Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.

The main objective of this thesis work was to gain understanding of the principles of biolubrication, focusing on synergistic effects between biolubricants. To this end surface force and friction measurements were carried out by means of Atomic Force Microscopy, using hydrophilic and hydrophobic model surfaces in salt solutions of high ionic strength (≈ 150 mM) in presence of different biolubricants. There was also a need to gain information on the adsorbed layers formed by the biolubricants. This was achieved by using a range of methods such as Atomic Force Microscopy PeakForce imaging, Quartz Crystal Microbalance with Dissipation, Dynamic Light Scattering and X-Ray Reflectometry. By combining data from these techniques, detailed information about the adsorbed layers could be obtained.The biolubricants that were chosen for investigation were a phospholipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) that all exist in the synovial joint area. First the lubrication ability of these components alone was investigated, and then focus was turned to two pairs that are known or assumed to associate in the synovial area. Of the biolubricants that were investigated, it was only the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) that was found to be an efficient lubricant on its own. Deposited DPPC bilayers on silica surfaces were found to be able to provide very low friction coefficients (≈ 0.01) up to high pressures, ≈ 50 MPa. A higher load bearing capacity was found for DPPC in the liquid crystalline state compared to in the gel state.The first synergy pair that was explored was DPPC and hyaluronan, that is known to associate on the cartilage surface, and we also noticed association between hyaluronan and DPPC vesicles as well as with adsorbed DPPC bilayers. By combining these two components a lubrication performance similar to that of DPPC alone could be achieved, even though the friction coefficient in presence of hyaluronan was found to be slightly higher. The synergy here is thus not in form of an increased performance, but rather that the presence of hyaluronan allows a large amount of the phospholipid lubricant to accumulate where it is needed, i.e. on the sliding surfaces.The other synergy pair was lubricin and COMP that recently has been shown to be co-localized on the cartilage surface, and thus suggested to associate with each other. Lubricin, as a single component, provided poor lubrication of PMMA surfaces, which we utilized as model hydrophobic surfaces. However, if COMP first was allowed to coat the surface, and then lubricin was added a low friction coefficient (≈ 0.03) was found. In this case the synergy arises from COMP facilitating strong anchoring of lubricin to the surface in conformations that provide good lubrication performance. / Huvudsyftet med det här avhandlingsarbetet var att öka förståelsen för den låga friktion som finns i vissa biologiska system, med fokus på synergistiska effekter mellan de smörjande molekylerna. För detta ändamål studerades ytkrafter och friktion med hjälp av atomkraftsmikroskopi. Mätningarna utfördes med hydrofila och hydrofoba modellytor i lösningar med hög salthalt (≈ 150 mM) i närvaro av smörjande biomolekyler. Det var också nödvändigt att få information om de adsorberade skikten av biomolekyler. Det åstadkoms med hjälp av en rad tekniker så som AFM PeakForce avbildning, kvartskristallmikrovåg, dynamisk ljusspridning och röntgen reflektometri. Genom att kombinera data från dessa tekniker erhölls detaljerad information om de smörjande skikten.De smörjande biomolekyler som valdes ut för studierna var en fosfolipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) vilka alla finns i synovialledsområdet. Först undersöktes den smörjande förmågan hos dessa komponenter var för sig, och sedan fokuserade vi på två par av biomolekyler som man vet eller antar bildar associationsstrukturer i synovialleder. Av de enskilda biomolekyler som undersöktes var det endast fosfolipiden 1,2-dipalmitoyl-sn-glycero-3-fosfokoline (DPPC) som visade sig vara en effektivt smörjande molekyl. Deponerade biskikt av DPPC på silikaytor gav upphov till mycket låga friktionskoefficienter (≈ 0.01) upp till höga pålagda tryck, ≈ 50 MPa. DPPC bilager i flytande kristallin fas visade sig ha högre lastbärande förmåga än DPPC bilager i geltillstånd.Det första synergistiska par som undersöktes var DPPC och hyaluronan vilka man vet associerar på broskytan, och vi visade att hyaluronan associerar med såväl DPPC vesiklar som med DPPC bilager. Genom att kombinera dessa två komponenter uppmättes en smörjande förmåga som var jämförbar med den som DPPC ensam uppvisar. Även om friktionskoefficienten var något högre i närvaro av hyaluronan. Synergieffekten här består inte av en bättre smörjande förmåga, utan istället gör närvaron av hyaluronan att de smörjande fosfolipiderna kan ansamlas i stora mängder där de behövs, dvs. på de glidande ytorna.Det andra synergiparet var lubricin och COMP vilka nyligen har visats vara lokaliserade på samma platser på broskytan, vilket tyder på att de associerar med varandra. På egen hand var lubricins smörjande förmåga av PMMA, våra hydrofoba modellytor, dålig. Emellertid, om COMP först adsorberades på PMMA och sedan lubricin tillsattes uppmättes en låg friktionskoefficient (≈ 0.03). I det här fallet består synergin av att COMP möjliggör en stark inbindning till ytan av lubricin i konformationer som ger god smörjande förmåga. / <p>QC 20141202</p> / Stiftelsen för strategisk forskning - SSF

The thesis entitled “Novel Redox Responsive Cationic Lipids, Lipopolymers, Glycolipids and Phospholipid-Cationic Lipid Mixtures: Syntheses, Aggregation and Gene Transfection Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel cholesterol based cationic lipids with ferrocene as the redox moiety, polyethylenimine based ferrocenylated lipopolymers and cholesterol based non-ionic glycolipids. The thesis also discusses the cationic phospholipid-cationic lipid mixtures as superior gene transfection agents. The work has been divided into six chapters. Chapter 1. Introduction Part A. Various Cholesterol based Systems for Applications as Biomaterials Liposomes composed of cationic lipids have become popular gene delivery vehicles. A great deal of research is being pursued to make efficient vectors by varying their molecular architecture. Cholesterol being ubiquitous component in most of the animal cell membranes is increasingly being used as a hydrophobic segment of synthetic cationic lipids. In this chapter we describe various cholesterol based cationic lipids and focus on the effect of modifying various structural segments like linker and the headgroup of the cationic lipids on gene transfection efficiency with a special emphasis on the importance of ether linkage between cholesteryl backbone and the polar headgroup. Interaction of cationic cholesteryl lipids with dipalmitylphosphatidycholine membranes is also discussed here. Apart from cholesterol being an attractive scaffold in the drug/gene delivery vehicles, certain cholesteryl derivatives have also been shown to be attractive room temperature liquid-crystalline materials. Part B. Diverse Applications of Ferrocene Derivatives This chapter gives a brief overview of ferrocene chemistry followed by description of major applications of ferrocenyl derivatives in a variety of fields like catalysis, materials chemistry, electrochemical sensors, medicinal chemistry etc. We discuss the use of ferrocene as an electrochemical and redox active switch to achieve control over supramolecular aggregation. It also reviews ferrocene based amphiphiles including surfactants, lipids and polymers with an emphasis on the role of ferrocene over aggregate formation and their utilization in biological applications. Chapter 2: Optimization of Redox Active Alkyl-Ferrocene Modified Polyethylenimines for Efficacious Gene Delivery in Serum 1a-c, n = 6, P8-C6-F1, P8-C6-F2, P8-C6-F3 2a-c, n = 11, P8-C11-F1 P8-C11-F2, P8-C11-F3 % ferrocene grafting, F1 = 15%, F2 = 25% and F3 = 50% Figure 1. Structure of the alkyl-ferrocene modified 800 Da Branched Polyethylenimine. In this chapter we present six new lipopolymers based on low molecular weight polyethylenimines (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and redox state of ferrocene in the lipopolymer on the self assembly properties were investigated in detail by transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements. The assemblies displayed a redox induced increase in the size of the aggregates. The coliposomes comprising of the lipopolymer and a helper lipid 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) showed excellent gene delivery capability in serum containing environment in two cancer cell lines (HeLa, U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI 25 KDa and even better than commercial Lipofectamine 2000 as evidenced from luciferase activity and EGFP expression analysis. Oxidation of ferrocene in lipopolymers led to reduced levels of gene transfection which was also followed by cellular internalization of fluorescently labeled pDNA using confocal microscopy. Cytotoxicity assay revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels. Overall, we have exploited the redox activity of ferrocene in PEI based polymeric gene carriers for trenchant control over gene transfection potential. RLU/mg protein HeLa Cells Figure 2. Maximum transfection efficacies of optimized redox lipopolymer/DOPE formulations by (A) Luciferase Assay and (B) Flow cytometry (GFP expression). Chapter 3. Membranes derived from Redox-active Cholesterol based Cationic Lipids and their Interactions with DNA and Phospholipid Membranes Figure 3. Molecular structures of the electroactive cholesterol based monomeric and gemini lipids. This chapter describes the synthesis and aggregation properties of two series of redox-active ferrocene containing monomeric and gemini cationic lipids with cholesterol as a hydrophobic domain. These cationic lipids are modified at their headgroup region using ferrocene terminated alkyl chains of differing length. All the four cationic lipids formed stable suspensions in water. Aggregation behavior of these cationic lipids in aqueous suspensions in their unoxidized and oxidized state was studied using TEM, DLS, zeta potential measurements and XRD studies. Cationic lipids with ferrocene in natural, reduced state were found form bigger sized vesicles which upon oxidation became smaller aggregates with increased zeta potential. XRD results indicate the existence of nice lamellar arrangements of the lipid bilayers. Thermotropic phase transition behavior of DPPC membranes incorporated with cationic ferrocene lipids was also studied using differential scanning calorimetry. Finally, we assayed pDNA (plasmid DNA) binding ability of all the four cationic lipids using ethidium bromide intercalation assay where all the cationic lipid formulations showed excellent DNA binding capability. In the experiments involving SDS-induced release of DNA, we observed that redox-active monomeric lipids (3a-b) were found to be more efficient in facilitating the release of DNA from the liposome-DNA complex in the presence of negatively charged SDS micelles than their gemini counterparts (4a-b). Chapter 4. Redox-responsive Gene Delivery by Ferrocene containing Cationic Cholesteryl Lipids in Serum This chapter describes the transfection efficacy of redox-active monomeric and gemini cationic lipids with cholesterol backbone. The transfection efficiency of all the lipids could be tuned by changing the oxidation state of the ferrocene moiety. Gene transfection capability was assayed in terms of EGFP expression using pEGFP-C3 plasmid DNA in three cancer cell lines of different origin, namely Caco-2, HEK293T and HeLa in the presence of serum. Figure 4. Effect of oxidation state of ferrocene on maximum transfection efficacies of monomeric and gemini lipids in three different cell lines (Caco-2, HEK 293T and HeLa). Cationic liposomal formulations with ferrocene in its reduced state were observed to be potent transfectants reaching the EGFP expression levels even better than commercial lipofectamine 2000 in the presence of serum as evidenced by flow cytometry. EGFP expression was further substantiated using fluorescence microscopy studies. All liposomal formulations containing oxidized ferrocene displayed diminished levels of gene expression and interestingly, these results were consistent for each formulation in all the three cell lines. Assessment of EGFP expression mediated by both reduced and oxidized ferrocene containing formulations was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Lipoplexes derived from different liposomal formulations with reduced and oxidized ferrocene were characterised using TEM, AFM, zeta potential and DLS measurements. Overall, we demonstrate here controlled gene transfection levels using redox driven, transfection efficient cationic monomeric and gemini lipids. Chapter 5: Synthesis of ‘Click Chemistry’ Mediated Glycolipids: Their Aggregation Properties and Interaction with DPPC Membranes This chapter describes the synthesis and aggregation properties of cholesterol based glycolipids along with their interaction with a model phosphatidylcholine membranes. Three series of non-ionic glycolipids with hydrophobic cholesterol backbone and various monosaccharide and disaccharide sugars as the hydrophilic polar domain have been synthesized. These were conjugated to the cholesteryl backbone via oligooxyethylene spacers of different lengths (n = 1, 3 and 4) using Cu (I) catalyzed Huisgen [3+2] cycloaddition, which is popularly known as „Click Chemistry‟. All the synthetic glycolipids (5a-d, 6a-d and 7a-d) formed vesicular aggregates in aqueous medium as confirmed by TEM and DLS. XRD studies with the cast films of lipids revealed that the bilayer width increased with increase in the length of oligoethylene spacer unit that has been incorporated between the hydrophobic and hydrophilic domains. Also, within the same series containing a particular oligoethylene unit, bilayer widths were found to be more for the lipids containing disaccharides as their headgroup than monosaccharides. Figure 5. Molecular structures of various cholesterol-based glycolipids. Calorimetry studies of the coaggregates containing naturally occurring 1, 2-dipalmitoylphosphatidylcholine (DPPC) and various mol-% of each of the glycolipids revealed that more than 30 mol-% of glycolipids are required to completely abolish the phase transition of DPPC membranes. These results were further supported by fluorescence anisotropy measurements of the co-aggregates using 1, 6-diphenylhexatriene (DPH) as a probe. Fluorescence anisotropy of the neat vesicles revealed that 9a and 9c were more rigid than DPPC vesicles in the solid-like gel phase, while the glycolipids with longer oxyethylene spacers (n = 3 and 4) were less rigid than the DPPC vesicles. Chapter 6. Hydrophobic Moiety Decides the Synergistic Increase in Transfection Efficiency in Cationic Phospholipid/Cationic Lipid mixtures This chapter describes the effect of inclusion of cationic lipid/cationic gemini lipids into the membranes of a cationic phospholipid on the gene delivery efficiency across HeLa and HEK293T cell lines. Although all the three cationic lipids have the same quaternary ammonium moiety as their headgroup, they differ from each other in terms of their hydrophobic moiety and in the number of cationic headgroups. Chol-N is a cholesterol based monocationic lipid, while 2C14-N and 2C14N-5-N2C14N are monomeric and gemini cationic lipids respectively with pseudoglycerol backbone consisting of tetradecyl (n-C14H29) chains. Each of the three cationic lipids under the current investigation, namely, Chol-N, 2C14-N and 2C14N-5-N2C14N were added in different ratios to EtDMoPC and the resultant mixed membranes were studied for the biophysical characterization and gene delivery efficacies. Figure 6. Molecular structures of cationic lipids used in this study. All the formulations were characterized using dynamic light scattering and zeta potential measurements to obtain their hydrodynamic diameters and surface charge properties respectively. Their DNA binding ability was also studied by measuring changes in zeta potential and gel electrophoresis of the lipoplexes formed by the coliposomal formulations and pDNA at different Lipid/DNA weight ratios. The gene delivery efficacies of various formulations were studied in terms of EGFP expression using pEGFP-C3 plasmid DNA in two different cell lines, namely HeLa and HEK293T. In the absence of serum we found that the formulation (EtDMoPC+2C14N-5-N2C14N) showed better transfection efficiency than the individual lipids. However, in the case of others, i.e., (EtDMoPC+Chol-N) and (EtDMoPC+2C14-N) formulations, there was a slight decrease in transfection efficiency compared to the individual lipids. In the presence of serum, the formulations (EtDMoPC+2C14-N) and (EtDMoPC+2C14N-5-N2C14N) showed significantly higher transfection efficacies compared to their individual lipids. Fusion assay using labelled cationic lipid formulations and unlabelled anionic liposomes revealed that lipoplexes prepared from EtDMoPC+ 2C14-N and EtDMoPC+ 2C14N-5-N2C14 exhibited much higher fusogenicity as compared to the lipoplexes prepared using EtDMoPC+Chol-N as well as the individual lipids. Thus, the liposome formulations which showed better transfection activity fused more readily with the anionic liposomes than did the formulations with poorer activity. Overall, we found that the hydrophobic domain of the cationic lipid/cationic gemini lipid that is added to cationic phospholipid has an important role on the transfection efficiency of the mixed formulations. Additionally the cytotoxicity studies revealed that each of these formulations was not significantly toxic making them viable for applications in vivo. (For structural formula pl see the abstract pdf file)

Le LPS est un puissant agent pro-inflammatoire bactérien, dont la partie lipide A est considérée comme le principe actif. Néanmoins, la chaîne O des LPS influence leur agrégation en solution aqueuse. Notre but a été de déterminer le rôle de la chaîne O sur les effets biologiques et physiopathologiques des LPS.Nos travaux, menés selon trois axes stratégiques complémentaires, ont donné lieu aux avancées suivantes :- développement d'un dosage innovant des LPS par LC-MS/MS et d'un ratio d'inactivation des LPS sur la base d'une utilisation combinée dudit dosage et du test LAL. Ce ratio traduit la capacité d'un organisme hôte à inactiver les LPS, notamment par leur transfert aux HDL par la PLTP. Ce ratio pourrait être utile dans l'évaluation des patients à haut risque.- la longueur de la chaîne O module l'inflammation induite par les LPS. Au-delà de leur concentration d'agrégation critique, les LPS forment des agrégats dotés d’une architecture et de propriétés physico-chimiques dépendant de leur chaîne O. Ces deux paramètres déterminent l'activité biologique des LPS et leur métabolisme ;- développement d'un double marquage innovant des LPS confirmant leur voie principale d'élimination : le transport inverse du LPS. Ce travail définit donc les effets physiopathologiques induits par les LPS comme résultant de deux composantes : leur activité biologique et leur métabolisme. Toute stratégie de recherche ou thérapeutique ciblant les LPS, devrait donc prendre en compte leur structure moléculaire, leur agrégabilité et la relation entre ces deux paramètres, déterminants majeurs de l'activité biologique des LPS et de leur métabolisme. / LPS is a potent bacterial pro-inflammatory agent, consisting of hydrophilic, polysaccharide part and of a lipid A which is considered like active moiety. Nevertheless, the O chain of LPS influences their aggregation in aqueous media. Therefore, our goal has been to determine the role of O chain on the LPS biological and physiopathological effects. Our work was organized according to three main axes, and led to the following findings :- development of a new LPS assay by LC-MS/MS. The combination of this new technique with LAL test allowed us to calculate an inactivation ratio which reflects the ability of host organism to inactivate LPS, especially through their transfer to HDL by PLTP. The ratio could be useful in predicting outcome of high risk patients.- the length of O chain modulates LPS-induced inflammation. Above their critical aggregation concentration, LPS form aggregates with an architecture and physiochemical properties dependent on their O chain. Both parameters determine LPS biological activity and their metabolism.- development of an innovative dual labelling of LPS as a new tool to explore LPS elimination pathway : the reverse LPS transport. This work brings evidence that the physiopathological effects of LPS depend on two parameters : their biological activity and their metabolism. Any strategy of research or therapeutic targeting LPS should take into account their molecular structure, their aggregability and the relation between the both parameters, which are major determinants of their biological activity and their metabolism.

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

Cette thèse expérimentale s’inscrit dans le domaine des sciences séparatives et se base sur la technique de SPLITT (SPLIT-flow Thin fractionation). Son objectif consiste en l’étude des mécanismes qui sont à l’origine de la séparation, en continu et sans membrane, d’objets de taille micrométrique dans des mini-séparateurs fluidiques (Step-SPLITT). Les expériences menées, en laboratoire et lors de vols paraboliques, ont révélé le couplage complexe comme l’influence des effets hydrodynamiques et du champ gravitationnel sur la migration transverse des espèces en écoulement. Des visualisations tridimensionnelles par holographie digitale ont corroboré nos résultats et dévoilé des comportements inattendus. Les capacités séparatives des Step-SPLITT ont rendu possible l’analyse et la séparation d’objets biologiques et biomimétiques. Enfin, cette étude complétée par une modélisation tridimensionnelle de l’écoulement nous a permis de mettre au point un nouveau prototype de séparateur. This experimental thesis belongs to the field of separative sciences and is based on the SPLITT technique (SPLIT-flow Thin fractionation). The objective is to study the mechanisms that are at the origin of continuous and membraneless separation of micron-size species in mini fluidic separators (Step-SPLITT). Experiments undertaken in laboratory and during parabolic flights revealed the complex coupling of the hydrodynamic effects and the gravitational field influencing the transverse migration of the flowing species. Three-dimensional visualizations performed by digital holography confirmed our results and disclosed unexpected behaviours. The separation capacities of Step-SPLITT made the analysis and the separation of biological and biomimetic species possible. In addition this study in conjunction with a three-dimensional flow modelling enabled us to develop a new prototype of separator.

Der hierarchisch strukturierte und hoch geordnete Aufbau von Calciumphosphat und Kollagen in Knochen und Zähnen wird von den Zellen mit Hilfe bestimmter Moleküle erreicht. Diese organischen Moleküle, zumeist Proteine, beeinflussen durch die räumliche Anordnung ihrer Ladung das Präzipitations- und Wachstumsverhalten der mineralischen Phase. Die in dieser Arbeit beschriebenen Computersimulationen zeigen, dass ein Calciumphosphatkomplex mit deprotoniertem Phosphat am stabilsten ist. Vermutlich nimmt die Bindungsenergie pro Oberfläche des Komplexes mit wachsender Größe bis zu einem Ca9(PO4)6 -Komplex (Posner Klaster) linear zu. Die Präzipitation von Calciumphosphat aus wässriger Lösung führt häufig zu amorphen Kugeln mit 50-500 nm Durchmesser, die sphärische Unterstrukturen von ca. 5 nm Durchmesser zeigen und bei großer Dichte zu einer amorphen Schicht verschmelzen. Geringe Unterschiede in der Präparation können aber schon zu stäbchenförmigen oder plättchenartigen Kristalliten führen. Phosphoserin ist eine der wichtigsten Aminosäuren bei der Anbindung von Proteinen an Calciumphosphat. Das Computermodell zeigt an der gesamten Oberfläche dieser Aminosäure ein deutliches elektrisches Potential, dies begünstigt die Wechselwirkung mit Ionen. FT-IR- und NMR-Untersuchungen zeigen, dass Phosphoserin bei Kopräzipitation mit Calciumphosphat höchstwahrscheinlich in die mineralische Phase eingebaut wird. Serin zeigt bei der Kopräzipitation ab 1 mM einen Einfluss auf die Morphologie von Calciumphosphat, während Phosphoserin schon bei 0,01 mM einen deutlichen Einfluss zeigt. Elektronenspray-Ionisations-Massenspektroskopie (ESI-MS) bestätigt die relativ zum Serin intensivere Wechselwirkung von Phosphoserin mit Calciumphosphat. Das wichtigste Protein zur Vermeidung ektopischer Mineralisierung ist Fetuin. Dieses Protein stabilisiert die transient auftretenden amorphen Calciumphosphatkugeln (ACP-Kugeln) und erlaubt so dem Körper deren Entsorgung. Fetuin verhindert das Verschmelzen von ACP-Kugeln, wenn diese in großer Dichte auftreten, wobei deren feine Unterstruktur erhalten bleibt. Trotz des starken inhibitorischen Verhaltens wird das Auflösen von Brushit durch die Anwesenheit von Fetuin praktisch nicht beschleunigt. Auch auf die Kinetik der Assemblierung von Kollagen zeigt Fetuin praktisch keinen Einfluss. Des Weiteren wurde das Nukleationsverhalten des häufigsten, nichtkollagenen Knochenproteins, dem Osteocalcin (OC), mittels ESI-MS beobachtet. Die Untersuchungen von Osteocalcin in Calciumphosphatlösung zeigten Komplexe mit bis zu 8 Ca2+, der größte identifizierbare Komplex bestand aus [OC Ca2 (PO4 )2 Na4 ]+. Um die Mineralisierung von Kollagen genauer zu untersuchen, wurden assemblierte Kollagenfibrillen in der Flüssigzelle eines Atomkraftmikroskops (AFM) mit Calciumphosphat nachmineralisiert. Hierbei wurde eine gleichmäßige Anlagerung der offenbar amorphen mineralischen Phase beobachtet. Die Inkubation der Fibrillen mit Phospholipidvesikeln führte zu einem Aufweichen der Fibrillen. Des Weiteren wurden Phospholipidvesikel hergestellt, um den Calciumphosphatniederschlag in einem räumlich stark begrenzten Abschnitt zu untersuchen. Die Vesikel wurden mit REM und AFM abgebildet und so verschiedene Präparationsmethoden verglichen. Es konnten plättchenförmige Kristallite an der Vesikelmembran gezüchtet werden, während bei Anwesenheit von Phosphoserin globuläre Objekte auftraten. Eine Arbeitshypothese wurde entwickelt, die das unterschiedliche Wachstumsverhalten von Calciumphosphat in wässriger Lösung mit einer positiv geladenen Hydrathülle um den Calciumphosphatkeim erklärt. Die Protonen stammen vom deprotonierten Phosphat des Mineralkeims und können sich auf Grund der adsorbierten Wassermoleküle nicht sofort in der Lösung verteilen. Diese Hülle aus H3O+ verhindert das beliebige Anlagern von Ionen an den Mineralkeim und lenkt so dessen Morphologie.

Cette thèse expérimentale s’inscrit dans le domaine des sciences séparatives et se base sur la technique de SPLITT (SPLIT-flow Thin fractionation). Son objectif consiste en l’étude des mécanismes qui sont à l’origine de la séparation, en continu et sans membrane, d’objets de taille micrométrique dans des mini-séparateurs fluidiques (Step-SPLITT). Les expériences menées, en laboratoire et lors de vols paraboliques, ont révélé le couplage complexe comme l’influence des effets hydrodynamiques et du champ gravitationnel sur la migration transverse des espèces en écoulement. Des visualisations tridimensionnelles par holographie digitale ont corroboré nos résultats et dévoilé des comportements inattendus. Les capacités séparatives des Step-SPLITT ont rendu possible l’analyse et la séparation d’objets biologiques et biomimétiques. Enfin, cette étude complétée par une modélisation tridimensionnelle de l’écoulement nous a permis de mettre au point un nouveau prototype de séparateur.<p><p>This experimental thesis belongs to the field of separative sciences and is based on the SPLITT technique (SPLIT-flow Thin fractionation). The objective is to study the mechanisms that are at the origin of continuous and membraneless separation of micron-size species in mini fluidic separators (Step-SPLITT). Experiments undertaken in laboratory and during parabolic flights revealed the complex coupling of the hydrodynamic effects and the gravitational field influencing the transverse migration of the flowing species. Three-dimensional visualizations performed by digital holography confirmed our results and disclosed unexpected behaviours. The separation capacities of Step-SPLITT made the analysis and the separation of biological and biomimetic species possible. In addition this study in conjunction with a three-dimensional flow modelling enabled us to develop a new prototype of separator. / Doctorat en sciences appliquées / info:eu-repo/semantics/nonPublished

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.

Der hierarchisch strukturierte und hoch geordnete Aufbau von Calciumphosphat und Kollagen in Knochen und Zähnen wird von den Zellen mit Hilfe bestimmter Moleküle erreicht. Diese organischen Moleküle, zumeist Proteine, beeinflussen durch die räumliche Anordnung ihrer Ladung das Präzipitations- und Wachstumsverhalten der mineralischen Phase. Die in dieser Arbeit beschriebenen Computersimulationen zeigen, dass ein Calciumphosphatkomplex mit deprotoniertem Phosphat am stabilsten ist. Vermutlich nimmt die Bindungsenergie pro Oberfläche des Komplexes mit wachsender Größe bis zu einem Ca9(PO4)6 -Komplex (Posner Klaster) linear zu. Die Präzipitation von Calciumphosphat aus wässriger Lösung führt häufig zu amorphen Kugeln mit 50-500 nm Durchmesser, die sphärische Unterstrukturen von ca. 5 nm Durchmesser zeigen und bei großer Dichte zu einer amorphen Schicht verschmelzen. Geringe Unterschiede in der Präparation können aber schon zu stäbchenförmigen oder plättchenartigen Kristalliten führen. Phosphoserin ist eine der wichtigsten Aminosäuren bei der Anbindung von Proteinen an Calciumphosphat. Das Computermodell zeigt an der gesamten Oberfläche dieser Aminosäure ein deutliches elektrisches Potential, dies begünstigt die Wechselwirkung mit Ionen. FT-IR- und NMR-Untersuchungen zeigen, dass Phosphoserin bei Kopräzipitation mit Calciumphosphat höchstwahrscheinlich in die mineralische Phase eingebaut wird. Serin zeigt bei der Kopräzipitation ab 1 mM einen Einfluss auf die Morphologie von Calciumphosphat, während Phosphoserin schon bei 0,01 mM einen deutlichen Einfluss zeigt. Elektronenspray-Ionisations-Massenspektroskopie (ESI-MS) bestätigt die relativ zum Serin intensivere Wechselwirkung von Phosphoserin mit Calciumphosphat. Das wichtigste Protein zur Vermeidung ektopischer Mineralisierung ist Fetuin. Dieses Protein stabilisiert die transient auftretenden amorphen Calciumphosphatkugeln (ACP-Kugeln) und erlaubt so dem Körper deren Entsorgung. Fetuin verhindert das Verschmelzen von ACP-Kugeln, wenn diese in großer Dichte auftreten, wobei deren feine Unterstruktur erhalten bleibt. Trotz des starken inhibitorischen Verhaltens wird das Auflösen von Brushit durch die Anwesenheit von Fetuin praktisch nicht beschleunigt. Auch auf die Kinetik der Assemblierung von Kollagen zeigt Fetuin praktisch keinen Einfluss. Des Weiteren wurde das Nukleationsverhalten des häufigsten, nichtkollagenen Knochenproteins, dem Osteocalcin (OC), mittels ESI-MS beobachtet. Die Untersuchungen von Osteocalcin in Calciumphosphatlösung zeigten Komplexe mit bis zu 8 Ca2+, der größte identifizierbare Komplex bestand aus [OC Ca2 (PO4 )2 Na4 ]+. Um die Mineralisierung von Kollagen genauer zu untersuchen, wurden assemblierte Kollagenfibrillen in der Flüssigzelle eines Atomkraftmikroskops (AFM) mit Calciumphosphat nachmineralisiert. Hierbei wurde eine gleichmäßige Anlagerung der offenbar amorphen mineralischen Phase beobachtet. Die Inkubation der Fibrillen mit Phospholipidvesikeln führte zu einem Aufweichen der Fibrillen. Des Weiteren wurden Phospholipidvesikel hergestellt, um den Calciumphosphatniederschlag in einem räumlich stark begrenzten Abschnitt zu untersuchen. Die Vesikel wurden mit REM und AFM abgebildet und so verschiedene Präparationsmethoden verglichen. Es konnten plättchenförmige Kristallite an der Vesikelmembran gezüchtet werden, während bei Anwesenheit von Phosphoserin globuläre Objekte auftraten. Eine Arbeitshypothese wurde entwickelt, die das unterschiedliche Wachstumsverhalten von Calciumphosphat in wässriger Lösung mit einer positiv geladenen Hydrathülle um den Calciumphosphatkeim erklärt. Die Protonen stammen vom deprotonierten Phosphat des Mineralkeims und können sich auf Grund der adsorbierten Wassermoleküle nicht sofort in der Lösung verteilen. Diese Hülle aus H3O+ verhindert das beliebige Anlagern von Ionen an den Mineralkeim und lenkt so dessen Morphologie.:1 Einführung 1.1 Biomineralisation 1.2 Calciumphosphat 1.3 Phosphoserin 1.4 Kollagen 1.5 Osteocalcin 1.6 Fetuin 1.7 Matrixvesikel 1.8 Fragestellung der Dissertation 2 Material und Methoden 2.1 Computermodellierung 2.2 Chemikalien und Lösungen 2.3 FT-IR-Messungen 2.4 UV/Vis-Messungen 2.5 Massenspektroskopische Experimente 2.6 REM 2.7 TEM 2.8 AFM 2.9 NMR 2.10 XRD 3 Ergebnisse und Interpretation 3.1 Calciumphosphat 3.2 Phosphoserin 3.3 Fetuin 3.4 Osteocalcin 3.5 Kollagen 3.6 Künstliche Vesikel 4 Abschließende Zusammenfassung Anhang Erläuterungen zu den Ergebnissen Glossar Abbildungsverzeichnis Tabellenverzeichnis Literaturverzeichnis Erklärung, Danke Publikationen Lebenslauf Index