within oocytes. During the study it was observed that: 1) relative abundance of p34cdc2 and MAPK mRNA was reduced after maturation, while cyclin B1 mRNA remained stable; meiosis block maintained the same pattern according to maturation stage; 2) p34cdc2 and MAPK proteins remained stable after maturation while cyclin B1 protein was undetected in immature oocytes and detected in the matured ones; meiosis block also maintained the same protein expression according to maturation stage; 3) MPF and MAPK activities were little affected by meiosis block and 4) all proteins were detected in the cytoplasm of immature (blocked or not) and matured (blocked or not) oocytes, and only showed a specific nuclear localizations in immature oocytes (blocked or not). According to the results it may be concluded that meiosis block maintained the same pattern of MPF and MAPK expression, activity and subcellular localization in immature and matured oocytes, suggesting that mRNA translation, kinase activation and protein distribution were controlled according to the maturation stage and were not affected by meiosis block.

A YakA é uma proteína quinase necessária para a regulação da resposta a diversos estresses em Dictyostelium e é uma efetora chave da transição do crescimento para desenvolvimento nesse organismo. O gene keaA foi isolado como um supressor do mutante yakA- em uma busca para revelar genes envolvidos na sobrevivência a estresse nitrosoativo. O gene keaA codifica uma proteína com seis repetições kelch na porção C-terminal, um domínio zf-C3HC4 na porção N-terminal, também chamado RING-finger, e uma sequência rica em cisteínas localizada na porção mediana da proteína. Mutantes deficientes em keaA foram avaliados revelando-se um papel para esse gene também no processo de desenvolvimento. A expressão de mRNA de keaA é induzida quando células selvagens crescem e a fonte de alimento começa a esgotar. A indução do mRNA de keaA também ocorre durante o desenvolvimento. Essa indução não é observada em células yakA- indicando que YakA regula KeaA. Células deficientes em keaA expressam baixos níveis de mRNA de pkaC, acaA e carA durante a agregação em baixa densidade celular, o que pode explicar porque as células deficientes em keaA apresentam o processo de desenvolvimento mais lento em baixa densidade celular. Células deficientes em keaA são mais resistentes a estresses nitrosoativo e oxidativo e keaA é necessário para a produção e detecção de AMPc. A análise da agregação de células deficientes em keaA durante o desenvolvimento multicelular indica que KeaA é necessário para que as células participem eficientemente desse processo. Células que super-expressam o domínio rico em cisteínas levam o mesmo tempo que as células selvagens para atingir o estágio de agregação. No entanto, essas células apresentam agregados, culminantes e corpos de frutificação menores. Células que super-expressam o domínio Kelch expressam altos níveis de acaA e carA depois de 8 horas de desenvolvimento, mas os níveis de pkaC são similares aos observados em células selvagens. Isso poderia indicar que o domínio Kelch induz a ativação da PKA. No entanto, essa interação não foi observada em experimentos de duplo híbrido. Adicionalmente, a expressão gênica em resposta a compostos que geram estresse oxidativo e nitrosoativo foi estudada utilizando-se microarranjos de cDNA. Os resultados revelaram um papel de keaA na resposta à pré-carência e no controle do ciclo celular / The YakA is a protein kinase required for the regulation of several stress responses in Dictyostelium and is a key effector of the transition from growth to development in this microorganism. The gene keaA was isolated as suppressor of the yakA- mutant in a screen targeted to reveal genes involved in the survival to nitrosoative stress. The keaA gene codes a protein with six kelch domain repeats at the C-terminus, a zf-C3HC4 domain at the N-terminus, also called RING-finger, and a cysteine-rich sequence located in the mid-portion of the protein. The analysis of mutants deficient in keaA revealed a role for this gene also in the development process. keaA mRNA expression is induced when wild type cells grow and the food source becomes scarce. An induction of keaA mRNA expression also occurs during development. This induction is not observed in yakA- cells, indicating that YakA regulates KeaA. keaA deficient cells express low levels of pkaC, acaA and carA mRNA during aggregation in low cell densities, which may explain why the keaA deficient cells present a delay in development in low cell densities. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. The analysis of agreggation of keaA deficient cells during multicellular development indicated that KeaA is required for the cells to efficiently participate in the process. Cells over-expressing the cisteine-rich domain took the same time as the wild-type cells to reach the aggregation stage. However, these cells present smaller aggregates, culminants and fruiting bodies. Cells over-expressing the Kelch domain express high levels of acaA and carA after 8 hours of development, but the levels of pkaC are kept similar to those in wild-type cells. This could indicate that the Kelch domain induces the activation of PKA. However, this interaction was not observed when we conducted tests in a two-hybrid system. Additionally, gene expression in response to compounds that generate redox stresses was studied using cDNA microarrays. The results revealed a role of keaA in response to pre-starvation and control of cell cycle

Mitochondria are critical for energy production and Ca2+ homeostasis and undergo fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by Drp1 (dynamin-related protein 1), a large GTPase tightly controlled by various posttranslational modifications, including phosphorylation. Bβ2 is a neuron-specific postnatally induced protein phosphatase 2A (PP2A) regulatory subunit that mediates PP2A translocation to the outer mitochondrial membrane (OMM) to promote mitochondrial fragmentation and sensitize neurons to various injuries. Opposing PP2A/Bβ2's effect on mitochondrial morphology and cell death is protein kinase A (PKA) anchored to the OMM via A kinase anchoring protein 1 (AKAP1). This dissertation describes how reversible phosphorylation of Drp1 at a conserved Serine residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase complex (PP2A/Bβ2) affects dendrite and synapse development in hippocampal neurons and synaptic plasticity and learning and memory in vivo. Inducing mitochondria fragmentation decreases dendritic arbor complexity, but increases spine and synapse number. Mitochondrial elongation induces opposite effects. L-carnitine increases mitochondria membrane potential and recapitulates the dendritic and synaptic effects of mitochondrial elongation. Epistasis experiments substantiate our hypothesis that PP2A/Bβ2 dephosphorylates and PKA/AKAP1 phosphorylates Drp1 to change mitochondrial shape and regulate mitochondria localization, dendrite outgrowth, and synapse development. Bβ2 null mice are viable and fertile, without obvious abnormalities. Bβ2 null mice demonstrate significantly larger cortical and hippocampal neuronal mitochondria than in wildtype. Bβ2 deletion decreases spine number on apical and basal cortical dendrites and hippocampal dendrites. Bβ2 null mice display significantly decreased input/output relationship in the hippocampus, consistent with a decrease in synapse number. In a combined context and cued fear-conditioning protocol, the hippocampal-dependent context recall trial revealed significant deficits in Bβ2 null and heterozygous mice. This deficit is also seen in hippocampal-dependent Barnes maze performance. These results are consistent with the reduced hippocampal long-term potentiation (LTP) found in Bβ2 null mice and demonstrate the importance of Bβ2 in hippocampal synaptic plasticity and memory. In conclusion, PP2A/Bβ2 and PKA/AKAP1 have important roles in mitochondria regulation and dendritic and synaptic development as seen in our results in vitro with rat hippocampal cultures and in vivo with Bβ2 null mice.

Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

cGMP-dependent protein kinases (PKG) are key players in intracellular second messenger signaling within many cellular systems throughout the body. Most notably PKG is known for its role in smooth muscle relaxation (Pfeiffer et.al, 1999). The Iα PKG isozyme has been identified as the primary effector of the nitric oxide pathway (and serves to be a novel drug target). To date the overall knowledge of structure and function of PKG is lacking in terms of the mechanisms of activation and the structural orientations that coordinate them. Recently, our laboratory has solved the crystal structure of the regulatory domain of PKG Iα, which revealed a previously unknown α-helical domain dubbed the Switch Helix (SW) (Osborne et.al, 2011). The SW domain was found to be a site of interprotomer communication via hydrophobic interactions between its C-terminus and hydrophobic residues, named the nest located on the opposing protomer. Synthetic peptides derived from the SW domain, named S-tides, dosedependently activate PKG Iα (Moon et.al, 2015). In addition, the amino acid residues of the nest are in proximity to the cGMP binding site B. It was hypothesized that the binding site for S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. The hypothesis of this thesis is the binding site for the S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. To test this hypothesis two aims were constructed: Aim 1: To develop an S1.5 analog that utilizes both the nest and the B-site to increase S-tide activity, Aim 2: To explore the intricacies of these modes of activation and how they interact with each other to obtain a better understanding of the interplay between these two sites. First, based on the most potent S-tide S1.5 (YEDAEAKAKYEAEAAFFANLKLSD, Ka=6 μM), two analogs were synthesized. The peptide S2.5 which lacked the amino acids LSD at the C-terminus showed a three-fold lower activation constant (Ka= 15 μM), although the molecule retained its helicity as demonstrated by circular dichroism. The second analog, S3.5 contained unnatural amino acid components from a molecular modeling approach in an effort to further increase the affinity by interacting with the adjacent cGMP binding site B. However, S3.5 showed further reduction in activity with an activation constant of 70 μM. These findings led us to conclude that the failure of the SAR approach indicates a different mode of S-tide activation as had been previously thought. Next, we investigated the role of the cGMP binding site B in the mechanism of S-tide mediated PKG Iα activation. Co-activation assays with cGMP and S1.5 demonstrated that cGMP activation is not altered in the presence of S1.5. Furthermore, S1.5 mediated activation is negatively affected in the presence of cGMP. These results suggest that the B-site of cGMP does not positively enforce the S1.5 activation kinetics. Next, we employed the PKG Iα mutant E292A, which cannot bind cGMP to the B-site (Moon et.al., 2018). Interestingly, this mutant retains the activation kinetics of PKG Iα WT when activated via S1.5 and cGMP. Thus, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with cGMP. Likewise, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with S1.5. To further support these findings, the PKG Iα mutant C42A, which showed crippled cGMP activation kinetics could be activated with S1.5 with a potency similar to wild type. Taken together, the results in this thesis demonstrate that in contrast to the initial hypothesis the binding sites for S-tides and cGMP, although in proximity, show no experimental support of a positive interaction. These findings are significant as they reveal that S1.5 mediated activation of PKG is truly independent of cGMP, thereby providing a molecular platform for the therapeutic development of these unique peptides.