The aim of this work is to make a literature search in the measurement of membrane voltage using the voltage-sensitive dyes and suggest a method of measuring the membrane voltage on the available cells using the voltage-sensitive dye Di-4-ANEPPS and RH237. The work contains an introduction to electrophysiology of cell, explains fluorescence and typical fluorescence characteristics. The thesis contains the description of a fluorescence microscope. The document was largely devoted to characterization and distribution of voltage-sensitive dyes. The output of a design solution is a real experiment.

La nano-organisation de la membrane cellulaire est essentielle à la régulation de certaines fonctions cellulaires. Dans cette thèse, les récepteurs EGF, CPεT et de la transferrine ont été marqués avec des nanoparticules luminescentes et ont été suivis à la fois dans leur environnement local dans la membrane cellulaire vivantes pour de longues durées et sous un flux hydrodynamique. Nous avons alors appliqué des techniques d'inférence bayésienne, d’arbre de décision et de clustering de données extraire des informations quantitatives sur les paramètres caractéristiques du mouvement des récepteurs, notamment la forme de leur confinement dans des microdomaines. L’application d’une force hydrodynamique sur les nanoparticules nous a alors permis de sonder les interactions auxquelles ces récepteurs sont soumis. Nous avons appliqué cette approche in vitro pour favoriser et mesurer la dissociation in vitro de paires récepteur / ligand à haute affinité entre des récepteurs membranaires et leurs ligands pharmaceutiques, telles que HB-EGF et DTR et l’avons ensuite appliqué à l’étude d’interactions à la membrane cellulaire. Nous avons ainsi mis en évidence trois modes différents d'organisation de la membrane et de confinement des récepteurs: le confinement de CPεTR est déterminé par l'interaction entre les récepteurs et les constituants lipidiques / protéiques des microdomaines, le potentiel de confinement de l'EGFR résulte de l'interaction avec les lipides et les protéines de l’environnement du radeau et de l’interaction avec la F-actine; les récepteurs de la transferrine diffusent librement dans la membrane, uniquement limités stériquement par des barrières d’actine, selon le modèle ‘picket-and-fence’. Nous avons de plus montré que les nanodomaines de type radeau sont rattachés au cytoskelette d’actine. Ce travail présente donc à la fois un aperçu quantitatif du récepteur membranaire, des mécanismes d’organisation à l’échelle nanométrique, et établit un cadre méthodologique avec lequel différents types de propriétés membranaires peuvent être étudiés. / In this thesis, EGF, CPεT and transferrin receptors were labeled with luminescent nanoparticles, , and were tracked both in their local environment in the cell membrane and under a hydrodynamic flow. Bayesian inference, Bayesian decision tree, and data clustering techniques can then be applied to obtain quantitative information on the receptor motion parameters. Furthermore, we introduced hydrodynamic force application in vitro to study biomolecule dissociation between membrane receptors and their pharmaceutical ligands in high affinity receptor- ligand pairs, such as HB-EGF and DTR. Finally, three different modes of membrane organization and receptor confinement were revealed: the confinement of CPεTR is determined by the interaction between the receptors and the lipid/protein constituents of the raft; the confining potential of EGFR results from the interaction with lipids and proteins of the raft environment and from the interaction with F-actin; transferrin receptors diffuse freely in the membrane, only sterically limited by actin barriers, according to the “picket-and-fence” model. We moreover showed that all raft nanodomains are attached to the actin cytoskeleton.

The current study investigates the effect of strain state, strain rate, and membrane planar area on phospholipid bilayer mechanoporation and failure. Using molecular dynamics, a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer was deformed biaxially to represent injury-induced neuronal membrane mechanoporation and failure. For all studies, water forming a bridge through both phospholipid bilayer leaflets was used as a failure metric. To examine the effect of strain state, 72 phospholipid structures were subjected to equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial tensile deformations at the von Mises strain rate of 5.45 × 108 s-1. The stress magnitude, failure strain, headgroup clustering, and damage behavior were strain state dependent. The strain state order of detrimentality in descending order was equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial with failure von Mises strains of 0.46, 0.47, 0.53, 0.77, and 1.67, respectively. Additionally, pore nucleation, growth, and failure were used to create a Membrane Failure Limit Diagram (MFLD) to demonstrate safe and unsafe membrane deformation regions. This MFLD allowed representative equations to be derived to predict membrane failure from in-plane strains. To examine the effect of strain rate, the equibiaxial and strip biaxial strain states were repeated at multiple strain rates. Additionally, a 144 phospholipid structure, which was twice the size of the 72 phospholipid structure in the x dimension, was subjected to strip biaxial tensile deformations to examine planar area effect. The applied strain rates, planar area, and cross-sectional area had no effect on the von Mises strains at which pores greater than 0.1 nm2 were detected (0.509 plus/minus 7.8%) or the von Mises strain at failure (0.68 plus/minus 4.8%). Additionally, changes in bilayer planar and cross-sectional areas did not affect the stress response. However, a strain rate increase from 1.4 × 108 to 6.8 × 108 s-1 resulted in a yield stress increase of 44.1 MPa and a yield strain increase of 0.17. Additionally, a stress and mechanoporation behavioral transition was determined to occur at a strain rate of ~1.4 × 108 s-1. These results provide the basis to implement a more accurate mechano-physiological internal state variable continuum model that captures lower-length scale damage.

Neuronal membrane disruption and mechanoporation are nanoscale damage mechanisms that critically affect brain cell viability during traumatic brain injury (TBI). These nanoscale cellular impairments are elusive in experiments and necessitate in silico approaches such as molecular dynamics (MD) simulations. Implementing MD, this research aims to investigate the effects of different key factors related to membrane deformation and damage, including force field resolutions, lipid compositions, and loading conditions. To examine the impact of force field resolution, MD deformation simulations were conducted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer membranes, using all-atom (AA), united-atom (UA), and coarse-grained Martini (CG-M) force fields. The mechanical responses of the three models progressively changed based on the coarse-graining level. The coarser systems exhibited lower yield stresses and failure strains, and higher mechanoporation damage. To study the influence of lipid components, tensile deformation was applied on seven lipid bilayers, each of which contained a different lipid type commonly found in human brain membrane. Larger headgroup structure, greater degree of unsaturation, and tail-length asymmetry decreased lipid packing, increased the area per lipid (APL), and decreased the failure strain of membrane. Lastly, the deformation behavior of a complex multicomponent MD bilayer (realistically representing human neuronal plasma membrane) under different strain rates and strain states was inspected. The yield stress increased with increasing strain rates and more equibiaxial strain states. Meanwhile, lower strain rates resulted in fewer but larger pores, as well as lower strain and APL at failure. Besides, more equibiaxial strain states exhibited more and larger pores, and lower failure strain. Similar failure APL was obtained regardless of strain states, suggesting that the membrane failed when reaching a critical APL value. In addition, the inclusion of cholesterol was shown to decrease the critical APL. The strain-state dependence results were then used to update the Membrane Failure Limit Diagram (MFLD) that indicates the planar strains for potential membrane failure. Overall, the study provides a non-invasive approach that aids in the current understanding of nanoscale neuronal damage dynamics and essential aspects affecting membrane mechanical responses, and furthermore lays the groundwork for future studies on brain injury biomechanics under various TBI scenarios.

O uso da cocaína e do crack tornou-se um problema de saúde pública importante no Brasil por conta de prejuízos significativos do ponto de vista médico, psicológico e social que ele acarreta. Estudos de gêmeos e, em famílias, sugerem que a dependência de cocaína é uma doença complexa, com participação importante de fatores genéticos. Os estudos genéticos sobre usuários de cocaína são poucos e padecem de problemas metodológicos, tais como, amostras pequenas, com alto grau de miscigenação populacional e um número limitado de marcadores genéticos pesquisados. Além disto, há pouco sendo feito no sentido de verificar como os genes já associados à dependência de cocaína interagem entre si, ou seja, de investigações sobre a epistasia genética. Com o intuito de aprofundar a investigação dos aspectos biológicos da dependência de cocaína, nós estudamos, através de um estudo casocontrole, uma amostra de inicial de 746 pacientes dependentes de crack/cocaína hospitalizados em clínicas especializadas para o tratamento de dependência química na cidade de São Paulo, que foram comparados a 891 controles normais, sem história prévia de abuso ilegal de substâncias. Os objetivos desta tese foram: 1) verificar a associação de três polimorfismos (rs1803274, rs4263329, rs4680662) para o gene da butirilcolinesterase (BCHE), uma enzima envolvida na metabolização da cocaína no desenho do tipo gene candidato; 2) testar a hipótese de interação entre o marcador funcional Val158Met do gene para a enzima catecol-O-metiltransferase (COMT) e os marcadores do tipo VNTR das regiões 3´UTR e Intron8 do gene do transportador da dopamina (DAT1) e; 3) numa análise de caráter exploratório, verificar a interação gene-gene de 40 polimorfismos em 12 genes com plausibilidade biológica para a dependência da cocaína. A análise estatística fez uso de modelos de regressão logística para a interação de marcadores nos dois genes, COMT e DAT1 e, do programa Multifactor Dimensionality Reduction (MDR) para a análise multivariada. A análise envolvendo os marcadores para o gene BCHE não se mostraram associados ao fenótipo da dependência de cocaína porém, encontrou-se uma associação do marcador funcional rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) nos usuários exclusivos de crack, a forma cheirada da cocaína quando comparados aos grupos de uso exclusivo da cocaína na forma cheirada ou, de uso das duas formas de administração. Os marcadores do tipo VNTR da DAT1 não interagiram em um modelo de regressão logística com o marcador Val158Met da COMT. Finalmente, os modelos construídos pelo programa MDR não forneceram interações gene-gene que tivessem uma previsibilidade além do acaso. Dentro de uma perspectiva genética, os estudos futuros para a dependência de cocaína devem aprimorar a caracterização fenotípica, por meio de subgrupos divididos por sintomas clínicos e pelo uso de fenótipos intermediários, fazer um rastreio minucioso dos marcadores ao longo dos genes de interesse e, usar de métodos analíticos para as interações gene-gene e gene-ambiente / The use of crack/cocaine has become a major public health problem in Brazil due to its manifold problems in the medical, psychological and social realms. Twin and family studies have documented the role played by genetic factors and environment in cocaine addiction. Genetic association studies in cocaine addiction are few and have methodological problems: small sample size, population stratification and a paucity of genetic markers have been studied so far. There is also a lack of knowledge on how the genes already shown to be associated with cocaine addiction interact, that is, genetic epistasis. In order to advance the knowledge of biological factors in cocaine addiction we investigated, by means of a case-control study, 746 patients with crack/cocaine dependence admitted to specialized clinics for the treatment of drug addiction in the city of São Paulo. They were compared to 891 control subjects with no previous history of illegal drug abuse. The objectives of this thesis were: 1) investigate the association of three SNPs (rs1803274, rs4263329, rs4680662) in the butirilcholinesterase gene (BCHE) that encodes an enzyme involved in cocaine metabolism; 2) test the hypothesis of an interaction between the functional marker Val158Met of the catechol-o-metiltransferase enzyme (COMT) gene and two VNTRs markers, 3´UTR and Intron8, of the dopamine transporter gene (DAT1) and, 3) in an exploratory analysis, investigate the gene-gene interaction of 40 polymorphisms in 12 genes with a biological plausibility for cocaine addiction. Logistic regression was used to assess COMT*DAT1 gene-gene interaction and, the Multifactor Dimensionality Reduction (MDR) program was used for the other multivariate analysis. Genetic variants for the BCHE gene were not associated with cocaine addiction but, an association was found between the functional marker rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) and crack users compared to those that snorted cocaine or used both forms of administration. The DAT1 VNTRs did not interact with the COMT Val158Met marker. Finally, the models generated by the MDR program did not provided any predictive gene-gene interaction better than chance. Future studies investigating genetic risk factors for cocaine dependence should improve phenotype characterization (clinically derived subgroups and use of endophenotypes) and should also make a thorough scan of the genetic markers along the genes of interest including gene-gene and gene-environment analysis

O uso da cocaína e do crack tornou-se um problema de saúde pública importante no Brasil por conta de prejuízos significativos do ponto de vista médico, psicológico e social que ele acarreta. Estudos de gêmeos e, em famílias, sugerem que a dependência de cocaína é uma doença complexa, com participação importante de fatores genéticos. Os estudos genéticos sobre usuários de cocaína são poucos e padecem de problemas metodológicos, tais como, amostras pequenas, com alto grau de miscigenação populacional e um número limitado de marcadores genéticos pesquisados. Além disto, há pouco sendo feito no sentido de verificar como os genes já associados à dependência de cocaína interagem entre si, ou seja, de investigações sobre a epistasia genética. Com o intuito de aprofundar a investigação dos aspectos biológicos da dependência de cocaína, nós estudamos, através de um estudo casocontrole, uma amostra de inicial de 746 pacientes dependentes de crack/cocaína hospitalizados em clínicas especializadas para o tratamento de dependência química na cidade de São Paulo, que foram comparados a 891 controles normais, sem história prévia de abuso ilegal de substâncias. Os objetivos desta tese foram: 1) verificar a associação de três polimorfismos (rs1803274, rs4263329, rs4680662) para o gene da butirilcolinesterase (BCHE), uma enzima envolvida na metabolização da cocaína no desenho do tipo gene candidato; 2) testar a hipótese de interação entre o marcador funcional Val158Met do gene para a enzima catecol-O-metiltransferase (COMT) e os marcadores do tipo VNTR das regiões 3´UTR e Intron8 do gene do transportador da dopamina (DAT1) e; 3) numa análise de caráter exploratório, verificar a interação gene-gene de 40 polimorfismos em 12 genes com plausibilidade biológica para a dependência da cocaína. A análise estatística fez uso de modelos de regressão logística para a interação de marcadores nos dois genes, COMT e DAT1 e, do programa Multifactor Dimensionality Reduction (MDR) para a análise multivariada. A análise envolvendo os marcadores para o gene BCHE não se mostraram associados ao fenótipo da dependência de cocaína porém, encontrou-se uma associação do marcador funcional rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) nos usuários exclusivos de crack, a forma cheirada da cocaína quando comparados aos grupos de uso exclusivo da cocaína na forma cheirada ou, de uso das duas formas de administração. Os marcadores do tipo VNTR da DAT1 não interagiram em um modelo de regressão logística com o marcador Val158Met da COMT. Finalmente, os modelos construídos pelo programa MDR não forneceram interações gene-gene que tivessem uma previsibilidade além do acaso. Dentro de uma perspectiva genética, os estudos futuros para a dependência de cocaína devem aprimorar a caracterização fenotípica, por meio de subgrupos divididos por sintomas clínicos e pelo uso de fenótipos intermediários, fazer um rastreio minucioso dos marcadores ao longo dos genes de interesse e, usar de métodos analíticos para as interações gene-gene e gene-ambiente / The use of crack/cocaine has become a major public health problem in Brazil due to its manifold problems in the medical, psychological and social realms. Twin and family studies have documented the role played by genetic factors and environment in cocaine addiction. Genetic association studies in cocaine addiction are few and have methodological problems: small sample size, population stratification and a paucity of genetic markers have been studied so far. There is also a lack of knowledge on how the genes already shown to be associated with cocaine addiction interact, that is, genetic epistasis. In order to advance the knowledge of biological factors in cocaine addiction we investigated, by means of a case-control study, 746 patients with crack/cocaine dependence admitted to specialized clinics for the treatment of drug addiction in the city of São Paulo. They were compared to 891 control subjects with no previous history of illegal drug abuse. The objectives of this thesis were: 1) investigate the association of three SNPs (rs1803274, rs4263329, rs4680662) in the butirilcholinesterase gene (BCHE) that encodes an enzyme involved in cocaine metabolism; 2) test the hypothesis of an interaction between the functional marker Val158Met of the catechol-o-metiltransferase enzyme (COMT) gene and two VNTRs markers, 3´UTR and Intron8, of the dopamine transporter gene (DAT1) and, 3) in an exploratory analysis, investigate the gene-gene interaction of 40 polymorphisms in 12 genes with a biological plausibility for cocaine addiction. Logistic regression was used to assess COMT*DAT1 gene-gene interaction and, the Multifactor Dimensionality Reduction (MDR) program was used for the other multivariate analysis. Genetic variants for the BCHE gene were not associated with cocaine addiction but, an association was found between the functional marker rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) and crack users compared to those that snorted cocaine or used both forms of administration. The DAT1 VNTRs did not interact with the COMT Val158Met marker. Finally, the models generated by the MDR program did not provided any predictive gene-gene interaction better than chance. Future studies investigating genetic risk factors for cocaine dependence should improve phenotype characterization (clinically derived subgroups and use of endophenotypes) and should also make a thorough scan of the genetic markers along the genes of interest including gene-gene and gene-environment analysis

Highly fluorescent conjugated polymers (CPs) are an important class of biomaterials used for various biological applications including labelling, sensing, and delivery of biological substances. Synthetic versatility and tunable emission make CPs a superior class of biomaterials. Understanding the structure-function relationship of CPs plays a vital role in designing high performing biomaterials. The cationic CPs are self-assembled to conjugated polymer nanoparticles (CPNs) in an aqueous environment due to their amphiphilicity. The physical and biophysical properties of CPNs are highly dependent on the chemical functionality and backbone structure of CPs. Modulation of the surface property and backbone structure of CPNs play an important role for efficient internalization of CPNs into cells. The goal of this dissertation is to understand the structure function relationship of CPNs in an aqueous environment and the change in their photo physical properties upon the self-assembly of CPNs with different backbone structure upon complexation with biologically significant polysaccharides and cell membrane. This work presents the self-assembly of a set of four cationic CPs with different connectivity and backbone structure upon complexation with a linear polyanion hyaluronic acid (HA). The study of photo physical properties changes upon the complexation with series of Glycosaminoglycans (GAGs) provides more insight about how the self-assembly behavior of cationic CPs changes upon the exposure to negatively charged polysaccharides. The understanding of the self-assembly of CPNs with negatively charged biologically important macromolecules under in vitro conditions can give us an idea of photophysical property changes of CPNs during the treatment of CPNs in the cellular environment. The study of the interaction of CPNs with cell membranes using scanning ion conductance microscopy (SICM)-based topography, potential mapping, and confocal microscopy imaging is presented. CPNs are able to induce transient pore like feature formation on the cell membrane during the cellular internalization process. A comparative study of cellular labelling and delivery of siRNA of five CPNs with guanidine motif is presented. The subcellular localization and delivery of siRNA were dependent on the side chain hydrophilicity. The CPNs fabricated with hydrophilic aminoethoxyethanol possesses excellent cellular imaging with higher siRNA delivery.

Polyunsaturated fatty acids (PUFA) have been used with some success in the treatment of canine atopic dermatitis (CAD). Correspondent in vitro studies revealed that PUFA play a crucial role in the exocytosis of mast cells. n3 PUFA such as α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), as well as the n6 PUFA linoleic acid (LA) have been shown to arrest the secretion of inflammatory mediators. Contrary, the n-6 PUFA arachidonic acid (AA) has been proven to promote the production of mast cell inflammatory mediators. However, we are still lacking a complete picture of the mode of action. The goal of this work was to further characterize the modulatory effects of PUFA supplementation on the plasma membrane lipid composition of mast cells. Furthermore the consequences of a membrane modulation of mast cells by PUFA on the localization and activity on of the membrane bound enzyme phospholipases D (PLD) were investigated. Canine mastocytoma cells (C2) were supplemented with one of the following PUFA: LNA, EPA, DHA, LA or AA. To investigate the influence of PUFA on the lipid composition of membrane microdomains, lipid rafts were separated from non-raft plasma membranes of mast cells for the first time using a detergent-free isolation technique. Results show that PUFA are significantly increased in rafts as well as in non-rafts microdomains (Publication 1). The incorporation of PUFA into the membrane goes along with an increase of the unsaturation status and the fluidity of the membrane. This rise in membrane fluidity may result in a reorganization of membrane signaling molecules and enzymes such as the PLD. To define the impact of a PUFA supplementation on PLD trafficking, C2 were transfected with green fluorescent protein (GFP) fusion plasmids encoding PLD1 or PLD2. Since the transfection ability of the suspension cell line C2 is limited, a special transfection protocol was established, suitable for non-adherent cell lines. Transfection succeeded using chicken egg white as coating material for the cell culture plates. The transfection efficiency rose to 50% versus 5% in uncoated plates. In addition to the obvious increase in the transfection efficiency, the new technique is simple and economic and might be suitable for a wide range of suspension cell lines (Publication 2). Using this optimized protocol the influence of PUFA on the trafficking of PLD isoforms was studied. LNA, EPA, DHA and LA but not AA prevented the stimulation-induced translocation of PLD1 to the plasma membrane. Since the translocation of PLD1 is important for mast cell exocytosis, LNA, EPA, DHA and LA do have an inhibiting effect on the stimulation-induced release of pro-inflammatory mediators. All PUFA tested boosted the total PLD activity. In order to rule out, which PLD isoform was affected by the PUFA, the mast cells were supplemented with DHA or AA in the presence of specific PLD isoform inhibitors. DHA completely abolished the inhibitiory effect of the PLD1 inhibitor but had no effect on the inhibitory effect of PLD2 inhibitor. On the other hand, AA suppressed the inhibitory effect of both PLD1 and PLD2 inhibitor (Publication 3). Taking together, the studies provide a mechanistic base for the role of PUFA in the exocytosis processes of mast cells. PUFA of the n3 and the n6 families impact the lipid composition of membrane microdomains, which in turn lead to a modulation of the physiochemical properties of the membrane. LNA, EPA, DHA and LA suppress the release of inflammatory mediators through their inhibitory action on the stimulation-induced translocation of the PLD1. Contrariwise, AA permits the stimulation-induced migration of PLD1 to the plasma membrane and increases the activity of both PLD isoforms. Therefore, LNA, EPA, DHA and LA but not AA inhibit the release of mast cell inflammatory mediators upon stimulation. / Mehrfach ungesättigte Fettsäuren (PUFA) können mit einigem Erfolg zur Behandlung der caninen atopischen Dermatitis (CAD) eingesetzt werden. In vitro-Studien zeigten, dass PUFA eine entscheidende Rolle in der Exozytose von Mastzellen spielen. N-3-PUFA wie α-Linolensäure (LNA), Eicosapentaensäure (EPA), Docosahexaensäure (DHA) sowie die n-6-PUFA Linolsäure (LA) können die Sekretion von Entzündungsmediatoren vermindern. Arachidonsäure (AA) als n-6 mehrfach ungesättigte Fettsäure hingegen fördert die Entzündungsmediatoren-Freisetzung aus den Mastzellen. Eine vollständige Aufklärung der Wirkungsweise fehlt aber weiterhin. Das Ziel dieser Arbeit war eine weitergehende Charakterisierung der modulierenden Effekte einer PUFA-Supplementierung auf die Lipidzusammensetzung der Plasmamembran von Mastzellen. Darüber hinaus wurden die Auswirkungen von PUFA auf die Lokalisation und Aktivität des Membran-gebundenen Enzyms Phospholipase D (PLD) untersucht. Canine Mastozytom-Zellen (C2) wurden mit einer der folgenden PUFA kultiviert: LNA, EPA, DHA, LA oder AA. Um den Einfluss von PUFA auf die Lipidzusammensetzung der Membran-Mikrodomänen zu untersuchen, konnten sowohl Lipid Raft als auch Nicht-Raft Plasmamembran-Anteile von Mastzellen zum ersten Mal mittels einer Detergenzien-freien Isolationsmethode getrennt werden. Hervorzuheben ist, dass PUFA signifikant vermehrt in Raft- sowie in Nicht-Raft Membranmikrodomänen eingelagert werden (Publikation 1). Die Integration von PUFA in die Membran geht mit einer Steigerung der Doppelbindungsanzahl und der Fluidität der Membran einher. Diese Erhöhung der Membranfluidität kann zu einer Reorganisation von membranären Signalmolekülen und Enzymen wie der PLD führen. Um die Auswirkungen einer PUFA-Supplementierung auf den intrazellulären Transport der PLD in C2 zu bestimmen, wurden die Zellen mit PLD1- oder PLD2-codierenden grün fluoreszierenden Protein-(GFP-)Fusionsplasmiden transfiziert. Da die Transfektionsfähigkeit der Suspensions-Zelllinie C2 begrenzt ist, wurde ein für nicht-adhärente Zelllinien geeignetes Transfektionsprotokoll etabliert. Mit Hühnereiweiß als Beschichtungsmaterial für die Zellkultur-Platten stieg die Transfektionseffizienz auf 50% im Vergleich zu 5% bei unbeschichteten Platten. Neben der deutlichen Erhöhung der Transfektionseffizienz ist die neu etablierte Technik einfach durchzuführen sowie wirtschaftlich und kann für eine Vielzahl von Suspension-Zelllinien geeignet sein (Publikation 2). Unter Verwendung dieses optimierten Protokolls wurde der Einfluss von PUFA auf die Translokation der PLD-Isoformen untersucht. LNA, EPA, DHA und LA, nicht aber AA verhindern die stimulationsinduzierte Translokation der PLD1 an die Plasmamembran. Die Translokation der PLD1 ist wichtig für die Mastzell-Exozytose. LNA, EPA, DHA und LA haben hier eine hemmende Wirkung auf die stimulationsinduzierte Freisetzung von proinflammatorischen Mediatoren. Alle getesteten PUFA verstärken die Gesamt-PLD-Aktivität. Um zu unterscheiden, welche PLD-Isoform durch PUFA beeinflusst ist, wurden die Mastzellen mit DHA oder AA in Gegenwart von PLD-Isoform-Inhibitoren supplementiert. DHA hebt die inhibitorische Wirkung des PLD1-Inhibitors vollständig auf, zeigte aber keinen Einfluss auf die hemmende Wirkung des PLD2-Inhibitors. Andererseits unterdrückt AA die hemmende Wirkung des PLD1- als auch des PLD2-Inhibitors (Publikation 3). Zusammenfassend bietet die Studie eine mechanistische Basis für die Rolle von PUFA bei Exozytose-Prozessen von Mastzellen. PUFA der n-3- und n-6-Familie beeinflussen die Lipidzusammensetzung von membranären Mikrodomänen, was wiederum zu einer Modulation der physikalisch-chemischen Eigenschaften der Membran führt. LNA, EPA, DHA und LA verhindern die Freisetzung von Entzündungsmediatoren durch ihre hemmende Wirkung auf die stimulationsinduzierte Translokation der PLD1. Umgekehrt erlaubt AA eine stimulationsinduzierte Migration der PLD1 zur Plasmamembran und steigert die Aktivität der beiden Isoformen der PLD. Somit hemmen LNA, EPA, DHA und LA, aber nicht AA die Freisetzung von Mastzell-Entzündungsmediatoren nach Stimulation.

Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.

Broadband spectral content is required to support ultrashort pulses. However this broadband content is subject to dispersion and hence the pulse duration of corresponding ultrashort pulses may be stretched accordingly. I used a commercially-available adaptive ultrashort pulse shaper featuring multiphoton intrapulse interference phase scan technology to characterise and compensate for the dispersion of the optical system in situ and conducted experimental and theoretical studies in various inter-linked topics relating to the light-matter interaction. Firstly, I examined the role of broadband ultrashort pulses in novel light-matter interacting systems involving optically co-trapped particle systems in which inter-particle light scattering occurs between optically-bound particles. Secondly, I delivered dispersion-compensated broadband ultrashort pulses in a dispersive microscope system to investigate the role of pulse duration in a biological light-matter interaction involving laser-induced cell membrane permeabilisation through linear and nonlinear optical absorption. Finally, I examined some of the propagation characteristics of broadband ultrashort pulse propagation using a computer-controlled spatial light modulator. The propagation characteristics of ultrashort pulses is of paramount importance for defining the light-matter interaction in systems. The ability to control ultrashort pulse propagation by using adaptive dispersion compensation enables chirp-free ultrashort pulses to be used in experiments requiring the shortest possible pulses for a specified spectral bandwidth. Ultrashort pulsed beams may be configured to provide high peak intensities over long propagation lengths, for example, using novel beam shapes such as Bessel-type beams, which has applications in biological light-matter interactions including phototransfection based on laser-induced cell membrane permeabilisation. The need for precise positioning of the beam focus on the cell membrane becomes less strenuous by virtue of the spatial properties of the Bessel beam. Dispersion compensation can be used to control the temporal properties of ultrashort pulses thus permitting, for example, a high peak intensity to be maintained along the length of a Bessel beam, thereby reducing the pulse energy required to permeabilise the cell membrane and potentially reduce damage therein.