Les interactions protéine-ligand interviennent dans de nombreux processus biochimiques et permettent notamment à certaines protéines sanguines d’assurer leur rôle de transport. Parmi ces protéines figurent notamment l’albumine, protéine la plus abondante du plasma, ou l’apolipoprotéine A1 (ApoA1), majoritaire au sein des lipoprotéines de haute densité (HDL). Dans un contexte diabétique, la glycation des protéines induit des modifications structurales affectant ainsi leur potentiel d’interaction.Le premier objectif de ce travail de thèse visait à déterminer l’impact de la glycation de l’albumine sur sa liaison au liraglutide, un médicament de plus en plus utilisé dans le traitement du diabète de type 2. Ensuite, la seconde partie de ce travail a consisté en la production d’une ApoA1 humaine recombinante fonctionnelle afin d’étudier ses propriétés d’interaction, sous forme libre ou associée aux phospholipides. La technique RMN (résonance magnétique nucléaire) a été utilisée sur les protéines préalablement marquées au fluor (pour le liraglutide) ou aux isotopes stables 13C/15N (pour l’ApoA1). La titration microcalorimétrique isotherme (ITC), méthode complémentaire à la RMN a été appliquée pour l’étude des interactions avec l’avantage de ne nécessiter aucun marquage. Différentes stratégies de clonage ont été explorées pour la surexpression de l’ApoA1 en bactérie Clearcoli.Les résultats obtenus démontrent une altération de l’affinité de l’albumine pour le liraglutide in vitro et in vivo, dépendante du degré de glycation. Ces résultats, enrichis d’une analyse lipidomique et peptidique, permettent d’expliquer les observations cliniques concernant la baisse de l’efficacité de médicaments liant l’albumine chez les patients ayant un diabète mal contrôlé. Concernant l’ApoA1, le choix de l’étiquette de fusion reste à optimiser, mais sa surexpression de manière soluble et abondante a été obtenue pour l’ApoA1 marquée et non marquée. / Protein-ligand interactions are involved in many biochemical processes. They are notably implicated in the role of transporter proteins in blood. Albumin, the most abundant plasma protein, and apolipoprotein A1 (ApoA1), which is the main component of high-density lipoprotein (HDL) belong to this class of proteins. In the context of diabetes, proteins are altered by glycation which leads to structural modifications and potentially affect their interactions.The first objective of this work was to determine the impact of albumin glycation on its binding to liraglutide, a drug increasingly used in the treatment of type 2 diabetes. Then, the second part of this work involved the production of recombinant functional human ApoA1 in order to study its interaction properties, in its lipid-free form or associated with phospholipids. The NMR (nuclear magnetic resonance) technique has been used on proteins previously labeled with fluorine (for liraglutide) or stable 13C/15N isotopes (for ApoA1). In addition, isothermal titration microcalorimetry (ITC), has been applied to the study of interactions with the advantage of not requiring any labeling. Various cloning strategies have been explored for the overexpression of ApoA1 in Clearcoli bacteria.The results demonstrate an alteration of the affinity of albumin for liraglutide in vitro and in vivo, depending on the degree of glycation. These results, supported by a lipidomic and peptide analysis, explain clinical observations concerning the decrease of efficacy of albumin-binding drugs in patients with poorly controlled diabetes. Regarding ApoA1, the choice of the fusion tag remains to be optimized, but both labeled and unlabeled ApoA1 were successfully overexpressed at high yields in a soluble form.

Submitted by CINTIA RAMOS CAMARGO null (cintiaramoscamargo@gmail.com) on 2017-10-26T19:26:25Z No. of bitstreams: 1 Tese _Cintia Ramos Camargo_.pdf: 5811868 bytes, checksum: a56e37c6cc9f843250f31549c5aa7e98 (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-10-31T18:52:56Z (GMT) No. of bitstreams: 1 camargo_cr_dr_sjrp.pdf: 5811868 bytes, checksum: a56e37c6cc9f843250f31549c5aa7e98 (MD5) / Made available in DSpace on 2017-10-31T18:52:56Z (GMT). No. of bitstreams: 1 camargo_cr_dr_sjrp.pdf: 5811868 bytes, checksum: a56e37c6cc9f843250f31549c5aa7e98 (MD5) Previous issue date: 2017-09-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A folha de louro verde brasileira, uma especiaria muito apreciada na cozinha local (Aniba riparia, Lauraceae), contém compostos químicos que apresentam derivados de benzoíla chamados riparinas, que possuem propriedades anti-inflamatórias, antimicrobianas e ansiolíticas. No entanto, não está claro qual o tipo de interação que as riparins desempenham com qualquer alvo molecular. Como um alvo rentável, a albumina de soro humano (HSA) é uma das principais proteínas extracelulares, com uma capacidade excepcional para interagir com várias moléculas, e também desempenha um papel crucial no transporte, distribuição e metabolismo de uma grande variedade de ligantes endógenos e exógenos. Para delinear o mecanismo de interação HSA-riparina, a espectroscopia e os métodos computacionais foram aplicados de forma sinérgica. Uma avaliação através de espectroscopia de fluorescência mostrou que a emissão, atribuída ao Trp 214, a 346 nm, diminuiu com as titulações das riparinas. Observou-se um mecanismo de supressão estática na ligação das riparinas à HSA. Os experimentos de fluorescência realizados em 298, 308 e 318 K possibilitaram a realização de análises termodinâmicas que indicassem uma reação espontânea na formação do complexo (ΔG <0). O experimento do balanço entálpico-entrópico e com um cálculo de modelagem molecular revelou que interações hidrofóbicas, ligação de hidrogênio e interações não específicas estão presentes para as riparinas I - III com a HSA. O conjunto de resultados das mudanças da fração de fluorescência obtidos através de Schatchard não foi conclusivo ao estabelecer que tipo de cooperatividade esteja presente na interação. Para esclarecer o complexo HSA-riparinas, a abordagem de Hill foi utilizada para distinguir o índice de afinidade e a constante de ligação. Observou-se uma correspondência entre as estruturas moleculares das riparinas, devido à presença do grupo hidroxila no anel B, com parâmetros termodinâmicos e índice de afinidade. Riparin III realiza uma ligação de hidrogênio intramolecular, que afeta o coeficiente de Hill e a constante de ligação. Portanto, a presença de grupos hidroxila é capaz de modular a interação entre riparinas e HSA. Os experimentos de competição de sítio indicaram o sítio I como sendo o mais acessado, e as ferramentas de modelagem molecular reforçavam os resultados experimentais detalhando a participação de resíduos. / The green Brazilian bay leaf, a spice much prized in local cuisine (Aniba riparia, Lauraceae), contains chemical compounds presenting benzoyl-derivatives named riparins, which have anti-inflammatory, antimicrobial and anxiolytic properties However, it is unclear what kind of interaction riparins perform with any molecular target. As a profitable target, human serum albumin (HSA) is one of the principal extracellular proteins, with an exceptional capacity to interact with several molecules, and it also plays a crucial role in the transport, distribution, and metabolism of a wide variety of endogenous and exogenous ligands. To outline the HSA– riparin interaction mechanism, spectroscopy and computational methods were synergistically applied. An evaluation through fluorescence spectroscopy showed that the emission, attributed to Trp 214, at 346 nm decreased with titrations of riparins. A static quenching mechanism was observed in the binding of riparins to HSA. Fluorescence experiments performed at 298, 308 and 318 K made it possible to conduct thermodynamic analysis indicating a spontaneous reaction in the complex formation (ΔG<0). The enthalpy-entropy balance experiment with a molecular modeling calculation revealed that hydrophobic, hydrogen bond and non-specific interactions are present for riparin I - III with HSA. The set of results from fractional fluorescence changes obtained through Schatchard was inconclusive in establishing what kind of cooperativity is present in the interaction. To shed light upon the HSA-riparins complex, Hill’s approach was utilized to distinguish the index of affinity and the binding constant. A correspondence between the molecular structures of riparins, due to the presence of the hydroxyl group in the B-ring, with thermodynamic parameters and index of affinity were observed. Riparin III performs an intramolecular hydrogen bond, which affects the Hill coefficient and the binding constant. Therefore, the presence of hydroxyl groups is capable of modulating the interaction between riparins and HSA. Site marker competitive experiments indicated Site I as being the most suitable, and the molecular modeling tools reinforced the experimental results detailing the participation of residues.

Este trabalho tem como objetivo sintetizar e caracterizar um novo complexo mononuclear de rutênio (III) e enrofloxacina (enro, fármaco antibacteriano da família das fluoroquinolonas), [Ru(enro)3].nH2O. Foram testadas várias rotas sintéticas e apenas a partir de uma delas obteve-se o composto desejado. O produto foi caracterizado pelas técnicas espectroscópicas de absorção na região do UV-visível e do infra-vermelho. Através desta última técnica foi possível determinar o modo pelo qual a enrofloxacina se coordena ao íon rutênio: a coordenação ocorre de modo bidentado através do oxigênio da piridona e do oxigênio do grupamento carboxilato. Outro objetivo deste trabalho foi investigar a interação do complexo mononuclear de rutênio (III) e norfloxacina, [Ru(nor)3].nH2O, com a albumina de soro humano (HSA), através da técnica de luminescência. Mais especificamente pelo estudo da supressão da luminescência dos resíduos de triptofano, aplicando-se o modelo de tratamento da supressão bimolecular de Stern-Volmer. O estudo de supressão de fluorescência mostrou, por meio de espectros de emissão da HSA, que com o aumento da concentração do complexo [Ru(nor)3].nH2O na solução de HSA, ocorre uma redução gradual da luminescência da HSA, devido a alterações da conformação da proteína, que sugerem alteração do microambiente próximos aos resíduos de triptofano. A partir do tratamento dos dados pode-se determinar tanto K_sv quanto a constante cinética do processo de supressão, que mostraram uma dependência com a temperatura sugerindo como mecanismo predominante de supressão o mecanismo dinâmico. Porém essa conclusão foi revista a partir da determinação dos tempos de vida do estado excitado da HSA, e pode-se concluir que o mecanismo predominante à temperatura ambiente é o mecanismo estático, porém com o aumento da temperatura ocorre a predominância do mecanismo do tipo dinâmico. Através da determinação dos parâmetros termodinâmicos, concluiu-se que as interações entre a HSA e o complexo são espontâneas, e forças de van der Waals e ligações de hidrogênio estão envolvidas na ligação entre a HSA e o supressor. / This work aims to synthesize and characterize a new mononuclear ruthenium (III) complex and enrofloxacin (enro, antibacterial drug of the fluoroquinolone family), [Ru(enro)3].nH2O. Several synthetic routes were tested, but only from one of them it was obtained the desired compound. The product was characterized by spectroscopic techniques of absorption in UV-visible and infra-red regions. Through this last technique, it was possible to determine the coordination mode of enrofloxacin to the ruthenium ion: the coordination occurs in a bidentate way through the pyridone oxygen and the oxygen of the carboxylate group. Another aim of this study was to investigate the interaction of mononuclear ruthenium (III) complex and the norfloxacin, [Ru(nor)3].nH2O, with the human serum albumin (HSA), through the technique of luminescence. More specifically, by the study of the quenching of luminescence of tryptophan residues, by applying the Stern-Volmers model of treatment of bimolecular suppression. The fluorescence quenching study showed, through the emission spectra of HSA, that increasing the complex concentration in HSA solution, there is a gradual reduction of the luminescence of HSA, due to the conformational changes of the protein that suggests the change of microenvironment near tryptophan residues. From the data processing it is possible to determine both K_sv and the kinetic constant of the suppression process, which showed temperature dependence, suggesting as the predominant mechanism of quenching the dynamic mechanism. However, this conclusion has been revised from the determination of the lifetimes of the excited state of HSA, and it can be concluded that the predominant mechanism at room temperature is the static mechanism, but with the temperatures increase, it occurs the predominance of the dynamic type mechanism. By determining the thermodynamic parameters, it was concluded that the interactions between HSA and the complex are spontaneous, and Van der Waals forces and hydrogen bonds are involved in the binding between HSA and suppressor.

Per-and polyfluoroalkyl substances (PFAS) are anthropogenic compounds that have been spread all over the world. The use of fluorotelomer compounds, short-chained homologues, and other PFASs with perfluorinated moieties has emerged recent years. One of these emerging compound classes is polyfluoroalkyl phosphate esters (PAPs), which have the ability to degrade into persistent PFCAs. The aim of this thesis was to assess the contribution of PAPs and other precursors to the exposure of PFCAs to humans and the environment. The main objective was to analyze a wide range of PFAS in human serum, wild bird eggs, indoor dust, waste water, and sludge. There was a significant contribution from selected precursors to the total amount of PFASs in the abiotic compartments indoor dust, waste water, and sludge. Levels of PAPs found in house dust exceeded those of PFCAs and perfluorosulfonic acids (PFSAs), revealing PAPs as a world-wide important exposure source. A net increase was during waste water treatment was observed for several PFASs in Swedish waste water treatment plants. Together with presence of precursor compounds and intermediates in the influent water and the sludge, this suggest that degradation of PFCA precursors contributed to the increase of PFCAs. Detection of precursors in human serum, together with slow declining trends of PFCAs, revealed an ongoing exposure of PFCAs to the general population of Australia. The diPAPs and the FTSAs were also detected in raptor bird eggs from Sweden from both the terrestrial and the freshwater environment. The precursors concentrations and patterns observed reveal that current regulatory measures are insufficient for the purpose of protecting humans and the environment from PFASs exposure.

As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (Ka = (6.70 ± 0.10) × 106 M-1) for HSA than resveratrol (Ka = (1.64 ± 0.07) × 105 M-1), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the π-π conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the π-π stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E = -1.432 KJ/mol) was more stable than the latter (E = -0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA.

Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.

京都大学 / 新制・論文博士 / 博士(工学) / 乙第13495号 / 論工博第4202号 / 新制||工||1786(附属図書館) / (主査)教授 米田 稔, 教授 高野 裕久, 教授 松井 康人 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Steady advancements in diagnostics over the past century have propelled the world of medicine into the more advanced era of preventative medicine, an era with a resoundingly clear message: early detection can save lives. For patients who suffer from either pancreatic cancer or malignant hyperthermia susceptibility, early or preoperative diagnosis, respectively can save lives and minimize morbidity and mortality, in addition to offering cost-savings to hospitals and healthcare systems. Fortunately, significant progress have been made in the fields of metabolomics and biomarker identification. Given the benefits carried by serum biomarkers as targets of screening and diagnostic tool development, we applied functional nucleic acid technology and in vitro selection directly in whole human serum to search for disease-specific biomarkers and associated detection probes without a priori knowledge of the biomarkers pursued. This endeavour simultaneously serves as a proof-of-concept study to establish whether in vitro selection can be successfully performed in human serum. We specifically focused on the derivation of RNA-cleaving DNAzymes (RCD) through in vitro selection, or SELEX (systemic evolution of ligands through exponential exposure). DNAzymes constructed with a fluorogenic signalling molecule were incubated with human serum with the goal of identification of a functional nucleic acid probe capable of detecting the presence of a disease-specific biomarker. Two independent protocols have been designed and executed for the identification of DNAzyme sequences capable of detecting pancreatic cancer and malignant hyperthermia susceptibility, respectively. The first exploration was performed in serum obtained from cancer patients, with the goal of identifying DNAzymes capable of distinguishing pancreatic cancer from other cancer types. To do so, we employed in vitro selection, Next-Generation Sequencing, and bioinformatic analysis. We successfully demonstrated the feasibility of performing in vitro selection with DNAzymes in human serum, evidenced by distinct round-to-round enrichment of a DNA library towards the identification of DNAzymes capable of detecting pancreatic cancer. Additionally, we isolated two DNAzymes capable of distinguishing pancreatic cancer serum from healthy patient serum in fresh collected serum samples. Based on the positive results gathered in the pancreatic cancer in vitro selection project, we subsequently endeavoured to replicate the demonstrated feasibility of performing in vitro selection in human serum. By selecting malignant hyperthermia as the pathology investigated, we simultaneously sought to diversify the scope of DNAzyme detection by establishing whether successful DNAzyme selection can be achieved in a non-acute disease state. Thus, the second exploration was performed in serum obtained from patients who underwent evaluation for malignant hyperthermia susceptibility using the gold-standard caffeine-halothane contracture test. The goal of this project rested on the identification of DNAzymes capable of distinguishing malignant hyperthermia susceptibility in serum and approximating the performance of the gold standard test. We successfully isolated four DNAzyme candidates which demonstrated clinically relevant thresholds of sensitivity and specificity following thorough sensitivity and specificity analysis. In doing so, we once again demonstrated the ability to perform in vitro selection in human serum. Given the complexity of molecular interactions observed over the course of two in vitro selection protocols in human serum, it became clear that distinguishing meaningful target-mediated interactions from non-specific interactions would require advanced bioinformatic analysis. Consequently, using principles of computational biology, we performed a deep exploration of Next-Generation Sequencing results obtained from sequencing our recovered DNA libraries to extract additional data that would inform on the next required steps required to identify a DNAzyme specific for the pathology pursued. In doing so, we identified a two-step method to evaluate the progress of the in vitro selection protocol undertaken, and offered a systematic approach for choosing candidate sequences to undergo further testing based on promising performance in silico. Using this approach, we successfully identified a DNAzyme sequence capable of acting as a general cancer detection probe, with promising potential for diagnostic application. Ultimately, this thesis serves as a feasibility study of a novel approach to both in vitro selection and biomarker identification technique by combining the latest nanotechnology techniques with clinical data and real patient serum samples, and advanced computational biology tools. Despite the inability to identify a highly sensitive and specific DNAzyme capable of advancing towards biosensor construction, several important strides and lessons have been acknowledged, establishing the feasibility of performing in vitro selection in human serum, and outlining strategies for addressing and anticipating challenges with this technique. The hope is for this work to inspire and inform future efforts to apply functional nucleic acid technology to solve current gaps in both the diagnostic and therapeutic branches of medicine, and with the help of computational biology continue to bridge the gap between basic science and clinical medicine. / Dissertation / Doctor of Philosophy (PhD)

Per- and polyfluoroalkyl substances (PFAS) encompass a large group of manufactured compounds that have been used in various production processes such as food packaging, commercial products, workplaces, homes, water supplies, and food. PFAS are persistent, resistant to degradation, and can bioaccumulate. Although an exposure limit that predicts adverse health effects has yet to be determined, the Center for Disease Control and Prevention’s 2015-16 health survey found average blood levels of 4.72 ng/ml for PFOS and 1.56 ng/ml for PFOA. The objective of this research was to evaluate the use of laminar flow tandem mass spectrometry following solid phase extraction (SPE) using weak anion exchange (WAX) properties on the detection and quantitation of PFAS compounds. Seven-point calibration standards applied to this research were prepared using certified reference materials (Wellington Laboratories, Ontario, CA), and calibrators were run without sample extraction. The concentrations varied slightly based on the PFAS analyte of interest. All samples and quality controls were prepared by spiking certified reference material (Wellington Laboratories) into pooled human serum (BioIVT, Westbury, NY, USA). A laminar flow QSight®220 ultra-high pressure liquid chromatography-tandem mass spectrometer (LC-MS/MS, PerkinElmer, Waltham, MA, USA) was equipped with a Selectra C18 100 x 2.1mm x 3μm (UCT, Bristol, PA, USA) column with a Brownlee C18 delay column (PerkinElmer) and followed the LC-MS/MS parameters developed for the method. Extraction was accomplished using a WAX SPE column (UCT, ECWAX053) by first conditioning the columns with 1 mL of methanol (Fisher Scientific, Fair Lawn, NJ, USA) followed by 1 mL of 100 mM pH 7 phosphate buffer (Acros Organics, Geel, Belgium, EU). Samples were loaded onto the column at a rate of 1-2 mL/min. The SPE cartridges were washed with 1 mL of 100 mM pH 7 phosphate buffer and 1 mL of millipore water (Millipore Milli- Q Ultrapure Type 1 water system, Millipore Sigma, Burlington, MA, USA), then dried under full flow for 5 minutes. Elution was carried out with 2.5mL of a 98:2 methanol: OptimaTM grade ammonium hydroxide (Fisher Scientific) solution. The eluted samples were then evaporated to dryness using a MULTIVAP® Nitrogen Evaporator (Organomation,Berlin,MA,USA) at 55°C and 5psi. All samples were reconstituted in 100 μL of a 96:4 methanol:water solution. The parameters assessed followed Academy Standards Board Standard 036: Standard Practices for Method Validation in Forensic Toxicology, including matrix interferences, limit of detection (LOD), limit of quantitation (LOQ), a recovery study, and a calibration model. The results of the study were gathered from the following eleven analytes: PFBA, PFBS, PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Depending on the analyte, a lower LOQ was established at 0.16 – 1.75ng/mL and an upper LOQ at 43.75 – 51.41 ng/mL. Based on the established linear calibration model an LOD in the range of 0.11 - 0.51 ng/mL was achieved. All eleven PFAS analytes showed an acceptable bias of ±20%. All analytes showed a between-run precision (%CV) in an acceptable range of ±20%. No matrix interferences were detected. The average recovery for SPE ranges from 77.64- 104.73% with recovery of 77.64% for PFBS, 83.89% for PFBA, and 95.64-104.73% for PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Utilizing the UCT WAX SPE column, good recovery for the PFAS compounds was demonstrated. Further, the extraction technique was efficient for high throughput analysis with the extraction time comparable to other traditional SPE methods. The total analytical run time of 11 minutes using the QSight®220 coupled with the UCT Selectra C18 100 x 2.1mm x 3μm column allowed for adequate re-equilibration and system washes to prevent carryover and contamination of these persistent pollutants with excellent chromatography. Having the ability to efficiently and accurately quantify PFAS compounds in biological matrices will allow for better understanding of prevalence, bioaccumulation in biological matrices, and will aid in understanding how these concentrations relate to various health outcomes.

<p>Aggregation of the<strong> </strong>Alzheimer’s Aβ peptide in the brain and blood plasma is controlled by endogenous Aβ binding proteins. The structural basis for the interaction between the Aβ peptide and the Aβ binding proteins is critical not only to understand how Aβ amyloids are controlled in vivo, but also to guide the design of novel Aβ-self association inhibitors. However, the current knowledge of the structures of the Aβ/Aβ binding protein complexes is still sparse. This thesis focuses mainly on the interaction of the Aβ peptide with Human Serum Albumin (HSA). It is known that HSA binds ~90% of the Aβ in human plasma and prevents the Aβ self-association into amyloid fibrils. However, the mechanism of Aβ self-association inhibition by albumin was not understood prior to our work. We have shown that albumin preferentially binds toxic Aβ oligomers and fibrils inhibiting their growth into larger Aβ assemblies through a “monomer competitor” mechanism. Using a combination of NMR, domain deletion mutants, dynamic light scattering and ultrafiltration we have investigated the stoichiomery and affinity of the Aβ oligomer: HSA complexes. Our results indicate that all three domains of HSA bind Aβ oligomers and fibrils with an affinity in the 1-100 nM range. Such binding site degeneracy explains how albumin minimizes competition by other ligands such as fatty acids and drugs. Moreover we have used the soluble and NMR suitable domain 3 of albumin to dissect further the determinants of the Aβ oligomer binding to albumin at subdomain and peptide resolution. We show that both subdomains of the HSA domain 3 (<em>i.e</em>. 3A and 3B) bind the Aβ oligomers. In addition, we identified a peptide sequence within subdomain 3B that displays significant potency in the inhibition of Aβ self-association.</p> / Doctor of Philosophy (PhD)