Rozprawy doktorskie na temat „Protein Dynamics - Confocal Microscopy”

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.<br><p>QC 20151217</p>

This thesis work aspires to present a new concept for the application of correlation techniques to the study of the cellular environment. By exploiting local analysis in combination to a fast fit-free technique (the phasor approach) we provide an exhaustive high-resolution analysis of structural and dynamic properties while maintaining a reasonable computation time. The dissertation will be articulated as follows: In CHAPTER 1 we aim to provide the reader with a description of the techniques that will be exploited during the rest of the dissertation together with the open questions and problematics that our techniques will try to answer to. In CHAPTER 2 we present the local analysis concept and its application to a correlation technique capable of measuring size and concentration (ICS). We will show how we coupled ICS to the phasor approach to create a technique (PLICS) for the assessment of size heterogeneity. PLICS will be demonstrated with simulations as well as with cellular samples and will be applied to the study of endocytic vesicles uptake and to the characterization of other organelles. In CHAPTER 3 the concept is extended to two-colors samples for the determination of local inter-structure distance (PLICCS). We will present a pattern analysis method we developed that exploits this information in order to evaluate the relative distribution of the structures imaged in the two channels, comparing it to a random distribution. This method will be validated with simulations and applied to the study of replication-transcription collisions. Successively, we will show that PLICCS can be converted to a localization algorithm for single particle tracking that will be used for tracking membrane receptors in living neurons. CHAPTER 4 will describe the extension of our local analysis to RICS, a correlation technique capable of measuring the diffusion coefficient of a fluorescent probe. The resulting algorithm (L-RICS) provides high resolution diffusion maps that will be used to characterize the diffusion of a fluorescent probe (GFP) within the nucleus and nucleolus of living cells. We will show that the algorithm can be implemented also in non-linear scanning systems. CHAPTER 5 will conclude the dissertation by introducing advanced correlation methods for the analysis of non-Brownian diffusion and their coupling to super-resolution techniques. In particular, we will present a super-resolution correlation technique (SPLIT) recently developed capable of analyzing the cellular environment and a microcamera-based approach (Airyscan comprehensive correlation analysis) we developed for the parallel implementation, in super-resolution, of several complementary correlation techniques.

<p>Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle.</p><p>Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles.</p><p><i>Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.</i></p>

The Microtubule (MT) network is a central component of the eukaryotic cell cytoskeleton. In the fission yeast S. pombe, a complex of three proteins specifically tracks MT +ends and stabilizes MTs in the cell. It is composed of the proteins Mal3, Tip1 and Tea2. Mal3, the S. pombe homologue of EB1, is a highly conserved ubiquitous protein found to be at the centre of many MT related processes. Tip1 is a CLIP170 homologue and Tea2 a kinesin-like motor protein. The mechanism by which they target the growing end of MTs and stabilize them is still unknown. A combination of biochemistry, electron microscopy and crystallography were used in an attempt to get a more precise understanding of the MT stabilization by this +Tip complex. Protein-A pull-down of the endogenous complex and analysis of its constituents by mass spectrometry revealed that Tea2 and Tip1 form a tight stoichiometric complex, making a much more labile interaction with Mal3. Biochemical experiments, light scattering and DIC microscopy demonstrate that Mal3 stabilizes the MT structure in a stoichiometric fashion by suppressing catastrophe events. 3D helical reconstruction of electron micrographs of Mal3 bound to the MT show that it most probably stabilizes the MT structure by bridging protofilaments together. Deletion mutant analysis suggests that contact with one of the protofilaments is via an interaction between the charged tails of tubulin and Mal3. Mal3 MT binding domain structure was solved by X-ray crystallography so that eventually it may be docked into a higher resolution electron microscopy map to provide a more precise structural insight on how Mal3 stabilizes the MT lattice. The EM analysis also shows that Mal3 regulates MT structure in vitro by restraining their protofilament number to 13, which is the number always found in vivo, and by driving the assembly of MTs with a high proportion of A-lattice. It is the first time that a protein is found to promote formation of A-lattice MTs. The fact that EB1 is such a ubiquitous protein reopens the question of MT structure in cells and has important implications for in vivo MT dynamics.

Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.

Inwardly rectifying potassium (Kir) channels are essential for controlling the excitability of eukaryotic cells, forming a key part of the inter-cellular signalling system in multi-cellular organisms. However, as prokaryotic (KirBac) channels are less technically challenging to study in vitro and have been shown to be directly homologous to eukaryotic channels, they are often studied in lieu of their mammalian counterparts. A vital feature of Kir and KirBac channels is their mechanism for opening and closing, or their gating: this study predominantly features observations of open and/or closed channel populations. A well-characterised member of the KirBac family, KirBac1.1, has been successfully expressed, purified into detergent micelles, and doubly labelled with fluorescent maleimide dyes in order to enable observation of confocal-in-solution Förster Resonance Energy Transfer (FRET) at the single molecule level. Results demonstrate single-molecule FRET signals from KirBac1.1 and therefore represent the first single-molecule FRET observations from a KirBac channel. Perturbation of the open-closed dynamic equilibrium was performed via activatory point mutations, changes in pH, and ligand binding. A protocol for reconstitution into nanodiscs was optimised in order to more closely approximate native conditions, and the single-molecule FRET observations repeated. This thesis presents a comparison between measurements made using the detergent solubilisation system and those made using nanodiscs.

Levels of macromolecules fluctuate both spatially and temporally in individual cells. Such heterogeneity could be exploited for bet hedging in uncertain environments, or be suppressed by negative feedback if perturbations are deleterious. For the master stress-response regulator in Escherichia coli, RpoS, both of these scenarios have been suggested. RpoS levels are also exceedingly low and controlled by the ClpXP protease, which reportedly displays extreme spatial heterogeneity. However, little is known quantitatively about RpoS dynamics. This is partly because no functional protein fusions exist, but also because the quantitative tools for studying fluctuations and localizations are limited, particularly ones that can be independently validated. Here I develop such methods and begin applying them to RpoS. Protein localization measurements increasingly rely on fluorescent protein fusions and are difficult to verify independently. I designed a non-intrusive method for validating localization patterns in live bacterial cells by exploiting post-division heterogeneity in downstream processes. Applying this assay to the ClpXP protease, widely reported to form biologically relevant foci, revealed in fact that the protease molecules are not specifically localized inside cells, as confirmed by four independent methods. I further evaluated 20+ commonly used fluorescent reporters and found that many cause severe mislocalization when fused to homo-oligomers, likely due to avidity effects. Further reinvestigating other foci-forming proteins strongly suggests that the previously reported foci were all caused by the fluorescent proteins used. For mRNAs – which are often present in low numbers per cell and major sources of non-genetic heterogeneity – existing single-cell assays have unknown accuracy: the experimental counting errors could completely over-shadow the natural variation. I therefore optimized and cross-evaluated two single-molecule mRNA detection methods. Several problems were identified and solutions discussed. I succeeded in building a functional RpoS protein fusion, and used bulk methods to show that the RpoS feedback loop is effectively not operating during exponential- phase growth. Mathematical analyses and initial experiments in a microfluidic device further suggest that the RpoS system has several unusual properties contributing towards extremely fast stress response. A stochastic analysis further suggests that the RpoS feedback loop cannot suppress spontaneous fluctuations, and preliminary experiments indicate that large deviations might indeed play important roles.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.<br><p>QC 20121017</p><br>The Human Protein Atlas

Optical modulation has shown the selective and sensitive signal improvement in high background systems in cell imaging; however, cell applications are still limited due to biocompatibility and delivery issues. Fluorescent proteins have a variety of optically accessible states that make them ideal candidates for investigation of modulatability. Combining the optical modulation technique with the biocompatibility of fluorescent proteins is a major advance. This work focuses on evaluation fluorescent proteins and their optical states for modulation, as well demonstrations of cellular imaging. Herein, we evaluate a green fluorescent protein with interesting photophysical properties favorable for optical modulation. Positive for optical modulation, further investigation of the state dictating modulation reveals the presence of a slow component on the order of milliseconds. To better understand the mechanism responsible modulation, blue fluorescent proteins are created to modify the chromophore environment. Extraction of photophysics confirm the alteration timescales of the modulated state. Motivated by the ability to improve imaging and decode hidden dynamics, demodulation of these proteins demonstrates the selective recovery of signal in the presence of high cellular background. The continued investigation of several other fluorescent proteins identifies modulatable proteins across the visible wavelength region. Additionally, solvent environmental factors show varying timescales which, when combined with mutagenesis, suggest a cis/trans isomerization coupled with a proton transfer. This information of the properties dictating optical modulation allows for the engineering of improved modulatable proteins to study cellular dynamics.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation, we developed a protocol using high throughput sequencing to measure steady-state RNA abundances, RNA polymerase elongation rates and RNA degradation rates simultaneously with high nucleotide-resolution genome-wide. Our data shows that RNA has similar lifetime at all positions throughout the length of the transcript. We also find that our polymerase elongation rates measured in vivo on a chromosome are generally slower than rates measured on plasmids by other groups. Studying nascent RNA will allow further understanding of RNA generation and degradation. To this end, we have developed a labeling protocol with a nucleoside analog that is compatible with high throughput sequencing.

In this project, self-assembly behaviour of colloidal particles was investigated by a combination of both computer simulations and experimental approach. In particular, a Brownian Dynamics algorithm was used to simulate either steep-repulsive spheres or spherocylinders in a shrinking spherical confinement. In accordance with literature, in the former case packed spheres were shown to crystallize into a distinctive icosahedral structure. In the latter study, spherocylinders clearly revealed a local tendency to form smectic layers. After the synthesis of micro-sized fluorescent-labelled silica spheres and rods, particle self-assembly in a spherical confinement was experimentally explored. While our selected method widely produced well-defined spherical supraparticles, it generally failed in inducing crystalline or liquid-crystalline ordering. This outcome was supposed to emerge due to fast compression of particles inside the confinement. In the last part of the project, Brownian Dynamics simulations of mixtures of rods and spheres in a spherical confinement were performed. Our preliminary investigation unveiled a modest tendency for rod-rich mixtures to form a binary smectic configuration. However, same-shape phase separation prominently occurred for increasing fractions of spheres. Notably, a quantitative analysis on the simulated configurations was accomplished by introduction of a novel binary smectic local order parameter.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Our understanding of the complexity of the brain is limited by the data we can collect and analyze. Because of experimental limitations and a desire for greater detail, most investigations focus on just one aspect of the brain. For example, brain function can be studied at many levels of abstraction including, but not limited to, gene expression, protein interactions, anatomical regions, neuronal connectivity, synaptic plasticity, and the electrical activity of neurons. By focusing on each of these levels, neuroscience has built up a detailed picture of how the brain works, but each level is understood mostly in isolation from the others. It is likely that interaction between all these levels is just as important. Therefore, a key hypothesis is that functional units spanning multiple levels of biological organization exist in the brain. This project attempted to combine neuronal circuitry analysis with functional proteomics and anatomical regions of the brain to explore this hypothesis, and took an evolutionary view of the results obtained. During the process we had to solve a number of technical challenges as the tools to undertake this type of research did not exist. Two informatics challenges for this research were to develop ways to analyze neurobiological data, such as brain protein expression patterns, to extract useful information, and how to share and present this data in a way that is fast and easy for anyone to access. This project contributes towards a more wholistic understanding of the fruit fly brain in three ways. Firstly, a screen was conducted to record the expression of proteins in the brain of the fruit fly, Drosophila melanogaster. Protein expression patterns in the fruit fly brain were recorded from 535 protein trap lines using confocal microscopy. A total of 884 3D images were annotated and made available on an easy to use website database, BrainTrap, available at fruitfly.inf.ed.ac.uk/braintrap. The website allows 3D images of the protein expression to be viewed interactively in the web browser, and an ontology-based search tool allows users to search for protein expression patterns in specific areas of interest. Different expression patterns mapped to a common template can be viewed simultaneously in multiple colours. This data bridges the gap between anatomical and biomolecular levels of understanding. Secondly, protein trap expression patterns were used to investigate the properties of the fruit fly brain. Thousands of protein-protein interactions have been recorded by methods such as yeast two-hybrid, however many of these protein pairs do not express in the same regions of the fruit fly brain. Using 535 protein expression patterns it was possible to rule out 149 protein-protein interactions. Also, protein expression patterns registered against a common template brain were used to produce new anatomical breakdowns of the fruit fly brain. Clustering techniques were able to naturally segment brain regions based only on the protein expression data. This is just one example of how, by combining proteomics with anatomy, we were able to learn more about both levels of understanding. Results are analysed further in combination with networks such as genetic homology networks, and connectivity networks. We show how the wealth of biological and neuroscience data now available in public databases can be combined with the Brain- Trap data to reveal similarities between areas of the fruit fly and mammalian brain. The BrainTrap data also informs us on the process of evolution and we show that genes found in fruit fly, yeast and mouse are more likely to be generally expressed throughout the brain, whereas genes found only in fruit fly and mouse, but not yeast, are more likely to have a specific expression pattern in the fruit fly brain. Thus, by combining data from multiple sources we can gain further insight into the complexity of the brain. Neural connectivity data is also analyzed and a new technique for enhanced motifs is developed for the combined analysis of connectivity data with other information such as neuron type data and potentially protein expression data. Thirdly, I investigated techniques for imaging the protein trap lines at higher resolution using electron microscopy (EM) and developed new informatics techniques for the automated analysis of neural connectivity data collected from serial section transmission electron microscopy (ssTEM). Measurement of the connectivity between neurons requires high resolution imaging techniques, such as electron microscopy, and images produced by this method are currently annotated manually to produce very detailed maps of cell morphology and connectivity. This is an extremely time consuming process and the volume of tissue and number of neurons that can be reconstructed is severely limited by the annotation step. I developed a set of computer vision algorithms to improve the alignment between consecutive images, and to perform partial annotation automatically by detecting membrane, synapses and mitochondria present in the images. Accuracy of the automatic annotation was evaluated on a small dataset and 96% of membrane could be identified at the cost of 13% false positives. This research demonstrates that informatics technology can help us to automatically analyze biological images and bring together genetic, anatomical, and connectivity data in a meaningful way. This combination of multiple data sources reveals more detail about each individual level of understanding, and gives us a more wholistic view of the fruit fly brain.

Anthracene was attached to light activated, ruthenium-based DNA disruptors to probe their distribution in cancer cells. The objective of this research is to understand the photophysical properties (Chapter 2), photoreactivity toward DNA and proteins (Chapter 3), and localization within cancer cells (Chapter 4) of ruthenium complexes that demonstrate promise as photodynamic therapy (PDT) agents. [(AnthbpyMe)(bpy)Ru(dpp)]2+ (1) and [(AnthbpyMe)2Ru(dpp)]2+ (2) absorb visible light with metal-to-ligand charge transfer (MLCT) transitions at 459 nm (16,000 M-1 cm-1 ) and 461 nm (21,000 M-1 cm-1 ), respectively. These species exhibit 3 MLCT emissions at λem = 661 nm and λem = 663 nm for 1 and 2, respectively, while the anthracene show emissions at 450 – 560 nm. The anthracene unit(s) quench the 3 MLCT to give quantum yields (lifetime) of Φem = 0.0059 [398(1) ns] and Φem = 0.0011 [414(1) ns] for 1 and 2, respectively. Voltammetry shows an irreversible anthracene oxidation at 1.23 – 1.28 V, RuIII/II oxidation at 1.53 – 1.55 V, and quasi-reversible reduction couples attributed to dpp0/-1 at 0.98 V. DNA gel shift assays demonstrate that complexes 1 and 2 modify DNA in the presence and absence of 3 O2 upon light activation to convert supercoiled DNA to a mixture of open circular (OC) DNA and a species that exhibit sa distinctly different migration rate than either OC and linear DNA. Binding constants, Kb, for complexes 1 and 2, toward DNA are 3.50 × 105 (3.50 × 104 ) and 4.50 × 103 (4.50 × 102 ) respectively. SDS-PAGE assays show that the complexes 1 and 2 modify bovine serum albumin (BSA) through an 3 O2-dependent mechanism upon light iii activation. The localization and PDT potency of the anthracene-Ru-dpp complexes are tested against F98 cells, which are rat glioma cells that simulate the infiltrative patterns of growth in cancer. Confocal microscopy demonstrates that complexes 1 and 2 internalize and localize primarily along the cell membrane and associate with dot-like vesicles within the cytoplasm. Complexes 1 and 2 show IC50 values of 107 µM and 85 µM, respectively, after 15 min of drug exposure and 1 h of PDT-treatment (λPDT = 455 nm).<br>Ph. D.

In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.

"KpOmpA est une protéine de la membrane externe de K. Pneumoniae. Elle fait partie de la famille des "outer membrane protein A", OmpA. KpOmpA est une protéine constituée de deux domaines: un domaine transmembranaire structuré en tonneau ß et une partie soluble, périplasmique. Le domaine transmembranaire de KpOmpA présente une homologie importante avec celle d'OmpA d'E. Coli dont la structure a été déterminée par cristallographie aux rayons X et spectroscopie RMN. OmpA d'E. Coli est responsable lors de la formation de biofilm. Elle a un rôle d'adhésine et d'invasine. Elle est la cible préférentielle du système immunitaire et est le récepteur de bactériophages. Il est admis que la plupart de ces fonctions sont dues aux boucles extracellulaires de ces protéines. Les différences majeures entre les protéines KpOmpA et OmpA d'E. Coli concernent les boucles extracellulaires de longueur plus importante dans le cas de KpOmpA. Elles jouent un rôle important au cours de l'activation des macrophages et des cellules dendritiques par la voie des récepteurs TLR2. Les boucles extracellulaires jouent un rôle essentiel au cours de l'activation du système immunitaire. Mieux définir la structure et la dynamique de ces boucles est d'une importance essentielle afin de mieux appréhender la fonctionnalité des boucles extracellulaires de KpOmpA. Les informations structurales connues actuellement (structure RMN déterminée dans le groupe IPBS RMN en 2009) ont été obtenus jusqu'à présent avec des échantillons de protéines recombinantes purifiées et repliées dans des micelles de détergent. Dans le présent travail, nous avons d'abord établi un protocole de reconstitution de la protéine dans une membrane phospholipidique et caractérisé nos échantillons par microscopie électronique. Des expériences de spectroscopie de force atomique sur molécule unique ont été réalisées pour caractériser le repliement de la protéine dans son environnement membranaire. Ces expériences suggèrent un nouveau rôle de KpOmpA au sein même de la membrane (collaboration D. Müller, ETH Zürich). Le domaine soluble périplasmique de la protéine a été exprimé indépendamment du domaine membranaire. Les premières expériences HSQC réalisées montrent une structuration de ce domaine. La structure de ce domaine par spectroscopie RMN est en cours de réalisation. Le comportement dynamique des boucles extracellulaires du domaine membranaire KpOmpA reconstitué dans des liposomes a été étudié par spectroscopie RMN à l'angle magique (MAS) et notamment par mesure des temps de relaxation. Nous avons montré que la dynamique intrinsèque de la protéine est indépendante de l'environnement (membrane vs micelle). Des expériences de protéolyse ménagée suivie par spectrométrie de masse (MALDI-TOF) ont été comparées avec les informations RMN afin d'évaluer plus précisément les niveaux de mobilité des différentes boucles extracellulaires. La préservation au cours de l'évolution des boucles extracellulaires semble lier à leur dynamique, ce qui suggère l'importance de ces boucles extracellulaires, en termes de séquence, longueur mais aussi de dynamique lors de la réponse immunitaire. "<br>KpOmpA is a two-domain membrane protein from Klebsiella pneumoniae belonging to the outer membrane protein A (OmpA) family. It is composed of a transmembrane ß-barrel with 8 ß-strands and a C-terminal, soluble periplasmic domain. The transmembrane domain presents a significant homology with E. Coli OmpA whose three dimensional structure has been determined by X-ray crystallography and by NMR. The E. Coli homologue can function as an adhesin and invasin, participate in biofilm formation, act as both an immune target and evasin, and serves as a receptor for several bacteriophages. It is assumed that most of these functions involve the four protein loops that emanate from the protein to the exterior of the cell. The difference between KpOmpA and E. Coli OmpA is mostly concentrated in these extracellular loops which are larger in the case of KpOmpA. KpOmpA was shown to activate macrophages and dendritic cells through the TLR2 dependent pathway, and these larger loops are supposed to play a specific role in the interactions with the immune system. Thus the structure and dynamics of these loops is of prime functional significance. The currently available information in this regard, including the NMR structure determined in the IPBS NMR group in 2009, have been obtained so far with recombinant protein samples purified and refolded in detergent micelles. In the present work we first established a reconstitution protocol that allowed the incorporation of the membrane protein in the more native environment of the lipid bilayer and characterised our samples by electron microscopy. SMFS experiments were used to probe the reconstituted KpOmpA unfolding-refolding pathways, exploring the folding mechanisms for ß-barrel proteins and suggesting a novel role for OmpA in the bacterial membrane (in collaboration with the group of D. Müller, ETH Zürich). The C-terminal periplasmic domain of KpOmpA was expressed and purified as a separate product and the feasibility of its structure elucidation by NMR was demonstrated by obtaining a high quality HSQC spectrum. The dynamic behaviour of the extracellular portion of the KpOmpA membrane domain reconstituted in liposomes has been investigated by solid state MAS NMR relaxation experiments. We confirmed that the previously observed gradient of dynamic along the molecule axis is an intrinsic property of the protein. Limited proteolysis and MALDI-TOF experiments were coupled with the NMR information in order to assess more precisely the different mobility levels in the loops. Evolutional preservation of the different loops regions is related to their observed flexibility, pointing towards immunologically important, variable, dynamic and accessible loops sections

Les neurones sont des cellules excitables capables de coder et transmettre l’information sous forme d’oscillations du potentiel membranaire. Cette activité électrique est produite par une modification des flux ioniques transmembranaires. Les neurones constituent un exemple d’oscillateur cellulaire dont la dynamique non linéaire permet l’apparition d’une activité électrique complexe. Dans ce système, les ions calciques sont des messagers intracellulaires importants. Ils servent de médiateur entre un signal électrique et un signal chimique, par une modulation de l’activité enzymatique de certaines protéines. Ils interviennent dans de nombreuses fonctions neuronales, dont l’excitabilité électrique. Un des mécanismes mis en place par les neurones pour contrôler l’homéostasie du calcium intracellulaire provient de protéines cytoplasmiques capables de lier les ions calciques. Ces protéines jouent un rôle de « tampon » du calcium. Cependant, toutes leurs fonctions n’ont pas encore été mises en évidence. C’est l’objectif de notre travail. Nous avons voulu comprendre le rôle joué par une protéine « tampon » particulière, la calrétinine, sur le mode de décharge électrique d’un neurone où elle est exprimée en abondance, le grain cérébelleux. Pour cela, nous avons utilisé une approche théorique et expérimentale. <p>Au niveau théorique, nous avons élaboré un modèle mathématique de l’activité électrique du grain cérébelleux, prenant en compte la chélation du calcium intracellulaire. Il permet de clarifier le rôle de la chélation du calcium intracellulaire sur les oscillations du potentiel membranaire. La modélisation de l’activité électrique du grain cérébelleux repose sur le formalisme développé par Hodgkin et Huxley pour l’axone géant de calmar. Dans ce contexte, l’application de la conservation de la charge au circuit équivalent de la membrane cellulaire fournit un système d’équations différentielles ordinaires, non linéaires. Dès lors, notre modèle nous a permis d’étudier l’impact des variations de la concentration de chélateur calcique sur les oscillations du potentiel membranaire. Nous avons ainsi pu constater qu’une diminution de la concentration en chélateur calcique induisait une augmentation de l’excitabilité électrique du grain cérébelleux, sans altérer le régime d’oscillations. Par contre, en augmentant fortement la concentration en chélateur calcique, nous avons montré que le grain cérébelleux changeait de dynamique oscillatoire, montrant des transitions d’un mode de décharge périodique régulier vers des oscillations en salve du potentiel membranaire.<p>Au niveau expérimental, nous avons vérifié les résultats prévus par le modèle théorique. Nous avons ainsi montré que des grains de souris transgéniques déficientes en calrétinine présentaient une excitabilité électrique accrue par rapport aux grains contrôles.<p>Puis, en restaurant un niveau de chélation calcique normal dans ces grains, par perfusion intracellulaire de chélateur calcique, nous montrons qu’ils retrouvent un niveau d’excitabilité normal. Ensuite, nous avons introduit dans des grains cérébelleux de souris sauvages, une forte concentration en chélateur calcique exogène. Conformément aux résultats théoriques, nous avons pu observer des transitions vers des oscillations en salve du potentiel membranaire. Enfin, nous avons montré que l’absence de calrétinine affecte les paramètres morphologiques du grain cérébelleux des souris transgéniques déficientes en calrétinine.<p>En conclusion, ces résultats suggèrent que le mode de décharge des cellules excitables peut être modulé d’une façon importante par les protéines liant le calcium. De ce fait, des changements dans le niveau d’expression et/ou dans la localisation subcellulaire des protéines liant le calcium pourraient aussi jouer un rôle critique dans la régulation de processus physiologiques contrôlés par l’excitabilité membranaire. De plus, les mécanismes que nous avons mis en évidence pourraient être à l’origine d’un nouveau principe de régulation de la signalisation dans les circuits neuronaux et pourraient jouer un rôle fonctionnel dans le contrôle du codage de l’information et de son stockage dans le système nerveux central.<br>Doctorat en sciences, Spécialisation physique<br>info:eu-repo/semantics/nonPublished

Les activités électriques ectopiques à l’origine des épisodes de fibrillation atriale pourraient être dues à des échanges calciques anormaux dans les cardiomyocytes (CM) de veine pulmonaire (VP). Le cycle du calcium des CM de VP a donc été caractérisé et comparé à celui des CM d’oreillette gauche (OG) et de ventricule gauche (VG). Des outils ont été développés pour mesurer la régularité d’organisation des réseaux de tubules transverses et la contractilité des CM de VP. Contrairement aux CM d’OG et de VG, l’organisation hétérogène du réseau de tubules dans la population des CM de VP conduit à une grande variabilité de forme des transitoires calciques et d’amplitude de contraction. Au sein des VP, ces différents types de CM sont regroupés en îlots. La fréquence des libérations calciques spontanées est également plus grande dans les CM de VP que dans ceux d’OG et de VG. La population des CM de VP présente une dynamique calcique aux caractéristiques uniques pouvant être source d’arythmies<br>Ectopic foci leading to atrial fibrillation episodes might be due to abnormal calcium handling by the pulmonary vein (PV) cardiomyocytes (CM). Therefore, the calcium cycle of PV CM was characterized and compared to those of left atria (LA) and left ventricle (LV) CM. Some tools have been developed to measure the organization of transverse tubular networks and contractility of PV CM. Unlike LA and LV CM, the heterogeneous organization of the tubular networks in the PV CM population leads to wide ranges of calcium transient shapes and contraction amplitudes. Within the whole PV, these different types of CM are gathered in islets. The frequency of spontaneous calcium release is also higher in PV CM than in LA and LV CM. The special features of the calcium handling properties of the PV CM population could be a source of arrhythmias

The ability of the biological control agents (BCAs) to colonize plant tissues is an important feature involved in microbe-assisted plant protection. Plant-microbe interaction research increased especially in the last decade thanks to technological revolution. Molecular methods and the development of advanced microscopic techniques allow researchers to explore gene expression and localization of beneficial microorganisms within plants. The green fluorescent protein (GFP) and its modified version, enhanced GFP (EGFP), more adapt for expression in mammalian cells and GC-rich actinomycetes like Streptomyces, have been widely used as markers to study gene expression, as well as plant-microbe interactions. Aside fluorescent protein approaches, fluorescence in situ hybridization (FISH) is another frequently used technique to visualize microbial colonization patterns and community composition by application of specific fluorescent probes. Firstly, we transformed five Streptomyces strains, which showed strong inhibition activity against Sclerotinia sclerotiorum, with the EGFP construct by the conjugation method. The conjugation efficiencies varied between the strains, but were comparable to the reference strain. The fitness of transformed strains was similar to wild-type; the transformants maintained similar sporulation, mycelium growth rate, and the ability to produce important secondary metabolites and lytic enzymes. Secondly, two transformed strains, Streptomyces cyaneus ZEA17I, and Streptomyces sp. SW06W, were used to study lettuce colonization dynamics by seed coating method. Their spatio-temporal dynamics were determined in sterile substrate. The strains were consistently recovered from lettuce rhizosphere and inner root tissues up to six weeks. Finally, the colonization pattern of lettuce by Streptomyces cyaneus ZEA17I was examined by both EGFP and FISH approaches combined with confocal laser scanning microscopy (CLSM). For FISH-CLSM analysis, universal bacteria and Streptomyces genus specific probes were used to label S. cyaneus ZEA17I. The consistent presence of the labeled strain at the lettuce root one week after sowing showed that Streptomyces spores could rapidly germinate and produce filamentous mycelium on lettuce. S. cyaneus ZEA17I was detected also on two-week-old roots, indicating the long-term survival ability of this strain in lettuce rhizosphere. Altogether, the antagonistic activity, rhizosphere and root competence showed by the Streptomyces conferred their potential to act as BCA. Further studies on the complex host-pathogen-antagonist interactions will provide additional knowledge to understand the modes and mechanisms of Streptomyces-mediated plant protection.

L’ADN est constamment soumis à de nombreux stress, menaçant son intégrité et, par conséquent, le bon fonctionnement cellulaire. Pour y faire face, la cellule dispose de tout un arsenal de voies de réparation. L’une des altération du génome les plus fréquentes est l’oxydation de la guanine en 8-oxoguanine (8-oxoG). La 8-oxoG possède un fort potentiel mutagène du fait de son appariement préférentiel avec l’adénine au lieu de la cytosine lors de la réplication. Ainsi, elle doit être détectée et réparée à temps pour éviter la fixation dans le génome de mutation par transversion G:C vers T:A. Cette lésion appairée à une cytosine est détectée et excisée par la 8-oxoguanine ADN-glycosylase (OGG1), ce qui initie la réparation par excision de base. Si le fonctionnement d’OGG1 a été largement étudié in vitro et que de nombreuses données structurales sont disponibles, très peu d’études se sont penchées sur la dynamique de cette protéine au sein du noyau cellulaire. Le but de ma thèse était donc de caractériser la dynamique de recherche de la 8-oxoG par OGG1 et d’identifier les éléments (résidus ou fonctions) régulant cette dynamique. Ainsi, j’ai pu montrer que l’interaction avec l’ADN était un élément majeur de la recherche de la 8-oxoG par OGG1, et que muter les résidus impliqués dans l’interaction avec l’ADN perturbait la dynamique d’OGG1 et sa capacité à trouver et exciser la 8-oxoG. De même, la reconnaissance de la 8-oxoG, mais également celle de la cytosine lui faisant face, jouent toutes deux un rôle important dans le fonctionnement de l’ADN-glycosylase et son recrutement à la zone de dommages. Enfin, le motif NNN, très conservé mais très peu caractérisé jusqu’ici semble être essentiel à l’association spécifique avec la 8-oxoG mais pas à la dynamique de recherche<br>DNA is constantly subjected to various stress, threatening its integrity, and consequently, the proper functioning of the cell. In order to preserve the genomic integrity, the cell can activate a large set of repair pathways. One of the most common genomic alteration is the base modification 8-oxoguanine (8-oxoG), an oxidized form of guanine. It is highly mutagenic, due to its tendency to pair with adenine instead of cytosine during replication. Thus, it needs to be detected and repaired on time to avoid G:C to T:A transversions. 8-oxoG paired with cytosine is recognized and excised by the 8-oxoguanine DNA-glycosylase (OGG1), which initiates the base excision repair pathway. Although OGG1 has been widely studied in vitro and many structural data are available, many questions remain concerning the dynamics of the protein within the cell nucleus. Hence, the goal of my PhD project was to characterize the dynamics of OGG1 searching for 8-oxoG and get new insights about the residues or functions of OGG1 that regulate these dynamics. I was able to show that the interaction between OGG1 and DNA is crucial for the efficient search of 8-oxoG, and that mutating the residues involved in such interaction impairs OGG1 dynamics and its ability to detect and excise 8-oxoG. Similarly, 8-oxoG detection, but also that of the facing cytosine, both play an important role in the function of the DNA-glycosylase and in its ability to accumulate at the sites of damage. Finally, the NNN motif, which is highly conserved but very poorly characterized, seems to be essential to the specific association with 8-oxoG, but not for the nuclear exploration by OGG1

L'objectif de la thèse était d'étudier la mobilité de traceurs particulaires dans des milieuxcomplexes par microscopie confocale à balayage laser (CLSM) combinée avec le suivi demultiple particules (MPT) et le recouvrement de fluorescence après photoblanchiment (FRAP).Tout d'abord, nous avons étudié la diffusion de particules dans les gels formés par des protéinesglobulaires. Dans ce but, des gels avec structures variés ont été préparés en faisant varier lesconcentrations en protéine et en sel. La structure a été caractérisée par l'analyse des imagesobtenues par CLSM en termes de fonction de corrélation de paires. La mobilité de particulesavec une large gamme de tailles (2nm - 1 micron) a été étudiée à la fois dans des gels homogèneset hétérogènes et reliée à la structure du gel.Deuxièmement, nous avons étudié des émulsions eau dans eau préparées en mélangeant dessolutions aqueuses de PEO et de dextran. Il a été montré que lorsque des particules colloïdalessont ajoutées, elles sont emprisonnées à l'interface eau-eau, car elles réduisent la tensioninterfaciale. La structure et le déplacement des particules à l'interface ont été déterminés parCLSM combinée avec MPT<br>The objective of the thesis was to investigate the mobility of tracer particles in complex media byConfocal Laser Scanning Microscopy (CLSM) combined with multiple particle tracking (MPT)and fluorescence recovery after photobleaching (FRAP).First, we investigated the diffusion of tracer particles in gels formed by globular proteins. Gelswith a variety of structures were prepared by varying the protein and salt concentrations. Thestructure was characterized by analysis of the CLSM images in terms of the pair correlationfunction. The mobility of particles with a broad range of sizes (2nm - 1μm) was investigatedboth in homogeneous and heterogeneous gels and related to the gel structure.Second, we studied water-in-water-emulsions prepared by mixing aqueous solutions of PEO anddextran. It is shown that when colloidal particles are added they become trapped at the waterwaterinterface because they reduce the interfacial tension. The structure and the displacement ofthe particles at the interface were determined using CLSM combined with MPT

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.

L’objectif de cette étude a été de formuler et caractériser des émulsions simples huile/eau d’intérêt pharmaceutique stabilisées par de la β-lactoglobuline (β-lg), de la gomme arabique (GA), de la gomme xanthane (GX) et des mélanges β-lg:GA et β-lg:GX. Les concentrations massiques totales des dispersions de biopolymères étaient de 1 % et ont été augmentées à 2,5 % si les émulsions formulées n’étaient pas stables. Le mélange β-lg:GA a été réalisé à pH 4,2 afin de permettre la formation de complexes par interactions électrostatiques attractives entre la β-lg et la GA. Deux ratios β-lg:GA ont été étudiés : 2:1 et 1:2. Enfin, le mélange β-lg:GX a été effectué à pH 7, où les deux biopolymères étant chargés négativement ne se complexent pas et à un ratio de 1:1. Une étude de stabilité des émulsions a été menée sur 6 mois. Les stabilités obtenues ont pu être classées par ordre croissant : GA 2,5 % &lt; β-lg:GA 2,5 % &lt; β-lg 2,5 % &lt; GX 1 % = β-lg:GX 1 %. Plusieurs mécanismes de stabilisation ont été mis en évidence grâce à l’étude des propriétés interfaciales des biopolymères, à l’étude des propriétés rhéologiques des émulsions et à des observations au microscope confocal à balayage laser des émulsions après marquage des biopolymères à la fluorescence. La β-lg et la GA sont toutes deux capables de s’adsorber à l’interface des globules huileux alors que la GX augmente la viscosité de la phase continue. L’association β-lg:GA conduit à la formation d’une double couche interfaciale stabilisante. Enfin, l’association β-lg:GX combine les mécanismes de stabilisation de la protéine, par adsorption interfaciale et de la gomme, par augmentation de la viscosité de la phase continue<br>The main objective of this study was to formulate and characterize oil-in-water simple emulsions of pharmaceutical interest stabilized by β-lactoglobulin (β-lg), gum arabic (GA), xanthan gum (XG), and mixtures of β-lg:GA and β-lg:XG. The total biopolymer final concentration in the dispersions was 1 (w/w) % and could be raised to 2.5 (w/w) % if the formulated emulsions were not stable. β-lg:GA mixing was performed at pH 4.2 to allow attractive electrostatic interactions between the two biopolymers and thus the formation of complexes. Two protein:polysaccharide ratios were investigated: 2:1 and 1:2. Conversely, β8lg:XG mixing was performed at pH 7, where both biopolymers are negatively charged, in order to avoid the complex formation, and with a 1:1 ratio. A stability study was conducted for emulsions over a 6-month period. The obtained stabilities could be classified increasingly: GA 2.5 % &lt; β-lg:GA 2.5 % &lt; β-lg 2.5 % &lt; XG 1 % = β-lg:XG 1 %. Several stabilization mechanisms were evidenced by the study of the biopolymer interfacial properties, the study of emulsion rheology and by confocal laser scanning microscopy observations with labeled fluorescent biopolymers. β-lg and GA were both able to adsorb at the interface of oil globule. XG enhanced the continuous phase viscosity. β-lg:GA mixing led to the formation of a stabilizing interfacial double layer. Finally, β-lg:XG association combined the stabilization mechanisms of both biopolymers, respectively: interfacial adsorption and enhancement of the continuous phase viscosity

Tese de doutoramento em Biofísica (Biofísica), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007<br>The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.<br>O núcleo celular é um organito complexo, dotado de um elevado grau de organização mas também uma natureza extremamente dinâmica. A utilização de proteínas fluorescentes como marcadores moleculares para visualização em células vivas, bem como as técnicas de photobleaching, têm sido essenciais na descoberta da natureza dinâmica do núcleo. Neste trabalho, estas ferramentas foram aplicadas no estudo da dinâmica e interacções moleculares dentro deste compartimento celular. As técnicas de Fluorescence Recovery After Photobleaching (FRAP) e Fluorescence Loss In Photobleaching (FLIP) foram utilizadas na análise do comportamento cinético dos componentes do spliceosoma SmE, U2AF65, U2AF35, SF1 e SC35 no interior do núcleo de células vivas. O mecanismo de recrutamento dos factores de splicing (SFs) para os locais de transcrição é ainda pouco conhecido. Os nossos resultados excluem a hipótese de haver um sinal associado à transcrição que seja responsável por este recrutamento. Sugerem ainda a formação de complexos multi-proteicos distintos do spliceosoma. A existência destes complexos foi confirmada por técnicas de Fluorescence Resonance Energy Transfer (FRET), que mostraram que os SFs podiam interagir uns com os outros mesmo na ausência de splicing activo. Foi ainda descoberta uma nova auto-interacção para o factor U2AF65, sugerindo os resultados no seu conjunto que a distribuição de SFs no núcleo é compatível com mecanismos de auto-organização. A mobilidade de mRNPs no núcleo foi estudada utilizando como marcadores moleculares duas proteínas que se ligam ao mRNA marcadas com GFP, PABPN1 e TAP. Foi desenvolvido um método de FLIP para quantificação da mobilidade das fracções ligadas e não ligadas ao mRNA e usado para testar a possibilidade de motores de miosina estarem envolvidos no movimento de mRNPs. Mostramos que tal não acontece e que a inibição de miosina parece antes afectar a transcrição. Um novo método de FLIP após foto-activação foi desenvolvido para estudar a dinâmica de trocas entre o núcleo e o citoplasma de proteínas nucleares, permitindo a estimação do tempo de permanência de moléculas dentro do núcleo. O método foi utilizado para investigar o papel dos diferentes domínios estruturais da proteína TAP na sua actividade de exportação nuclear.<br>Fundação para a Ciência e Tecnologia (BD/21518/99); European Commission (“RNOMICS” QLG2-CT-2001-01554 and “Integrated Technologies for in vivo Molecular Imaging” LSHG-CT-2003-503259)

Indiana University-Purdue University Indianapolis (IUPUI)<br>Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.

碩士<br>國立陽明大學<br>生醫光電工程研究所<br>93<br>Since the first invention in 1970 by Ashkin, optical tweezers have become a versatile non-invasive tool extensively used in controlling tiny biological samples, especially cytoskeleton related proteins. In optical tweezers, the precise measurement of axial motion of a trapped object in optical tweezers is not as well developed as that of the lateral motion. In this thesis we have introduced differential confocal microscopy (DCM), developed by Lee in 1997, into optical tweezers for the axial detection. DCM has advantageous features such as high depth resolution, large dynamic range, and long working distance. Combined with optical tweezers, we can detect the axial movement of an optically trapped latex bead. The resolution of axial position detection is 16 nm, which is limited by the background fluctuations of the whole system. We have detected about 50 nm scale of thermal fluctuation of a latex bead of 3 µm in diameter, trapped in a beam with a power of 27 mW through 1.3-NA objective lens. From the thermal fluctuations of the bead, we calculate the axial tweezers stiffness to be 2.34 µN/m at this power. We also find that the axial stiffness is linearly proportional to the laser power. With this setup, we measured the effect of myosin–actin binding to the fluctuations of a trapped bead. The result shows the fluctuations of a bead are lager with the presence of protein in solution. However, as myosin–actin bonds formed, the fluctuations of the myosin-coated bead gradually decreased when we elevated the bead from actin-coated dish bottom. Also, we have observed the motion retardation of bead, caused by the tension of myosin–actin binding between the bead and the bottom. We have successfully detected the binding effect of proteins to the motion of a trapped latex bead. In our experiment, the dynamic range of detection is lager than 3 µm, which makes the system suitable for the application of protein elasticity research.

Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems. Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization of two proteins of interest fused to green fluorescent protein (GFP) and DivIVA, a cell division protein from Bacillus subtilis. We demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions over four orders of magnitude. FRAP data was analyzed using a combination of various image processing techniques and analytical models. This combined approach made it possible to estimate cell morphology parameters such as length, diameter, the effective laser probe volume, as well as to the mobile protein concentration in vivo, the number of bound molecules at the cellular poles, and the biophysical parameter koff. Cells not only utilize molecular interactions in the intracellular environment, but also express proteins, polysaccharides and other complex molecules to mediate interactions with the surrounding extracellular environment. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Further analyses suggest that the extracellular matrix differs between the cheA1 and the cheY1 deletion mutants, despite similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013<br>A membrana celular é composta por uma ampla variedade de lípidos e proteínas. Através do estabelecimento de interacções entre lípidos e proteínas, quer membranares quer solúveis, podem ser originadas heterogeneidades espaciais, levando à formação de ‘jangadas lipídicas’. Estes domínios transitórios desempenham um papel fundamental em diferentes cadeias de sinalização celular. Apesar da investigação desenvolvida na área, a membrana celular é ainda hoje um dos componentes celulares menos bem compreendidos. Nas últimas décadas têm vindo a surgir um conjunto de diferentes metodologias in vitro que permitem o estudo de variados processos biológicos sob condições definidas e controladas. Neste sentido, foram desenvolvidos variados modelos membranares minimalistas, como GUVs (giant unillamelar vesicles), GPMVs (giant plasma membrane vesicles) and SLBs (supported lipid bilayers), permitindo a análise das interacções lípido-proteína até a um nível unimolecular. Quando correctamente aplicados estes modelos permitem simplificar o sistema isolando virtualmente o fenómeno ou a partícula de interesse retendo, porém, as suas características fundamentais. Apesar da vantagem evidente do uso destes modelos, estas abordagens experimentais são tediosas, envolvendo geralmente o uso de amostras de elevado volume e em que a variação de certas características membranares (como a difusão lipídica) é apenas possível por alteração da composição lipídica ou da temperatura. As monocamadas lipídicas são historicamente descritas como o primeiro sistema lipídico observado, formando-se por distribuição espontânea de moléculas lipídicas sobre a interface água - ar e formação de filmes finos sobre superfícies. Dado o seu design único, a mobilidade e compactação lipídica podem ser facilmente ajustadas neste sistema modelo por compressão da monocamada depositada na interface. Apesar de o seu uso em ensaios de ligação proteica, este sistema tem sido negligenciado na investigação membranar recente devido à sua restrita aplicação. Mais concretamente, as tinas de Langmuir comercialmente disponíveis não são compatíveis com microscopia confocal, estando assim limitadas à monitorização da pressão superficial (π). Além disso, dado o volume de cada amostra relativamente elevado (cerca de 100 mL), são necessárias elevadas quantidades de proteína purificada, comprometendo assim a sua aplicação em estudos biológicos. Neste estudo, foi utilizada uma nova tina miniaturizada de área fixa, desenvolvida no grupo por Chwastek (Chwastek & Schwille, 2013). As suas dimensões reduzidas permitem não só o uso de amostras de pequeno volume (cerca de 200 μL) como também conferem a versatilidade necessária para o seu acoplamento a um microscópio confocal. Deste modo, é possível aplicar técnicas de imagiologia de alta resolução (permitindo a eliminação de ruído de fundo, o controlo de profundidade de campo e a compilação de secções ópticas de amostras espessas oferecido pelo microscópio confocal), assim como espectroscopia de correlação de fluorescência (FCS – técnica espectroscópica cuja sensibilidade advém do uso de baixas concentrações de sonda e na consequente medição de flutuações espontâneas da intensidade de fluorescência que resultam de desvios estocásticos do sistema relativamente ao seu equilíbrio térmico). O objectivo global deste projecto prende-se com a melhor compreensão da interacção estabelecida entre proteínas e monocamadas lipídicas. Mais concretamente foi medida a ligação a monocamadas lipídicas de duas proteínas estruturalmente bem caracterizadas: a estreptavidina – proteína tetramérica solúvel com elevada afinidade de ligação para a biotina (KD ≈ 10-15 M); e a toxina da cólera – enterotoxina, secretada pela bactéria Vibrio cholerae, constituída por um anel homo-pentamérico de subunidades B (CtxB5), responsável pela sua ligação ao receptor membranar gangliósido GM1 (KD ≈ 10-9 ou 10-10 M), e uma subunidade A com actividade de ribosilação que é parcialmente internalizada pelas células do hospedeiro, perturbando cascatas celulares de sinalização e desencadeando a infecção colérica. Foram realizadosensaios de ligação proteica a monocamadas lipídicas homogéneas e com separação de fases, na ausência ou presença dos respectivos ligandos específicos, de modo a distinguir e caracterizar os diferentes comportamentos proteicos. Em última análise, procurou-se desenvolver e optimizar um ensaio universal e quantitativo de ligação proteica a membranas lipídicas. Previamente aos estudos das interacções proteína-monocamanda, procedeu-se à caracterização morfológica das monocamadas lipídicas, assim como da mobilidade lipídica, recorrendo a microscopia confocal e a FCS, respectivamente. Observou-se pois que a fluidez e homogeneidade global das monocamadas é acompanhada pelo aumento linear do coeficiente de difusão em função da densidade lipídica no intervalo estudado (50 a 90 MMA – área molecular média em Å2), tal como se prevê do modelo de área livre (free area model). Mais, os valores obtidos para o ponto crítico do DMPC (36.0 ± 4.7 e 37.2 ± 13.5 Å2 em misturas marcadas com sondas diferentes) encontram-se dentro do erro dos valores descritos na literatura. Apesar da robustez demonstrada pelo sistema, é porém necessário ter em conta a possibilidade de ocorrerem desvios em extremos de densidade lipídica (50 e 100 MMA), por formação de fases S (sólida) e G (gás), respectivamente. Após validação da metodologia proposta, procedeu-se ao estudo da influência da compactação lipídica na ligação proteica à monocamada – quer com a estreptavidina, quer com a CtxB5 observou-se uma preferência para a ligação a monocamadas de baixa densidade lipídica e, correspondentemente baixa π e elevada difusão lipídica. Estes resultados são ainda suportados pelas experiências realizadas em misturas lipídicas com separação de fase – ambas as proteínas apresentam uma elevada afinidade para a fase fluida LE (liquid expanded) em comparação com os domínios compactos LC (liquid condensed). Adicionalmente, resultados de titulações de monocamadas com CtxB5 apontam para a mesma tendência visto que a 90 MMA se obtêm intensidades de fluorescência proteica ao nível da monocamada superiores que a 50 MMA. Este comportamento já tinha sido proposto no início da década de 80 tendo por base a análise de variações de π por adição proteica a monocamadas lipídicas (Phillips et al, 1975; Fidelio et al, 1981; Cumar et al, 1982). Porém, no caso da estreptavidina observa-se ainda um comportamento extraordinário de acumulação nas bordas de domínios quando a diferença na organização lipídica entre diferentes fases é muito elevada. A especificidade de ligação foi apenas possível para a CtxB5 por incorporação de GM1 na monocamada. Titulações de monocamadas demonstraram que a presença de uma baixa concentração do gangliósido (0.1 mol %) é suficiente para aumentar fortemente a ligação da proteína à monocamada. Além disso, em sistemas lipídicos homogéneos a proteína retém apreferência por baixas densidades lipídicas. Porém, na presença de separação de fases, a presença de GM1 promove a ligação de CtxB5 a domínios LC, dado que este se incorpora preferencialmente em domínios rígidos. Apesar da elevada reprodutibilidade associada às medições em lípidos, a aplicação do sistema a proteínas tem um erro associado bastante elevado que impede ainda uma quantificação exacta da sua ligação aos lípidos. É de salientar que para além da elevada sensibilidade do sistema de detecção usado, as monocamadas lipídicas formadas sobre a interface água-ar são altamente sensíveis a diferentes factores externos (p.e. oxidação lipídica, formação de menisco e não homogeneidades da compactação lipídica aquando da deposição lipídica, evaporação da subfase, deposição proteica, etc.) que ainda não são totalmente controladas. Deste modo, é ainda necessário proceder à optimização do método de modo a poder determinar-se KD da ligação de proteínas à superfície lipídica. Este projecto resultou pois na obtenção de novos dados semi-quantitativos de que a compactação lipídica desempenha um papel importante na interacção proteína-membrana. A separação de fases surge pois como um mecanismo de regulação da ligação proteica, não só por controlo da mobilidade lipídica mas também por segregação de ligandos. Em suma, a abordagem aqui proposta surge como uma alternativa poderosa para estudos de interacções membrana-proteina, permitindo o fácil ajuste de parâmetros membranares como a compactação lipídica, difícil de regular em sistemas de bicamadas. A sua combinação potente com a microscopia confocal e espectroscopia de correlação de fluorescência resulta não só numa elevada resolução temporal da mobilidade de partículas lipídicas, como também na alta resolução espacial da ligação proteica. Deste modo, o trabalho apresentado contribuiu para a aproximação ao desenvolvimento de um novo ensaio quantitativo global de ligação proteica a membranas lipídicas.<br>Cellular membranes are composed of a wide variety of lipids and proteins which can lead to spatial heterogeneity and formation of so-called ‘lipid rafts’, which play an important role in signaling processes. To investigate these features of lipid bilayers various in vitro models have been developed. Although a variety of experimental assays exist, they very often require large samples volume, are complicated and the membrane features can be varied only by changes in lipid composition or temperature. Here, a fixed-area miniaturized monolayer trough was used. Due to its unique design, lipid mobility in monolayers is easily accessible while keeping other factors constant. The system utilized in this study allows the use of small sample volumes (200 μL) and can be combined with a confocal microscope and thus providing access to high resolution imaging and sensitive fluorescence correlation spectroscopy (FCS) technique. The goal of the project was to design a quantitative assay by which protein-monolayer interactions can be studied. Thus, monolayers were investigated in terms of morphology and lipid mobility. Further, interactions of well-known ligand-protein pairs were studied: cholera toxin (Ctx)/ganglioside GM1 and streptavidin/biotin. The influences of phase separation and presence of lipid ligands were investigated. It was shown that the membrane affinities of cholera toxin B (CtxB5) and streptavidin depend on the surface density of lipid molecules. Moreover, FCS measurements indicate a correlation between higher protein binding and increased lipid mobility. When phase separated lipid monolayers were used, both proteins bound preferentially to liquid expanded phase (LE). However, in case of CtxB5, in the presence of the ganglioside, the protein mostly binds to the liquid condensed phase (LC). In summary, protein binding to lipid monolayers was studied by means of fluorescence microscopy and spectroscopy. During the studies, an appropriate methodology was developed and several experimental scenarios were tested.

Purpose: To locate protein sorption on the surface and inside the matrix of soft contact lens materials and intraocular lenses (IOL). Methods: The proteins albumin and lysozyme were investigated as they are highly abundant in blood serum and tears, respectively. Proteins were conjugated with organic fluorescent probes and using confocal laser scanning microscopy (CLSM) the sorption profile to contact lenses and IOL could be determined. Radiolabeled protein was used for quantification purposes. • Albumin sorption to etafilcon A and lotrafilcon B was determined (Chapter 3) • Different fluorescent probes were used for conjugation and the impact on albumin sorption behaviour was investigated (Chapter 4) • Lysozyme sorption to nine different pHEMA-based and silicone hydrogel contact lenses was determined using two fluorescent probes (Chapter 5) • The efficiency of protein removal from contact lenses using contact lens care regimens was investigated (Chapter 6) • Albumin sorption to IOL materials was quantified and imaged using a modified CLSM technique (Chapter 7) Results: Albumin and lysozyme sorption profiles differed between materials, and were influenced by the fluorescent probes used for conjugation. After one day of incubation, both proteins could be located within all contact lens materials, except for lotrafilcon A and lotrafilcon B, which primarily allowed deposition on the lens surface. An increase in protein accumulation was found for most materials over the maximum investigated period of 14 days, using CLSM and radiolabel techniques. The efficiency of contact lens care regimens to remove lysozyme and albumin depended on the lens material, care regimen and protein type investigated. PMMA and silicone IOLs showed protein exclusively on the surface, while a hydrophilic acrylic IOL allowed penetration into the lens matrix over time. Despite the albumin penetration depth into hydrophilic acrylic, the highest albumin levels were determined for the silicone IOL. Conclusions: CLSM provides detailed information that can describe the protein distribution in transparent biomaterials, with scanning depths up to a few hundred microns. However, the CLSM data are primarily of qualitative value, which necessitates a quantitative technique (e.g. radiolabeling) to determine the total protein content.