The thesis argues, in lie main, for both a negative and positive agenda to Wittgenstein's rule-following remarks in both his Philosophical Investigations and Remarks on the foundations of Mathematics. The negative agenda is a sceptical agenda, different than as conceived by Kripke, that is destructive of a realist account of rules and contends that the correct application of a rule is not fully determined in an understanding of the rule. In addition to these consequences, this negative agenda opens Wittgenstein to Dummett's charge of radical conventionalism (a charge that also, but differently, applies to certain mid-period views and this is addressed in the first chapter). These negative consequences are left unresolved by Kripke's sceptical solution and, notably, are wrongly assessed by those that dissent from a sceptical reading (e.g., McDowell). The positive agenda builds on these negative considerations arguing that although there is no determination in the understanding of a rule of what will count as a correct application in so far unconsidered situations, we are still able to follow a rule correctly. This seems to involve an epistemic leap, from an underdetermined understanding to a determinate application, and, in respect of this appearance, involves what Wittgenstein calls following a rule "blindly" in an epistemic sense. Developing this view, of following a rule blindly, involves developing an account of an alternative rational response to rule instruction, one that need not involve a role for interpreting or inferring, but all the same allows for correctness in rule application in virtue of enabling agreement in rule application.

O Transtorno Bipolar é uma doença crônica e grave que apresenta curso episódico e alto impacto sobre a funcionalidade do paciente. Possui potencial para alto grau de severidade, recorrência e intensidade e estima-se que sua prevalência ao longo da vida seja de 2,4% da população mundial. A fisiopatologia deste transtorno ainda não é bem compreendida e ainda o fármaco de primeira escolha é o lítio, embora tampouco seja claro seu mecanismo de ação. O sistema purinérgico principalmente devido a suas ações sobre neuroproteção, inflamação e ativação microglial se destaca como uma via promissora de novos alvos terapêuticos, especialmente o receptor purinérgico P2X7 (P2X7R). Dessa maneira, o objetivo desta tese é caracterizar o envolvimento do sistema purinérgico na fisiopatologia do transtorno bipolar. De maneira geral, avaliamos metabólitos do sistema purinérgico em soro de pacientes e controles, correlacionando com dados clínicos. Avaliamos a frequência do polimorfismo do P2RX7 1513A>C em pacientes e controles. Realizamos um modelo animal farmacológico de mania e investigamos o envolvimento do P2X7R neste modelo e por último buscamos melhor compreender a ação neuroprotetora do lítio verificando a participação purinérgica. Nossos resultados primeiramente nos mostraram uma diminuição nos níveis de adenosina em pacientes em comparação a controles e uma importante correlação inversa entre os níveis de adenosina e a piora dos sintomas. Demonstramos ainda que pacientes com transtorno bipolar apresentam diminuição na frequência do alelo 1513C e o potencial aumento na frequência do genótipo 1513AA e 1513AA/AC em relação aos controles configurando um pior prognóstico de aumento da forma mais ativa do receptor. Também mostramos que o P2X7R possui um papel importante no estabelecimento do modelo de mania, modulando principalmente a via dopaminérgica que é refletida na mudança comportamental observada pelo modelo e por último, o lítio foi capaz de prevenir a resposta de insulto nas células neuronais (linhagem PC-12) e não foi capaz de prevenir a ativação mediada pelo P2X7R em células microgliais (linhagem N9), indicando uma resposta celular diferenciada. Podemos concluir que de maneira translacional conseguimos demonstrar a participação do sistema purinérgico na fisiopatologia do transtorno bipolar, sugerindo ainda um possível biomarcador tanto para diagnóstico como para progressão da doença, os níveis séricos de adenosina, e ainda um potencial biomarcador genético de predição e diagnóstico da doença, o polimorfismo do P2X7R 1513A>C, assim como contribuímos para o entendimento do mecanismo de ação do lítio e definimos um potencial novo alvo terapêutico para o transtorno, o receptor P2X7. / Bipolar Disorder is a severe and chronic psychiatric disorder that presents an episodic course and high impact on the patient's functionality. It has potential for a high degree of severity, recurrence and intensity. The prevalence throughout life of bipolar disorder is 2.4% of the world population. The pathophysiology of this disorder is still not well understood and the drug of first choice is lithium, although its mechanism of action is not clear. The purinergic system mainly due to its actions on neuroprotection, inflammation and microglial activation stands out as a promising pathway for new therapeutic targets, especially the purinergic receptor P2X7 (P2X7R). Thus, the purpose of this thesis is to characterize the involvement of the purinergic system in the pathophysiology of bipolar disorder. In general, we evaluated purinergic system metabolites in serum from bipolar disorder patients and healthy controls, correlating with clinical data. We evaluated the frequency of P2RX7 1513A>C polymorphism in bipolar disorder patients and in healthy subjects. We performed a pharmacological animal model of mania and investigated the involvement of the P2X7 purinergic receptor in this model and finally we sought to better understand the neuroprotective action of lithium by verifying the purinergic participation. Our results primarily showed a decrease in adenosine levels in patients compared to controls and a significant inverse correlation between adenosine levels and worsening of symptoms. We also demonstrated that patients with bipolar disorder present a decrease in the frequency of the 1513C allele and the potential increase in the frequency of genotype 1513AA and 1513AA/AC in relation to controls, setting a worse prognosis of the most active form of the receptor. We also showed that P2X7R plays an important role in the establishment of the mania model, modulating mainly the dopaminergic pathway that is reflected in the behavioral change observed by the model. Finally, lithium was able to prevent the insult response in neuronal cells (PC-12 lineage) and was not capable of preventing P2X7R-mediated activation in microglial cells (N9 lineage), indicating a differentiated cellular response. We can conclude that in a translational way we could demonstrate the participation of the purinergic system in the pathophysiology of bipolar disorder. We also suggested a possible biomarker for both diagnosis and progression of the disorder, the serum adenosine levels, and a potential genetic marker for prediction and diagnosis, the polymorphism of P2X7R 1513A>C. Lastly we contributed to the understanding of lithium action mechanism and defined a potential new therapeutic target for the disorder, the P2X7 receptor.

Florida citrus represents approximately 70 percent of the industry production in the United States; therefore, any associated agricultural and industrial contamination is of concern and a focus of attention. The use of synthetic organic chemicals has become a farmer's necessity in order to supply consumers with high quality products, free of pest damage. However, industrial citrus wastes and chemical residual levels worry not only government agencies but also consumers since they indicate a serious habitat risk. This study assesses citrus industrial processes and the paths that chemical substances follow from the time the citrus seed is planted until consumers get a final product as either fresh fruit or processed product. The study is built on information from United States Environmental Protection Agency (US EPA) manuals, Dade County Environmental Resources Management (DERM) inspection records, United States Food and Drug Administration (US FDA) regulations, Florida standards, journal publications, and research reports. Pollution prevention (P2 or prevention-of-pollution) alternatives are identified; alternatives are proposed, evaluated, and included. Strategies are described and pollution prevention opportunities proposed to minimize citrus wastes generation, chemical residuals in products, their environmental impact and health risk aspects while maximizing product quality.

Parmi les nouvelles stratégies thérapeutiques, la thérapie génique contre le VIH connaît actuellement un regain d'intérêt. La description du premier cas de guérison avéré après greffe de cellules souches mutantes pour le corécepteur majeur du VIH (CCR5) a relancé l'intérêt de la communauté pour développer des stratégies de thérapies géniques ciblant CCR5. Durant ma thèse, j'ai développé une stratégie basée sur l'inhibition de l'entrée du virus dans les lymphocytes T CD4 humains. Nos gènes thérapeutiques comprennent un inhibiteur de fusion membranaire (peptide C46) et un inhibiteur d'expression de CCR5. L'expertise de l'équipe dans les modèles de souris humanisées m'a permis de valider l'intérêt pré-clinique de cette stratégie dans un modèle de transfert de lymphocytes T modifiés. J'ai pu montrer que les lymphocytes T modifiés possédaient un avantage sélectif massif in vivo comparé à des cellules non protégées, ainsi qu’une protection totale contre la délétion induite par le VIH. De plus, dans une autre étude, j'ai montré que la dynamique virale dans les modèles de souris humanisées par transfert de CSH est plus proche des patients. Cette étude nous montre qu'il n'est pas nécessaire d'invoquer la réponse immune, pour expliquer les profils de réplication virale observés dans la phase primaire de l'infection. Ce résultat suggère donc qu'une partie de la dynamique virale est dépendante du nombre de cellules à infecter et du nombre de virions disponibles. L'ensemble des résultats de ma thèse a permis (i) de faire progresser notre compréhension de la dynamique virale, et (ii) de valider la combinaison d'inhibiteurs d'entrée dans un vecteur lentiviral. / Among new therapeutic strategies to fight the infection, gene therapy targeting CCR5 will be an asset. Indeed, the first patient to be cured from the infection received hematopoietic progenitors deprived of CCR5. This finding has ignited a flurry of research on genetic means to decrease CCR5 from the cell surface.During my PhD, I developed a gene therapy strategy based on entry inhibition of HIV in CD4 T cells. Our therapeutic genes are a membrane fusion inhibitor (the C46 peptide) and a CCR5 expression inhibitor, based on a super-agonist ligand. The experience of the team in humanized mice models allowed the validation of the strategy in vivo in a model of gene-modified T cell transfer. I first showed that the combination of the transgenes was able to prevent HIV infection in vitro in a dose dependent manner. Most importantly, I then showed that this protection conferred modified T cells with a huge selective advantage in vivo. This advantage was associated with a complete protection of CD4 T cells from HIV-induced deletion. Moreover, in another study, I showed that the viral dynamics in mouse models humanized by transferring HSC is closer to patients. This study also teaches us that it is not necessary to invoke the immune response to HIV, to explain the profiles of viral replication observed early after infection. This result suggests that part of the viral dynamic is dependent on the target/virus ratio. Together, results collected during my PhD thesis provides new information on viral dynamic and establish the interest of combining two viral entry inhibitors for gene therapy of HIV infection.

O Transtorno Bipolar é uma doença crônica e grave que apresenta curso episódico e alto impacto sobre a funcionalidade do paciente. Possui potencial para alto grau de severidade, recorrência e intensidade e estima-se que sua prevalência ao longo da vida seja de 2,4% da população mundial. A fisiopatologia deste transtorno ainda não é bem compreendida e ainda o fármaco de primeira escolha é o lítio, embora tampouco seja claro seu mecanismo de ação. O sistema purinérgico principalmente devido a suas ações sobre neuroproteção, inflamação e ativação microglial se destaca como uma via promissora de novos alvos terapêuticos, especialmente o receptor purinérgico P2X7 (P2X7R). Dessa maneira, o objetivo desta tese é caracterizar o envolvimento do sistema purinérgico na fisiopatologia do transtorno bipolar. De maneira geral, avaliamos metabólitos do sistema purinérgico em soro de pacientes e controles, correlacionando com dados clínicos. Avaliamos a frequência do polimorfismo do P2RX7 1513A>C em pacientes e controles. Realizamos um modelo animal farmacológico de mania e investigamos o envolvimento do P2X7R neste modelo e por último buscamos melhor compreender a ação neuroprotetora do lítio verificando a participação purinérgica. Nossos resultados primeiramente nos mostraram uma diminuição nos níveis de adenosina em pacientes em comparação a controles e uma importante correlação inversa entre os níveis de adenosina e a piora dos sintomas. Demonstramos ainda que pacientes com transtorno bipolar apresentam diminuição na frequência do alelo 1513C e o potencial aumento na frequência do genótipo 1513AA e 1513AA/AC em relação aos controles configurando um pior prognóstico de aumento da forma mais ativa do receptor. Também mostramos que o P2X7R possui um papel importante no estabelecimento do modelo de mania, modulando principalmente a via dopaminérgica que é refletida na mudança comportamental observada pelo modelo e por último, o lítio foi capaz de prevenir a resposta de insulto nas células neuronais (linhagem PC-12) e não foi capaz de prevenir a ativação mediada pelo P2X7R em células microgliais (linhagem N9), indicando uma resposta celular diferenciada. Podemos concluir que de maneira translacional conseguimos demonstrar a participação do sistema purinérgico na fisiopatologia do transtorno bipolar, sugerindo ainda um possível biomarcador tanto para diagnóstico como para progressão da doença, os níveis séricos de adenosina, e ainda um potencial biomarcador genético de predição e diagnóstico da doença, o polimorfismo do P2X7R 1513A>C, assim como contribuímos para o entendimento do mecanismo de ação do lítio e definimos um potencial novo alvo terapêutico para o transtorno, o receptor P2X7. / Bipolar Disorder is a severe and chronic psychiatric disorder that presents an episodic course and high impact on the patient's functionality. It has potential for a high degree of severity, recurrence and intensity. The prevalence throughout life of bipolar disorder is 2.4% of the world population. The pathophysiology of this disorder is still not well understood and the drug of first choice is lithium, although its mechanism of action is not clear. The purinergic system mainly due to its actions on neuroprotection, inflammation and microglial activation stands out as a promising pathway for new therapeutic targets, especially the purinergic receptor P2X7 (P2X7R). Thus, the purpose of this thesis is to characterize the involvement of the purinergic system in the pathophysiology of bipolar disorder. In general, we evaluated purinergic system metabolites in serum from bipolar disorder patients and healthy controls, correlating with clinical data. We evaluated the frequency of P2RX7 1513A>C polymorphism in bipolar disorder patients and in healthy subjects. We performed a pharmacological animal model of mania and investigated the involvement of the P2X7 purinergic receptor in this model and finally we sought to better understand the neuroprotective action of lithium by verifying the purinergic participation. Our results primarily showed a decrease in adenosine levels in patients compared to controls and a significant inverse correlation between adenosine levels and worsening of symptoms. We also demonstrated that patients with bipolar disorder present a decrease in the frequency of the 1513C allele and the potential increase in the frequency of genotype 1513AA and 1513AA/AC in relation to controls, setting a worse prognosis of the most active form of the receptor. We also showed that P2X7R plays an important role in the establishment of the mania model, modulating mainly the dopaminergic pathway that is reflected in the behavioral change observed by the model. Finally, lithium was able to prevent the insult response in neuronal cells (PC-12 lineage) and was not capable of preventing P2X7R-mediated activation in microglial cells (N9 lineage), indicating a differentiated cellular response. We can conclude that in a translational way we could demonstrate the participation of the purinergic system in the pathophysiology of bipolar disorder. We also suggested a possible biomarker for both diagnosis and progression of the disorder, the serum adenosine levels, and a potential genetic marker for prediction and diagnosis, the polymorphism of P2X7R 1513A>C. Lastly we contributed to the understanding of lithium action mechanism and defined a potential new therapeutic target for the disorder, the P2X7 receptor.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

The purpose of this study was to investigate the relationship between selective serotonin reuptake inhibitor (SSRI) medication on auditory skills in clinically depressed subjects. Experimental subjects prescribed an SSRI were tested in a medicated and an unmedicated condition, and the test results were compared. Furthermore, the experimental group was compared with a control group consisting of normal subjects. Test measures included pure tone audiometry, tympanometry, acoustic reflex thresholds, and auditory electrophysiologic measures such as auditory brainstem and auditory late responses. An assessment scale for depression (Beck Depression Inventory-II) was also used. Results indicated statistically significant differences for the BDI-II between the control and experimental groups for both conditions. Electrophysiologic measures indicated a significantly shorter latency for auditory late potential P1 at 55 dBnSL, and a significantly larger amplitude at 45 dBnSL for the N1/P2 component for the unmedicated group. Although the other measures showed trends, they did not reach significance.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

Thesis (M.C.P.)--Massachusetts Institute of Technology, Dept. of City and Regional Planning, 1961. / Includes bibliographical references (leaves 171-178). / In examining the present status of water resource planning in the Pacific Northwest, numerous critical inadequacies become readily apparent. One method of minimizing some of these inadequacies is through administrative reorganization. Realizing this there have been many different reorganization proposals put forth. Along with these proposals has come much propaganda. In order to obtain a clearer picture comparative criteria were established. Upon doing this it was decided to limit the analysis to the compact, the authority and the inter-agency approaches. Upon applying the criteria to these three structures, the following conclusions were reached: 1) the compact would probably be worse than the existing setup, 2) the inter-agency committee has some value and not too many dangers, 3) the authority fulfills by far the most criteria but because of certain possible dangers should not be strived for, 4) that the criteria are useful in discovering which structures may be unacceptable but they do not tell which is the optimum one. Much is dependent upon the type of planners the structures attract. / by James Myron Bowers. / M.C.P.

No description available.