Les liposomes sont des nanovecteurs polyvalents et prometteurs quant à leur utilisation dans plusieurs domaines. Il y a une décennie, un nouveau type de liposome constitué d’amphiphiles monoalkylés et de stérols est né fortuitement dans notre groupe. Ils sont nommés Stérosomes puisqu’ils contiennent une grande proportion de stérols, entre 50 et 70 mol %. Les objectifs de cette thèse sont de développer de nouvelles formulations de Stérosomes ayant des caractéristiques spécifiques et d’acquérir une compréhension plus profonde des règles physicochimiques qui dictent leur comportement de phase. Nous avons spécifiquement examiné le rôle de motifs moléculaires des stérols, de la charge interfaciale et de la capacité à former des liaisons H dans les interactions intermoléculaires menant à l’autoassemblage. Le comportement de phase a été caractérisé par calorimétrie différentielle à balayage (DSC), par spectroscopie infrarouge (IR) et par spectroscopie de résonance magnétique nucléaire du deutérium (²H NMR). Premièrement, nous avons établi certaines corrélations entre la structure des stérols, leur tendance à former des bicouches fluides en présence d'amphiphile monoalkylé et la perméabilité des grandes vésicules unilamellaires (LUV) formées. La nature des stérols module les propriétés de mélange avec de l’acide palmitique (PA). Les stérols portant une chaîne volumineuse en position C17 sont moins aptes à induire des bicouches fluides que ceux qui ont une chaîne plus simple, comme celle du cholestérol. Un grand ordre de la chaîne alkyle de PA est un effet commun à tous les stérols investigués. Il a été démontré que la perméabilité des LUV peut être contrôlée en utilisant des stérols différents. Cependant, ces stérols n’ont aucun impact significatif sur la sensibilité des Stérosomes au pH. Afin de créer des liposomes qui sont sensibles au pH et qui ont une charge positive à la surface, des Stérosomes composés de stéarylamine et de cholestérol (Chol) ont été conçus et caractérisés. Il a été conclu que l’état de protonation de l’amine, dans ce travail, ou du groupe carboxylique, dans un travail précédent, confère une sensibilité au pH et détermine la charge à la surface du liposome. Les premiers Stérosomes complètement neutres ont été fabriqués en utilisant un réseau de fortes liaisons H intermoléculaires. Le groupe sulfoxyde est capable de former de fortes liaisons H avec le cholestérol et les molécules d’eau. Une bicouche fluide métastable a été obtenue, à la température de la pièce, à partir d'un mélange équimolaire d’octadécyl méthyl sulfoxyde (OMSO) et de Chol. Ce comportement distinct a permis d’extruder le mélange pour former des LUV à la température de la pièce. Après 30 h, le temps de vie de la phase métastable, des Stérosomes stables et imperméables existaient toujours sous une forme solide. Un diagramme de température-composition a été proposé afin de résumer le comportement de phase des mélanges d’OMSO/Chol. Finalement, nous avons élaboré des Stérosomes furtifs en incorporant du polyéthylène glycol (PEG) avec une ancre de cholestérol (PEG-Chol) à l’interface de Stérosomes de PA/Chol. Jusqu’à 20 mol % de PEG-Chol peut être introduit sans perturber la structure de la bicouche. La présence du PEG-Chol n’a aucun impact significatif sur la perméabilité de la LUV. L'encapsulation active de la doxorubicine, un médicament contre le cancer, a été réalisée malgré la faible perméabilité de ces LUV et la présence du PEG à l’interface. L’inclusion de PEG a modifié considérablement les propriétés de l’interface et a diminué la libération induite par la variation de pH observée avec des LUV nues de PA/Chol. Cette formulation inédite est potentiellement utile pour l’administration intraveineuse de médicaments. / Liposomes are promising and versatile nanocarriers suitable for potential applications in many fields. A decade ago, a new type of liposomes formed from monoalkylated amphiphiles and sterols was born somehow fortuitously in our group. They are referred to as Sterosomes, because they contain a large proportion of sterols, between 50 and 70 mol %. The objectives of the present thesis are to develop novel Sterosome formulations with specific features, and to gain a deeper understanding of the physicochemical rules that dictate their phase behavior. We have specifically examined the role of the molecular features of sterols, of the interfacial charges and of the H-bond capacity in the intermolecular interactions leading to the self-assembly. The phase behavior was characterized by differential scanning calorimetry (DSC), infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy of deuterium (2H NMR). First, we have established some correlations between the structure of the sterols, the propensity to form fluid bilayers, and the permeability of the resulting large unilamellar vesicles (LUVs). The nature of the sterol modulates the properties of the mixture with palmitic acid (PA). Sterols bearing a bulky tail chain at C17 are less capable to induce fluid bilayers than those with a non-bulky tail chain, like that of cholesterol. A large ordering of the alkyl chain of PA is an effect exhibited by all of the investigated sterols. It is shown that the permeability of the LUVs can be controlled using different sterols. However, these sterols have no significant impact on the pH-sensitivity of Sterosomes. In order to create liposomes that are pH-sensitive and that have a positive surface charge, Sterosomes composed of stearylamine and cholesterol (Chol) were designed and characterized. It is concluded that the protonation/deprotonation state of the amine (in this work) and carboxylic acid (in previous work) groups confers the pH-sensitivity and determines the surface charge of the liposomes. The first completely neutral Sterosomes were crafted based on the creation of strong intermolecular hydrogen bond networks. The sulfoxide group was capable of forming strong hydrogen bonds with cholesterol and water molecules. In an equimolar octadecyl methyl sulfoxide (OMSO)/Chol mixture, a metastable fluid bilayer was obtained at room temperature. This distinct phase behavior allowed extruding the mixtures to form LUVs at room temperature. After 30 h, the life-time of the metastable phase, stable and impermeable Sterosomes still existed in the solid form. A temperature–composition diagram was proposed to summarize the phase behavior of OMSO/Chol mixtures. Finally, a further step was made to prepare “stealth” Sterosomes by incorporating polyethylene glycol (PEG) with a cholesterol anchor (PEG-Chol) at the interface of PA/Chol Sterosomes. Up to 20 mol % PEG-Chol can be introduced without disturbing the bilayer structure. The presence of PEG-Chol had no significant impact on the permeability of the resulting LUVs. Active-loading of an anti-cancer drug, doxorubicin, can be achieved despite the low permeability of these LUVs and the presence of the PEG at the interface. The inclusion of PEG modified considerably the interface properties and decreased significantly the pH-triggered release observed with naked PA/Chol LUVs. This novel formulation is potentially useful for the application of intravenous administration in the drug delivery field.

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Pat. mit Multipler Sklerose haben ein erhöhtes Risiko, einen schweren Verlauf einer SARS- CoV-2 Infektion zu entwickeln. Durch das fehlregulierte Immunsystem und durch die mit dem Krankheitsbild verbundenen vielen körperlichen Einschränkungen treten bei MS Pat. häufiger schwere Infektionen auf. Eine Anti-CD20-Antikörpertherapie depletiert vor allem B-Zellen, zu einem geringen Teil auch T-Zellen. Mit dieser immunsupprimierenden Therapie haben Pat. ein nochmals erhöhtes Infektionsrisiko. Zur B-Zell depletierenden Therapie (BZDT) werden aktuell Ocrelizumab, Ofatumumab und Rituximab verwendet. Durch eine Impfung könnten die MS Pat. mit BZDT vor einer SARS-CoV-2 Infektion geschützt werden bzw. ein schwerer Verlauf durch sie verhindert werden. Die Beurteilung der Effektivität einer Impfung kann durch die Analyse der B-Zell spezifischen und T-Zell spezifischen Impfantwort untersucht werden. Ziel der Studie ist es, herauszufinden, ob Pat. trotz der MS-Erkrankung und einer Anti-CD20 Antikörpertherapie eine Impfantwort entwickeln und ob die Pat. dadurch von einer SARS- CoV2-Impfung profitieren.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1. Einleitung 1.1 Multiple Sklerose 1.1.1 Epidemiologie 1.1.2 Ätiologie und Risikofaktoren 1.1.3 Pathophysiologie 1.1.4 Symptome 1.1.5 Verlaufsformen 1.1.6 Diagnostik 1.1.7 Prognosefaktoren 1.1.8 Therapie 1.2 SARS-CoV-2 1.2.1 Epidemiologie 1.2.2 Ätiologie und Risikofaktoren 1.2.3 Infektion und ihre Immunantwort 1.2.4 Impfung 2. Fragestellung und Zielsetzung 3. Material und Methode 3.1 Einschlusskriterien 3.2 Methode 3.2.1 B-Zell Antwort 3.2.2 T-Zell Antwort 3.3 Statistik 3.4 Pat.kollektiv 4. Ergebnisse 4.1 Ergebnisse nach der Grundimmunisierung 4.1.1 B- und T-Zell Antwort nach der Grundimmunisierung 4.1.2 Einfluss einer SARS-CoV-2-Infektion auf die B- und T-Zell Antwort nach der Grundimmunisierung 4.2 Ergebnisse nach der Booster Impfung 4.2.1 B- und T-Zell Antwort nach der Booster Impfung 4.2.2 Einfluss einer SARS-CoV-2-Infektion auf die B- und T-Zell Antwort nach der Booster Impfung 4.3 SARS-CoV-2 spezifische B- und T-Zell Antwort im Verlauf 4.4 Bedeutung des zeitlichen Beginns einer BZDT auf die SARS-CoV-2 spezifische Impfantwort 5. Diskussion 6. Zusammenfassung 7. Summary 8. Literaturverzeichnis 9. Danksagung 10. Anhang (einschließlich Originalpublikationen) / Patients with multiple sclerosis generally face an elevated risk of experiencing a severe course of SARS-CoV-2 infection. This heightened susceptibility results from the dysregulated immune system and various physical limitations associated with the disease. Anti-CD20 therapy primarily leads to B-cell depletion and, to a lesser extent, T-cell depletion. Consequently, patients undergoing BCDT, which includes ocrelizumab, ofatumumab, and rituximab, are at an even greater risk of infection due to this immunosuppressive therapy. Vaccination emerges crucial of protecting MS patients undergoing BCDT from SARS-CoV-2 infection or mitigating the severity of the disease. The effectiveness of a vaccination can be assessed by analyzing the B-cell specific and T-cell specific vaccination response. The study aims to ascertain whether patients with MS, despite the disease and anti-CD20 antibody therapy, develop a vaccination response and whether they indeed benefit from a SARS-CoV-2 vaccination.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1. Einleitung 1.1 Multiple Sklerose 1.1.1 Epidemiologie 1.1.2 Ätiologie und Risikofaktoren 1.1.3 Pathophysiologie 1.1.4 Symptome 1.1.5 Verlaufsformen 1.1.6 Diagnostik 1.1.7 Prognosefaktoren 1.1.8 Therapie 1.2 SARS-CoV-2 1.2.1 Epidemiologie 1.2.2 Ätiologie und Risikofaktoren 1.2.3 Infektion und ihre Immunantwort 1.2.4 Impfung 2. Fragestellung und Zielsetzung 3. Material und Methode 3.1 Einschlusskriterien 3.2 Methode 3.2.1 B-Zell Antwort 3.2.2 T-Zell Antwort 3.3 Statistik 3.4 Pat.kollektiv 4. Ergebnisse 4.1 Ergebnisse nach der Grundimmunisierung 4.1.1 B- und T-Zell Antwort nach der Grundimmunisierung 4.1.2 Einfluss einer SARS-CoV-2-Infektion auf die B- und T-Zell Antwort nach der Grundimmunisierung 4.2 Ergebnisse nach der Booster Impfung 4.2.1 B- und T-Zell Antwort nach der Booster Impfung 4.2.2 Einfluss einer SARS-CoV-2-Infektion auf die B- und T-Zell Antwort nach der Booster Impfung 4.3 SARS-CoV-2 spezifische B- und T-Zell Antwort im Verlauf 4.4 Bedeutung des zeitlichen Beginns einer BZDT auf die SARS-CoV-2 spezifische Impfantwort 5. Diskussion 6. Zusammenfassung 7. Summary 8. Literaturverzeichnis 9. Danksagung 10. Anhang (einschließlich Originalpublikationen)

Master of Science / Department of Agronomy / David B. Mengel / Nitrogen fertilizers play an essential role in agricultural production in Kansas, particularly in row crops such as corn (Zea mays L.) and grain sorghum (Sorghum bicolor (L.) Moench). A good portion of the corn and grain sorghum grown in Kansas is typically grown using no-till production systems. These systems leave a large amount of surface residue on the soil surface, which can lead to ammonia volatilization losses from surface applied urea-containing fertilizers and immobilization of N fertilizers placed in contact with the residue. Leaching and denitrification can also be a problem on some soils. Current nitrogen prices, as well as concerns over environmental stewardship, are forcing producers to make smarter choices in the fertilizer products used as well as when and how the materials are applied, to optimize their nitrogen use efficiency. A common practice throughout Kansas is to apply N fertilizers prior to planting, sometimes up to 6 month prior to planting. What affect does this practice have on nitrogen availability to the growing crop? Current Kansas State University (KSU) soil test fertilizer recommendations assume 50% nitrogen use efficiency. This means of every pound of nitrogen applied only half will be utilized by the plant and turned into valuable grain. Possible solutions to help increase nitrogen use efficiency are the use of nitrogen additives which are currently on the market and claim to reduce nitrogen loss through denitrification and volatilization as well as the use of timing and application of fertilizers to further increase nitrogen use efficiency. The objective of this study is to evaluate different N fertilizer products, as well as additives and application practices and determine whether specific combinations can improve yield and N use efficiency of no-till corn and grain sorghum. The long-term goal of this study is to quantify some of these relationships to assist farmers in selecting specific combinations that could enhance yield and profitability. In this study five tools for preventing N loss were examined: fertilizer placement, or placing N below the soil surface or in bands on the residue-covered soil surface to reduce immobilization and/or volatilization; use of a urease inhibitor Agrotain (NBPT) that blocks the urease hydrolysis reaction that converts urea to ammonia and potentially could reduce ammonia volatilization; the use of a commercially available additive, Agrotain Plus, that contains both a nitrification inhibitor (DCD) and a urease inhibitor to slow both urea hydrolysis and the rate of ammonium conversion to nitrate and subsequent denitrification or leaching loss; use of a commercial product NutriSphere-N, which claims urease and nitrification inhibition; and the use of a polyurethane plastic-coated urea to delay release of urea fertilizer until the crop can use it. The ultimate goal of using these practices or products is to increase N uptake by the plant and enhance yield. An important measurement that was developed for this research was the use of a greenleaf firing index which used the number of green leaves below the ear at pollination as a key measurement in determining the effectiveness of fertilizer placement, application method, application timing and the use of nitrogen additives. If significant differences in lower leaf nitrogen stress are found, the potential exists to further develop this index and correlate differences observed with key parameters of nitrogen uptake such as ear-leaf nitrogen concentration, total nitrogen uptake and grain yield. Results observed from this research show that the potential to increase nitrogen use efficiency and reduce nitrogen loss do exist with the use of certain nitrogen additives, application methods and application timing. When conditions are conducive for nitrogen loss the use of currently available tools to protect nitrogen from volatilization, immobilization and/or denitrification loss significantly increased yields in the corn experiments. Results from the grain sorghum research indicate that when N losses limit yield, the use of products and practices enhance yield. In locations where nitrogen loss is minimal or low yields limit nitrogen response, the use of these practices was not found to be helpful.

Well-regulated Ca2+ signalling is essential for every living organism, and disruption of this signalling can lead to diseases including heart failure, neurological disorders and diabetes. Intracellular Ca2+ levels are regulated by influx of extracellular Ca2+ through channels located in the cell membrane. In addition, release of Ca2+ from intracellular stores also plays an important role in controlling intracellular Ca2+ concentration. Of the three types of intracellular Ca2+ stores that have been characterised those with D-myo-Inositol 1,4,5 trisphosphate receptors (InsP3Rs) showed a close relationship with cell proliferation. Hence, selective blockage of InsP3Rs will allow better understanding of Ca2+ signalling and might also unveil novel treatment for cancers, in the long term. There were no selective InsP3Rs antagonists known at the start of these studies. Based on the crystal structure of InsP3Rs bound to InsP3 and SAR studies of InsP3, we designed and tested several InsP3 analogues.1 Compound 15, 16 and 23 acted as InsP3R antagonists, though their selectivity for InsP3Rs was not completely determined. Furthermore, we also attempted to improve the potency of 16 via substitution at the 1-postion phosphate. By considering the interaction formed between adenophosphostins and InsP3Rs compounds (53-55) were designed and synthesised. In addition, analogues of compound 92, selected from an in silico screen, have led to the discovery of another novel scaffold that acts as an InsP3R antagonist.

The applications of membranes are increasing rapidly in various fields of engineering and science. The geometric, material, force and contact non-linearities complicate their analysis, which increases the demand for computationally efficient methods and interpretation of counter-intuitive behaviours. The first part of the present work studies the free and constrained inflation of circular and cylindrical membranes. The membranes are assumed to be in contact with a soft substrate, modelled as a linear spring distribution.Adhesive and frictionless contact conditions are considered during inflation,while only adhesive contact conditions are considered during deflation. For a circular membrane, peeling of the membrane during deflation is studied, and a numerical formulation of the energy release rate is proposed. The second part of the thesis discusses the instabilities observed for fluid containing cylindrical membranes. Limit points and bifurcation points are observed on primary equilibrium branches. The secondary branches emerge from bifurcation points, with their directions determined by eigenvectors corresponding to zero eigenvalues at the bifurcation point. Symmetry has major implications on stability analysis of the structures, and the relationship between eigenvalue analysis and symmetry is highlighted in this part of the thesis. In the third part, wrinkling in the pressurized membranes is investigated,and robustness of the modified membrane theory and tension field theory is examined. The effect of boundary conditions, thickness variations, and inflating media on the wrinkling is investigated. It is observed that, with a relaxed strain energy formulation, the obtained equilibrium solutions are unstable due to the occurrence of pressure induced instabilities. A detailed analysis of pressure induced instabilities in the wrinkled membranes is described in the thesis. / <p>QC 20160518</p>

Liposomal delivery systems hold considerable promise for improvement of cancer therapy provided that critical formulation design criteria can be met. The main objective of the current project was to enable quality by design in the formulation of liposomal delivery systems by developing comprehensive, mechanism-based mathematical models of drug loading, binding and release kinetics that take into account not only the therapeutic requirement but the physicochemical properties of the drug, the bilayer membrane, and the intraliposomal microenvironment. Membrane binding of the drug affects both drug loading and release from liposomes. The influence of bilayer composition and phase structure on the partitioning behavior of a model non-polar drug, dexamethasone, and its water soluble prodrug, dexamethasone phosphate, was evaluated. Consequently, a quantitative dependence of the partition coefficient on the free surface area of the bilayer, a property related to acyl chain ordering, was noted. The efficacy of liposomal formulations is critically dependent on the drug release rates from liposomes. However, various formulation efforts to design optimal release rates are futile without a validated characterization method. The pitfalls of the commonly used dynamic dialysis method for determination of apparent release kinetics from nanoparticles were highlighted along with the experimental and mathematical approaches to overcome them. The value of using mechanism-based models to obtain the actual rate constant for nanoparticle release was demonstrated. A novel method to improve liposomal loading of poorly soluble ionizable drugs using supersaturated drug solutions was developed using the model drug AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin), a poorly soluble camptothecin analogue. Enhanced loading with a drug to lipid ratio of 0.17 was achieved and the rate and extent of loading was explained by a mathematical model that took into account the chemical equilibria inside and outside the vesicles and the transport kinetics of various permeable species across the lipid bilayer and the dialysis membrane. Tunable liposomal release kinetics would be highly desirable to meet the varying therapeutic requirements. A large range of liposome release half-lives from 1 hr to 892 hr were obtained by modulation of intraliposomal pH and lipid composition using dexamethasone phosphate as a model ionizable drug. The mathematical models developed were successful in accounting for the change in apparent permeability with change in intraliposomal pH and bilayer free surface area. This work demonstrates the critical role of mechanism-based models in design of liposomal formulations.

Molecular association is part of many important processes in living cells. Computational methods for calculating binding free energies allows for a quantitative examination of biomolecular structures and hypotheses drawn from biochemical experiments. Here, binding free energy calculations for tRNAs and release factors binding to mRNA codons on the ribosome, sugars binding to lectins and purine analogs binding to the purine riboswitch are presented. The relative affinities between cognate and non-cognate tRNAs for different states involved in codon reading on the ribosome were determined. The calculations show that tRNA discrimination varies between different conformations of the 30S subunit, where the existence of both low and high selectivity states provides an efficient common mechanism for initial selection and proofreading. The simulations reveal a desolvation mechanism for the 30S conformational switch with which the accuracy of peptide bond formation can be amplified. When an mRNA stop codon (UAA, UAG or UGA) is located in the ribosomal A-site release factors bind to the ribosome and the synthesized protein is released. RF1 is specific for UAA and UAG whereas RF2 is specific for UAA and UGA. The free energy calculations and an analysis of the performed simulations show the mechanisms for how RF1 and RF2 are able to read the stop codons with different specificities. Also mitochondrial release factors were investigated. Vertebrate mitochondria have four stop codons, UAA, UAG, AGA and AGG and two release factors mtRF1 and mtRF1a. The calculations show how the specificities of both mtRF1 and mtRF1a agree with RF1 and that none of them are likely to read the non-standard stop codons AGA and AGG. The linear interaction energy method has also been examined for the RSL and PA-IIL lectins and for the purine riboswitch. The standard parameterization of the method works well for RSL, but fails for PA-IIL and the purine riboswitch due to compositions of the active sites in these systems. The development of new parameterizations to overcome these problems leads to a better understanding of both the method and the binding mechanisms in these systems.

Inflammatory processes are infamous for their destructive effects on tissues and joints in a variety of diseases. Within the body, inflammation is a highly regulated biological response whose purpose is to promote tissue regeneration following injury. However, in certain settings, inflammation persists and leads to progressive tissue destruction. This thesis focused on modulating inflammatory signaling in both contexts. Part I investigated the effects of a model pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), on the in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). In contrast, Part II describes the development and in vivo evaluation of the first intra-articular controlled release system for the temporomandibular joint (TMJ), which silences inflammatory signaling and thus mitigates the painful joint damage seen in inflammatory TMJ disease. The following specific aims were addressed: (1) to determine the concentration of TNF-α that enhances in vitro osteogenic differentiation of MSCs; (2) to determine the temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (3) to determine the impact of bone-like extracellular matrix (ECM) on the concentration and temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (4) to evaluate the biocompatibility of intra-articular microparticles in the rat TMJ; (5) to develop a microparticle-based formulation for sustained release of a model anti-inflammatory small interfering ribonucleic acid (siRNA); and (6) to evaluate the therapeutic efficacy of intra-articular microparticles delivering siRNA in an animal model of TMJ inflammation. These studies led to the development of powerful strategies to rationally control inflammation to promote bone regeneration and mitigate joint damage in the setting of disease, both of which will ultimately improve the quality and specificity of therapies available in modern medicine. / Only volume 2 has been digitized.