Thesis advisor: Thomas C. Chiles / For ten weeks during the summer of 2005, I was a Summer Undergraduate Research Fellow in an immunology laboratory at the Mayo Clinic. My research focused on the BLyS/APRIL system and the receptor TACI on the surface of B cells. Going into my summer research, I had very little experience in immunology. Throughout the process of writing this thesis, I have sought to improve upon my knowledge of immunology, building a cohesive story that begins with basic biology and ends with the results of the summer's experiments. The first part of this thesis covers topics in general immunology, and narrow down in focus to cover the function and development of lymphocytes and B cell maturation and activation. In the second part of the thesis, the background for my research is described in more detail, and topics such as autoimmunity and cancer, lipid rafts, cell polarization, the BLyS/APRIL system for B cell survival, and TACI are covered extensively. The final portion of this thesis discusses the experimental logic, a background on materials and methods, and the results of the experiments I conducted over the summer. By reading this thesis, anyone with a background in biology should become familiar with basic subjects in immunology, advanced concepts in the study of lymphocytes, the ligands BLyS and APRIL, and the receptor TACI in B cells. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program.

Thesis advisor: Thomas Seyfried / Lipids, in all their forms from structural components of the membranes (phosphoglycerides, glycolglycerolipids) to signaling molecules (IP3, DAG, prostaglandins, etc.,) post-translational modification of proteins (palmitoylated, farnesylated, prenylated, and GPI anchoring) play an essential role in cancer cell survival, proliferation, and metastasis. Alteration in structural lipids can impair transport, and signaling cascades. Abnormalities in lipids, such as cardiolipin (Ptd2Gro), impair mitochondrial function, bioenergetics, and could play a role in precipitatting the high incidence of spontaneous tumors in VM/Dk mice. This thesis explores the role of glucose and glutamine in their incorporation into lipids in the VM-M3 murine glioblastoma cell line as well as the inheritance of brain cardiolipin fatty acids abnormalities in VM/Dk mice. I used labeled [14C]-U-D-glucose and [14C]-U-L-glutamine to examine the profile of de novo lipid biosynthesis in the VM-M3 cell line. The major lipids synthesized included phosphatidylcholine (PtdCho), phosphatidylethanolamine (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin (CerPCho), bis(monoacylglycero)phosphate (BMP) / phosphatidic acid (PtdOH), cholesterol (C), Ptd2Gro, and the gangliosides. The data show that the incorporation of labeled glucose and glutamine into synthesized lipids was dependent on the type of growth environment, and that the VM-M3 glioblastoma cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. In addition, this thesis also explores and evaluates the abnormality of Ptd2Gro fatty acid composition in VM mice in comparison to B6 mice. Although previously reported, I confirmed the finding in the abnormal cardiolipin fatty acid composition in the VM mice. The abnormal brain cardiolipin fatty acid composition was found to be inherited as an autosomal dominant trait in reciprocal B6 x VM F1 hybrids for both male and female. Impaired cognitive awareness under hypoxia observed for the VM mice and reciprocal F1 hybrids is associated with abnormalities in neural lipid composition. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A associação lipídio/citocromo c é importante e deve ser estudada, pois repercute na atividade peroxidática da proteína abordada e pode contribuir para o processo apoptótico, ou morte programada da célula, e também desempenha um papel significativo na cadeia respiratória. A natureza e a especificidade da interação do citocromo c com bicamadas lipídicas têm sido bastante investigadas ultimamente, mas informações detalhadas e precisas sobre tais assuntos ainda não existem. É aceito que ocorre primeiramente uma interação eletrostática entre a proteína citocromo c e as membranas fosfolipídicas. Em seguida, há uma interação hidrofóbica. Entretanto, ainda não é bem compreendido o papel da cadeia fosfolipídica. A associação do citocromo c com membranas lipídicas induz mudanças no estado de spin do átomo de ferro. A interação entre as vesículas carregadas e o citocromo c induz mudanças estruturais na proteína, as quais são refletidas no seu centro ativo, ou grupo heme. As mudanças do campo cristalino no sítio do ferro hemínico de forte para fraco são acompanhadas por mudanças do estado de spin de baixo para alto, respectivamente. Neste trabalho, estuda-se sistematicamente a natureza da interação entre o citocromo c e a cadeia fosfolipídica. As mudanças estruturais no grupo heme foram correlacionadas com a natureza do lipídio, ou seja, com a carga da cabeça e com o tamanho e o tipo da cadeia fosfolipídica. Foram utilizados treze lipídios diferentes, naturais e sintetizados, com cabeças polares negativas e neutras e com cadeias carbônicas saturadas e insaturadas de diferentes comprimentos. Para tal investigação, utilizamos as técnicas: Ressonância Paramagnética Eletrônica (RPE) Onda Contínua (CW) e Pulsada (PW) e Dicroísmo Circular Magnético (MCD). As técnicas enunciadas avaliam as mudanças de estado de spin e a simetria do citocromo c nos seus estados férrico e ferroso. A interação lipoprotéica lipídio/citocromo c foi avaliada com lipídios diferentes, inclusive com o lipossomo PCPECL, que mimetiza a membrana interna da mitocôndria nos eucariontes. A partir dos resultados experimentais, sugerimos um modelo para esse tipo de associação. / This association lipid/cytochrome c is interesting to study in order to understand the peroxidase activity of this protein, that plays an important role in the respiratory chain and in the apoptosis process or the programmed cell death. The nature and specificity of the interaction of cytochrome c with lipid bilayers have been major goals in recent studies, but detailed information on that issue is not yet widely available. In this regard, it is generally accepted that the electrostatic interaction is an important factor in the association of cytochrome c with phospholipid membranes, followed by a hydrophobic interaction. However, the role played by the phospholipid chain is not well understood. The association of cytochrome c with negative membranes induces a change in the heme iron spin state. The interaction between the charged vesicles and cytochrome c leads to structural changes in the active central or heme group. The changing of the crystalline field of the heme iron from strong to weak is accompanied by spin states changes from low to high spin, respectively. These facts concerned us to investigate more systematically the nature of the interaction between cytochrome c and the phospholipid chains. The lipid-induced effects in the heme iron crystalline field are correlated to the nature of the charged head group and to the size and type of the phospholipid chain. Thirteen different lipids, nature and synthetic, were used, with negative and neutra1 polar head group and saturated and unsaturated acyl chains with different length. This work investigates the change of heme iron spin state and symmetry of ferric cytochrome c using Continuous Wave (CW) and pulsed (PW) Electron Paramagnetic Resonance (EPR) and Magnetic Circular Dichroism (MCD) techniques. These techniques analyze the spin state change and the symmetry of the iron cytochrome c in its ferric and ferrous states. The effect of the different lipids were analyzed, including PCPECL membrane that mimetics the inner mitocondrial membrane in eukaryotes.

Foram estudadas, quanto ao perfil lipídico, duas populações, compreendendo indivíduos acima dos 20 anos de idade: -trabalhadores da Termomecânica, indústria metalúrgica de São Bernardo do Campo: 1.616 do sexo masculino e 230 do sexo feminino; -pacientes atendidos no ambulatório do Hospital das Clínicas da Universidade Estadual de Campinas (UNICAMP): 664 do sexo feminino e 317 do sexo masculino. Os objetivos foram: I. Avaliar se a determinação isolada da colesterolemia total, sem considerar a dos triglicérides e a do HDL-colesterol e o cálculo do LDL-colesterol pela fórmula de Friedewald, para rastreamento populacional ou para decisão de tratamento, implica importante porcentagem de aparecimento de casos classificados inadequadamente, considerando o LDL-colesterol como \"padrão ouro\". 2. Analisar se a determinação da trigliceridemia associada à da colesterolemia total, além de mostrar os casos que apresentam hipertrigliceridemia isolada, auxilia a melhor identificação dos casos quanto ao LDL-colesterol. 3. Avaliar, com base na determinação isolada da colesterolemia total, qual a chance de os pacientes, por não lerem seu HDL-colesterol dosado, estar em maior risco lipídico para doença ateroselerótica por apresentar essa fração do colesterol abaixo do desejável. As conclusões foram: I. A dosagem isolada do colesterol total pode levar a chance importante de classificar enganosamente um paciente (em relação ao LDL-colesterol calculado). Para o rastreamento, os falsos negativos variaram de 9 por cento a 24 por cento e os falsos positivos, de 10 por cento a 24 por cento. Para a situação de decisão de tratamento, a variação dos falsos negativos foi de 14 por cento a 25 por cento, enquanto a dos falsos positivos foi de 1 por cento a 9 por cento. 2. A determinação dos triglicérides associada à do colesterol total auxilia a melhor identificação dos casos que apresentam LDL-colesterol elevado, somente quando a colesterolemia se encontra abaixo ue 240 mg/dl. Para níveis de colesterol totai iguais ou acima desse valor, a determinação também dos triglicérides não auxilia a melhor identificação do risco lipídico pelo LDL-colesterol ou a necessidade de tratamento dessa variável do perfil lipídico. A não possibilidade do diagnóstico de hipertrigliceridemia isolada (acima de 200 mg/dl) quando da determinação somente do colesterol total apresentou baixas chances nas populações avaliadas neste estudo, de 0,5 por cento a 3 por cento. 3. A não determinação do HDL-colesterol associa risco de erro pequeno de não identificação de paciente com risco de doença coronária por nível baixo dessa fração do colesterol (abaixo de 35 mg/dl), com colesterol total em níveis desejáveis (abaixo de 200 mg/dl), que variou de 3 por cento a 10 por cento. / Lipid profile of two populations composed of individuais older than 20 years, was studied: - metallurgic employees from São Bernardo do Campo (Termomecânica): 1,616 males and 230 females; - outpatients seen in the Hospital das Clínicas from Campinas State University (UNICAMP): 664 females and 317 males. The objectives were: I. To evaluate if the isolated determination of serum total cholesterol, without triglyceridcs, HDL-cholesterol and calculation of LDL-cholesterol by Friedewald\'s formula, for population screening or treatement decision implicates in high percentage of misclassification, considering LDL-cholesterol as the gold standard. 2. To analyze if triglycerides associated to total cholesterol determination helps in better identification of cases according to LDL-cholesterol, parallel to the definition of the percentage of cases presenting isolated hypertriglyceridemia. 3. To evaluate what is the chance of, according to isolated total choleslerol determination, patients being misclassified due a low HDL-cholesterol. The conclusions were: I. Isolated determination of total cholesterol can induce to an important chance of misclassifying a patient (according to calculated LDL-cholesterol). For screening, false negatives ranged from 9 per cent to 24 per cent and false positives from 10 per cent to 24 per cent. For treatment decision situation, false negatives varied from 14 per cent to 25 per cent, while false positives varied from 1 per cent to 9 per cent. 2. Triglycerides determination associated to total cholesterol helps in better identification of cases presenting elevated LDL-cholesterol, only when the cholesterolemia is below 240 mg/dL. For total cholesterol equal or above this value, triglycerides determination does not improve better identification of lipid risk depending on LDL-cholesterol during screening or treatment imposition. The impossibility of diagnosing isolated hypertriglyeeridemia (above 200 mg/dL) when only total cholesterol determination is done present low chance in both studied populations varying from 0.5 per cent to 3 per cent. 3. In a patient presenting a desirable total cholesterol level (below 200 mg/dL), not dosing HDL-cholesterol associates low risk of misclassification depending on a low level or this lipoprotein fraction (below 35 mg/dL): from 3 per cent to 10 per cent.

by Hui Yee Han, Ellen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 68-71). / acknowledgements --- p.i / abstract --- p.ii / table of contents --- p.iii-vi / list of figures --- p.vii / LIST OF TABLES --- p.viii / chapter / Chapter chapter i : --- introduction --- p.1 / Chapter chapter ii : --- background knowledge --- p.2 -33 / Chapter 2.1 --- Definition of Free Radical --- p.2 / Chapter 2.2 --- Oxygen Derived Radicals and Their Generation In Vivo --- p.2 -9 / Chapter 2.2.1 --- Production of Singlet Oxygen --- p.4 / Chapter 2.2.2 --- Production of Superoxide Radical (O2-) and Hydrogen Peroxide (H2O2) --- p.4 -8 / Chapter I. --- Endogenous Production --- p.4-7 / Chapter II. --- Exogenous Production --- p.7-8 / Chapter 2.2.3 --- Generation of Hydroxy Radical (OH) through H2O2 and O2- --- p.8 -9 / Chapter 2.3 --- Free Radical Damage and Lipid Peroxidation --- p.9 -13 / Chapter 2.4 --- Lipid Peroxidation and Atherosclerosis --- p.13 -14 / Chapter 2.5 --- Antioxidant --- p.15-21 / Chapter 2.5.1 --- Primary Preventive Antioxidants --- p.15-17 / Chapter 2.5.2 --- Secondary Radical Scavenging Antioxidants --- p.18-21 / Chapter I. --- Lipid Soluble Radical-Scavenging Antioxidants --- p.18-19 / Chapter II. --- Water Soluble Radical-Scavenging Antioxidants --- p.20 -21 / Chapter 2.6 --- Measurement of Oxygen-Derived Radical in Vivo --- p.21 -26 / Chapter 2.7 --- Principle of the TRAP assay --- p.27-33 / Chapter 2.7.1 --- Oxygen Consumption Method --- p.29 -30 / Chapter 2.7.2 --- Chemiluminescence Method --- p.31-33 / Chapter chapter III： --- materials and methods --- p.34 -43 / Chapter 3.1 --- Instrumentation and Materials --- p.34 / Chapter 3.2 --- Method --- p.34-43 / Chapter 3.2.1 --- Establishment of Chemiluminescence Method for Determination of TRAP --- p.34 -36 / Chapter I. --- Preparation of Luminometer --- p.35 / Chapter II. --- Preparation of Sample before Analysis --- p.3 5 / Chapter III. --- Manual Operation of the Chemiluminescence Method --- p.35-36 / Chapter IV. --- Calculation of TRAP --- p.36 / Chapter 3.2.2 --- Evaluation of the Chemiluminescence Method --- p.37 -42 / Chapter I. --- Linearity --- p.37 / Chapter II. --- Recovery --- p.37-38 / Chapter III. --- Precision --- p.39 / Chapter IV. --- Interference Experiment --- p.39 -41 / Chapter V. --- Effect of Storage on TRAP --- p.42 / Chapter 3.2.3 --- "Determination of Analytical, Intraindividual and Interindividual Biological Variation of TRAP in A Group of Healthy Chinese" --- p.42-43 / Chapter CHAPTER IV: --- ANALYTICAL RESULTS --- p.44-56 / Chapter 4.1 --- Method Evaluation --- p.44-51 / Chapter 4.1.1 --- Linearity --- p.44 / Chapter 4.1.2 --- Recovery --- p.45 / Chapter 4.1.3 --- Within-Day and Between-Day Precision --- p.46-47 / Chapter 4.1.4 --- Interference --- p.47-48 / Chapter 4.1.5 --- Effect of Storage on TRAP --- p.48-51 / Chapter 4.2 --- "Analytical, Intraindividual and Interindividual Variation of TRAP in A Group of Healthy Chinese Population" --- p.52 -56 / Chapter 4.2.1 --- Difference in TRAP value obtained from the 22 subjects over time --- p.52 -54 / Chapter 4.2.2 --- The Effect of Genders on Trap --- p.55 / Chapter 4.2.3 --- "Determination of Analytical, Intraindividual and Interindividual Variation of TRAP in A Group of Healthy Chinese" --- p.55-56 / Chapter CHAPTER V: --- DISCUSSION --- p.57-67 / Chapter 5.1 --- Validation of the Method Performance --- p.57 / Chapter 5.2 --- Effect of Storage on TRAP --- p.57 / Chapter 5.3 --- Interference of Hemolysis and Lipemia on TRAP assay --- p.58-60 / Chapter 5.3.1 --- Effect of Hemolysis on TRAP --- p.58-59 / Chapter 5.3.2 --- Effect of Lipemia on TRAP --- p.59-60 / Chapter 5.4 --- Possible Sources of Variation in TRAP Over Time --- p.60 -63 / Chapter 5.5 --- Usefulness of the Variation Data of TRAP obtained from a Group of Healthy Chinese --- p.64 -67 / reference --- p.68 -71

No description available.

Bacterial infections are and likely always will be a serious and costly complication to treatment in a healthcare environment. However consistent rises in the number of both healthcare associated and antibiotic resistant infections over the last of decades has the potential to turn a serious problem into a catastrophe. Control of infections in hospital wards has improved over the last five years but data from the European Centre for Disease Prevention and Control suggests a stale mate. While the numerical rise in drug resistant organisms has slowed, the severity of drug resistance appears to be on the increase with the prolific emergence of multiple drug resistant isolates. On the front lines of the threat that these organisms represent are some of the most susceptible. In hospitals those who are already sick are more vulnerable, those with co-morbidities, those with surgical or other wounds, the very old and the very young. Children especially show high susceptibility as they are often incapable of communicating clinical complications in the way an adult might. This coupled with higher commonality of specific aetiologies in children such as scalds, open wounds that are prone to infection without proper treatment, creates population in need. Antibiotics are often thought to be part of the problem in drug resistance, indeed to an extent they are. However their real downfall may be improper use. In order to improve treatment outcomes and simultaneously decrease antimicrobial resistance a combination of rapid diagnosis and prophylaxis can be utilised to decrease selection of resistance. As such, this study focuses on the development of a novel vesicle based sensor system for the detection of bacterial infections in burn wounds. Additionally an organometallic antimicrobial system has been developed with the potential for surface attachment. Work with the vesicle based biosensor demonstrates high sensitivity to both Staphylococcus aureus and Pseudomonas aeruginosa. The toxins involved in activation of the sensor have been determined in both cases and an in-depth study into the activity of the staphylococcal agents of lysis (Phenol Soluble Modulins and delta haemolysin), shows a high degree of plasticity and tunability in the sensors function. Work with the zinc based antimicrobial reveals a highly complex system which demonstrates possible functions as a not only an antimicrobial but as a sensor system in its own right.

Due to the escalating challenge of antibiotic resistance in bacteria over the past several decades, interest in the identification and development of antibiotic alternatives has intensified. Antimicrobial peptides (AMPs), which serve as part of the innate immune systems of most eukaryotic organisms, are being researched extensively as potential alternatives. However, the mechanism behind their bactericidal capabilities is not well understood. Previous studies have suggested that AMPs may first attach to the cell membranes, leading to pore formation caused by peptide insertion, lipid removal in the form of peptide-lipid aggregates, or a combination of both mechanisms. In addition to the lack of mechanistic knowledge, a significant hurdle in AMP-based drug development is their potential cytotoxicity to mammalian cells. Understanding AMP interactions with eukaryotic model membranes would allow therapeutics to be tailored for preferential action toward specific classes of bacterial membranes. In this study, we developed novel methods of quartz crystal microbalance with dissipation monitoring (QCM-D) data analysis to determine the fundamental mechanism of action between eukaryotic and bacterial membrane mimics and select membrane-active AMPs. A new technique for creating supported membranes composed entirely of anionic lipids was developed to model Gram-positive bacterial membranes. Atomic force microscopy (AFM) imaging was also used to capture the progression of AMP-induced changes in supported lipid membranes over time and to validate our method of QCM-D analysis. QCM-D and AFM were used to investigate the molecular-scale interactions of four peptides, alamethicin, chrysophsin-3, sheep myeloid antimicrobial peptide (SMAP-29) and indolicidin, with a supported zwitterionic membrane, which served as a model for eukaryotic cell membranes. Since established methods of QCM-D analysis were not sufficient to provide information about these interaction mechanisms, we developed a novel method of using QCM-D overtones to probe molecular events occurring within supported lipid membranes. Also, most previous studies that have used AFM imaging to investigate AMP-membrane interactions have been inconclusive due to AFM limitations and poor image quality. We were able to capture high-resolution AFM images that clearly show the progression of AMP-induced defects in the membrane. Each AMP produced a unique QCM-D signature that clearly distinguished their mechanism of action and provided information on peptide addition to and lipid removal from the membrane. Alamethicin, an alpha-helical peptide, predominantly demonstrated a pore formation mechanism. Chrysophsin-3 and SMAP-29, which are also alpha-helical peptides of varied lengths, inserted into the membrane and adsorbed to the membrane surface. Indolicidin, a shorter peptide that forms a folded, boat-shaped structure, was shown to adsorb and partially insert into the membrane. An investigation of rates at which the peptide actions were initiated revealed that the highest initial interaction rate was demonstrated by SMAP-29, the most cationic peptide in this study. The mechanistic variations in peptide action were related to their fundamental structural properties including length, net charge, hydrophobicity, hydrophobic moment, accessible surface area and the probability of alpha-helical secondary structures. Due to the charges associated with anionic lipids, previous studies have not been successful in forming consistent anionic supported lipid membranes, which were required to mimic Gram-positive bacterial membranes. We developed a new protocol for forming anionic supported lipid membranes and supported vesicle films using a vesicle fusion process. Chrysophsin-3 was shown to favor insertion into the anionic lipid bilayer and did not adsorb to the surface as it did with zwitterionic membranes. When introduced to supported anionic vesicle films, chrysophsin-3 caused some vesicles to rupture, likely through lipid membrane disruption. This study demonstrated that molecular-level interactions between antimicrobial peptides and model cell membranes are largely determined by peptide structure, peptide concentration, and membrane lipid composition. Novel techniques for analyzing QCM-D overtone data were also developed, which could enable the extraction of more molecular orientation and interaction dynamics information from other QCM-D studies. A new method of forming supported anionic membranes was also designed, which may be used to further investigate the behavior of bacterial membranes in future studies. Insight into AMP-membrane interactions and development of AMP structure-activity relationships will facilitate the selection and design of more efficient AMPs for use in therapeutics that could impact the lives of millions of people per year who are threatened by antibiotic-resistant organisms.

Supported lipid bilayers (SLBs) are one of the most common model membranes used in the field of cell membrane biology as they provide a well-defined model membrane platform for determination of molecular-level interactions between different biomolecules (e.g. proteins, peptides) and lipid membrane. Compared to model organisms, the use of SLB is preferable since it mimics cell plasma membrane in a very simple and well-controlled way. Therefore, molecular structure of membrane and experimental conditions (e.g. solution chemistry, temperature, and pH) can be easily adjusted to the required conditions of any systematic research. In addition, SLBs are typically easy to form, cheap and very reproducible and they are compatible with different surface characterization techniques, such as quartz crystal microbalance with dissipation (QCM-D), ellipsometry and atomic force microscopy (AFM). This study demonstrates that QCM-D analysis of SLBs serve as powerful tool to investigate and characterize the mechanisms of interactions between lipid membrane and gold nanoparticles (NPs), environmentally relevant polymers, and disease-inducing peptides. Due to many critical applications of gold NPs in drug delivery and diagnostics, understanding of membrane-NP interactions is crucial especially for determination of NPs cytotoxicity. In this study we focus on membrane disruption as one of the different mechanisms by which metal NPs induce cytotoxicity. The use of SLB is beneficial for this goal as it elucidates the unique mechanism of membrane disruption without interference of other mechanisms taking place simultaneously in biological cells. For NP-membrane interaction studies, a SLB composed of L-α-phosphatidylcholine (egg PC) was formed on a SiO2-coated crystal and QCM-D analysis was performed to obtain information about mass and viscoelastic changes of SLB resulting from interactions with gold NPs. For better understanding of the mechanisms of NP-membrane interactions, we systematically changed the NPs properties and the experimental conditions. In order to understand the effect of NP size, gold NPs with diameters of 2,5,10, and 40 nm were tested and compared to each other. NPs were tested in their citric acid-stabilized state as well as in the presence of poly (methacrylic acid) (PMAA), representing an organic coating that could become associated with NPs in the environment. The results indicated that when dissolved in water, gold NPs with the dimeters of 2, 5, 10, and 40 nm did not perturb the membrane, but in the presence of environmentally relevant polymer, the larger nanoparticles were found to disrupt the membrane. In order to elucidate the effect of surface chemistry, 10 nm - gold NPs with various functionalizations (i.e. anionic, cationic and non-ionic ligands) were tested. Control experiments were designed to test the effect of NPs in the absence of humic substances which means the NPs were dissolved in water. In these cases, regardless of the type of NP functionalization, no substantial bilayer mass changes were observed. This suggests that the charge and chemistry of the ligands had a minor effect on NP-membrane interactions. Furthermore, in both the control and humic acid experiments, there were small dissipation changes (less than 1 unit) indicating that the overall membrane structure was not perturbed. In order to mimic environmentally-relevant conditions, mass and viscoelasticity of SLB was characterized in the presence of four different natural polymers, also known as natural organic materials (NOMs): Fulvic and humic acids extracted from Suwannee River (SRFA and SRHA), which had relatively lower molecular weights and a commercial humic acid (HA) and the humic acid extracted from Elliott soil (ESHA) with higher molecular weight. The results showed that NOMs with lower molecular weights, adsorbed to the bilayer, while higher molecular weight components, did not induce any changes to the bilayers. In addition, the NPs in SRFA and SRHA increased the mass of the bilayer by 20-30 ng, while the NPs in HA and ESHA changed the mass of the bilayer by < 10 ng. It was concluded that the presence of humic substances as well as their physical and chemical properties exert a direct impact on the interactions between cell membrane and the nanoparticles. In addition to the field of NP toxicity, SLBs play a pivotal role in the field of neurodegenerative diseases, such as Alzheimer’s disease (AD), in which the pathological cascade of events starts from interactions of a misfolded peptide with cell membrane. In this thesis, we confirm the validity of QCM-D analysis of SLB as an important platform for investigation of amyloid β (the peptide associated with AD) interactions with lipid membrane. Adsorption of Aβ peptide to cell membrane is known to take place on the so-called “lipid raftâ€� which are membrane microdomains enriched with cholesterol, sphingomyelin and ganglioside. The formation of SLBs containing lipid rafts is not only important for the field of AD research, but also it is important for other in vitro studies of cell biology as the lipid rafts are responsible for a variety of biological functions such as association of some membrane proteins and cellular signaling. However, the presence of lipid raft components such as sphingomyelin and cholesterol makes the formation of the bilayer more challenging which leads to adsorption of intact vesicles on the substrate without formation of the bilayer. In this study, the formation of lipid bilayer composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM) was investigated using QCM-D. A challenge was that the raft-containing vesicles remained intact on the SiO2 crystal. Therefore, different experimental conditions were tested to induce vesicle fusion, such as pH, temperature, osmotic pressure, and vesicle size. The key parameter in forming the bilayer was found to be applying osmotic pressure to the vesicles by having the vesicles exterior concentration of NaCl higher than interior concentration. When this concentration gradient was applied to the vesicles before flowing them on the substrate, vesicle rupture was favored and formation of a complete bilayer could occur. Here, we report the effects of each tested variable on the adsorption and fusion of the raft-containing vesicles, and the results are discussed based on the mechanisms of vesicle-vesicle and vesicle-substrate interactions.After developing the robust method for formation of SLB with lipid rafts, we used that as a template to characterize the mechanism of interactions between Aβ peptide and cell membrane which leads to onset of AD. The mechanism of Aβ toxicity leading to AD has not fully discovered yet, due to the complexity of the process including several steps of Aβ peptide adsorption on membrane, conformational change from disordered in solution to a membrane-bound α-helix structure and then formation of β-sheet aggregates that serve as fibrillation seeds. In this study, we showed that QCM-D technique as a promising tool to conduct systematic studies on the mechanism of interactions between Aβ peptide with lipid membrane. To our knowledge, this was the first time QCM-D was utilized for characterization of Aβ fibrillation starting from monomer states until formation of mature fibrils. The data indicated that peptide-membrane interactions follow a two-step kinetic pathway starting with the adsorption of small (low-n) oligomers until covering all the adsorption sites on the surface. In the second step, the membrane structure is destabilized as the result of interaction with oligomers which leads to lipid loss from the surface. Consistency of the results with the data obtained via other techniques substantiates QCM-D technique as a robust approach to answer the remaining unanswered questions in the field of Alzheimer’s disease.

Membrane proteins are important targets that represent more than 50% of current drug targets. However, characterisation of membrane proteins falls behind compared to their soluble counterparts. The most challenging part of membrane protein research is finding a suitable membrane mimetic that stabilises them in solution and maintains their native structure and function. The recently developed saposin-A (SapA) based lipid nanoparticle system seems to be advantageous over existing membrane mimetic system. It provides a native-like lipid bilayer, high incorporation yield and more importantly size adaptability. SapA lipid nanoparticles have been applied to structural studies and two high-resolution structures of membrane proteins were previously obtained using cryo-electron microscopy. This thesis aimed to study small-to-medium sized membrane proteins in SapA lipid nanoparticles using NMR spectroscopy. We first explore the mechanism of SapA lipid nanoparticle formation for the purpose of establishing an incorporation protocol that can be applied to most membrane proteins. The effect of pH and the presence of detergents on the opening of SapA was investigated in Chapter 2. A proposed energy diagram describing the mechanism of SapA opening is reported with which we were able to develop a protocol that can generate different sizes of SapA-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) nanoparticles. In addition, we also showed that SapA can form lipid nanoparticles with various lipid compositions, showing the versatility of the system. In Chapter 3, we validated the ability of SapA lipid nanoparticles to be used as a membrane mimetic. A -barrel model protein, bacterial outer membrane protein X (OmpX), was incorporated into SapA-DMPC nanoparticles and a 2D 15N-1H correlation NMR spectrum was recorded. Our result was compared to the NMR parameters of the same protein in MSP nanodiscs from the literature, and it was concluded that SapA lipid nanoparticles indeed provide a lipid bilayer environment similar to MSP nanodiscs. Because of high incorporation yield, we were able to incorporate OmpX into different lipid compositions to investigate the effect of lipid head groups and aliphatic chains on the membrane protein's chemical environment. Next, the applicability of SapA lipid nanoparticles was expanded to -helical transmembrane proteins in Chapter 4. Two microbial rhodopsins, Anabaena sensory rhodopsin (ASR) and Natronomonas pharaonis sensory rhodopsin II (pSRII), were tested. The parameters for expression and purification of ASR were first screened for the optimal yield. Although incorporation of ASR resulted in inhomogeneous particles due to imperfect experimental procedure, pSRII in SapA-DMPC nanoparticles showed high sample quality. The 2D NMR spectrum of pSRII in SapA-DMPC nanoparticles shows distinct differences to pSRII in detergent micelles, suggesting substantial effects from the membrane mimetic on the conformation of the membrane protein. Despite the good NMR spectral quality considering the large particle size, perdeuteration of pSRII and the lipids will be necessary for further investigation. With the SapA lipid nanoparticles established, we aimed to use it for the study of a biologically important G protein-coupled receptor, 1-adrenergic receptor (1AR), discussed in Chapter 5. The possibility of expressing 1AR using a cell-free expression system was explored first. Although a good amount of the protein was obtained, only a fraction of it was functional. Therefore, a conventional baculovirus-insect cell expression system was used to produce selective isotope labelled 1AR for NMR studies. NMR spectra of 1AR in SapA-DMPC nanoparticles with activating ligands and an intracellular binding partner were recorded and compared to the spectra of the same protein in detergents. This revealed a more active-like conformation of ligand-bound 1AR in the lipid bilayer, suggesting that certain parts of the protein are sensitive to the membrane mimetic used. This emphasises the importance of using a native-like membrane mimetic to capture the full properties of membrane proteins. In conclusion, I demonstrate in this thesis that SapA lipid nanoparticles are a versatile membrane mimetic system that can accommodate membrane proteins with different sizes and folds. This system is also compatible with solution NMR spectroscopy enabling structure and dynamics studies of biologically important membrane proteins. We believe SapA lipid nanoparticles will have a significant impact on membrane protein research in the future.