Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Thesis advisor: Gabor J. Kalman / Mixtures of charged particles, where the components have different charge numbers (Z_A ), masses (m_A ) and densities (n_A ), with A = 1, 2 denoting the components, occur in Nature in a great variety. To be sure, even the simplest plasmas are necessarily multicomponent systems, consisting of negative and positive charges. This feature is, however, obscured within the centrally important and popular OCP (one component plasma) or jellium models, where the role of one of the components is reduced to providing a neutralizing background. When this background is inert, one is led to the Coulomb OCP model, while when the background is polarizable (such as an electron gas surrounding heavy particles), to a Yukawa OCP (YOCP), with a screened Yukawa potential replacing the Coulomb potential between the dynamically active particles. There are, however situations of physical importance, where the OCP description is inadequate and a genuine two component description of a plasma composed of two species is required. This Thesis focuses on the study of the dynamics of many-body systems consisting of two components of like charges (all the Z_A -s being of the same signature) in a neutralizing background. The methodology is based upon parallel attacks through theoretical analysis and Molecular Dynamics (MD) simulations, the latter yielding the capability of instant verification of the former. The investigation involves the study of the partial (i.e. species by species) structure functions S_AB (k, ω) and current-current correlation functions L_AB (k, ω). The Fluctuation–Dissipation Theorem (FDT) con- nects these quantities to the total and partial response functions χ_AB (k, ω) (matrices in species space), which are instrumental in the description of the collective mode excitations of the system. This analysis has revealed an entirely novel feature: both S_11 (k, ω) and S_22 (k, ω) exhibit very sharp and deep (several orders of magnitude) minima in the strongly coupled liquid phase at robust characteristic frequencies of the system, which are virtually coupling independent. The FDT then demands that these anti-resonances show up as well in the imaginary part of the partial density response function χ_AB (k, ω). Our theoretical analysis, based on the Quasi-Localized Charge Approximation (QLCA), has confirmed that this is indeed the case. These anti-resonant frequencies being related to the dissipative part of the response, require a physical description of the principal source of dissipation. This has been identified as the inter-species momentum transfer, governed by drag between the microscopic current fluctuations of the two species. The description of this effect was incorporatedv in the QLCA formalism, making it possible to derive a closed analytic representation of the fluctuation spectra in the frequency domain of interest and compare them with the results of the MD simulations. Other important novel concepts, such as the idea of coupling dependent effective mass, fast vs. slow sound, the mechanism of tran- sition from short-range to long-range interaction have been identified and analyzed. Furthermore, the investigation of the dynamics has led to the first comprehensive description of the mode structures of classical binary Coulomb and Yukawa mixtures at arbitrary coupling values, which has been a longstanding problem in statistical plasma physics. Focusing on the longitudinal excitations, we describe the transition from weak coupling (where one is acquainted with the RPA result yielding only the single plasmon mode in the Coulomb case or a single acoustic mode in the Yukawa case) to strong coupling, with a doublet of modes that arise from the complex rel- ative motion between the two components, as affected by the interaction with the background. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Physics.

Human p-glycoprotein (P-gp) is an ATP-binding cassette transporter that actively transports a diverse set of substrates at the plasma membrane. Specifically, P-gp is expressed most highly at important blood tissue barriers on the lumenal side of endothelial cells and secretory tissues asymmetrically where it provides generalized protection against xenobiotics due to its promiscuous substrate binding pocket. Substrates typically interact with P-gp within the inner leaflet of the plasma membrane before being effluxed through large conformation changes driven by ATP binding and hydrolysis. Since many small molecule drugs are substrates of P-gp and P-gp has the ability to transport chemically and structurally diverse molecules, delivery of bioavailable small molecule therapies and treatment of diseases beyond blood-tissue barriers may be difficult. In cancer, expression of P-gp may confer a multidrug resistance phenotype due to upregulation of the MDR1 gene, which encodes P-gp, in response to treatment with chemotherapies. Treatments of diseases beyond blood-tissue barriers and some cancers may be more complex given the protective role of P-gp coupled with it promiscuous substrate binding site.<br>Many studies of P-gp have been centered around understanding the structure function relationship of how P-gp effluxes small molecules across the plasma membrane. Here we have used a transient Vaccinia virus expression system to rapidly express many mutants of P-gp in human cells for analysis. Transient expression using the Vaccinia system was optimized to produce a large amount of protein while avoiding significant cell death. Optimization of the Vaccinia expression system has also helped to show that changes in P-gp surface expression are not correlated to changes in substrate accumulation within cells expressing P-gp, a topic that has yet to be addressed within the field of P-gp study. Reduced surface expression of P-gp to 68% maintained the same level of reduced cellular accumulation of two substrates, calcein-AM and rhodamine 123, relative to a WT P-gp control. Further study of P-gp mutations revealed a Y998A mutation had a 90% reduction of surface expression but the same reduction of cellular accumulation of rhodamine 123 further supporting that changes in surface expression do not correlate to changes in substrate transport.<br>We then sought to demonstrate how flexibility in transmembrane helix (TMH) 12 of P-gp affected overall stability and transport ability in vitro. TMH 12 in inward facing conformations shows a region of decreased hydrogen bonding in the backbone of the helix leading to a “kink” present in many crystal structures of C. elegans and mouse P-gp as well as in an occluded structure of human P-gp. Outward facing crystal structures of C. elegans, mouse, and human P-gp show TMH 12 where the backbone of the helix is fully hydrogen bonded and ordered. The change in hydrogen bonding pattern and the presence of the kink in TMH 12 suggest the importance of flexibility in the function of TMH 12. Clustal Omega was used to align the primary structure of P-gp between 8 species and a conserved sequence of 996-PDYAKA-1001 was identified aligning with the kink observed in crystallographic data. The kinked nature of this region led to our development of a rigid poly-alanine mutation and a flexible poly-glycine mutation based on the propensity of these amnio acids to form helices. The more flexible poly-glycine mutation obtained no significant transport while the poly-alanine mutation maintained some ability to transport fluorescent substrate relative to a WT control. Crosslinking of the nucleotide binding domains (NBDs) revealed a decrease of NBD dimerization likely correlating to decreased transport. Thus, some degree of flexibility within the kink region is critical for substrate transport as rigid and flexible mutations of this region abrogate transport of fluorescent substrates.<br>While the substrate binding pocket it located towards the interior of P-gp within the lipid bilayer, it has been theorized that substrates may interact with P-gp at the lipid-protein interface of the inner leaflet near portals for substrate entry formed by pairs of helices either side of the protein. To test this hypothesis, aromatic residues on TMH 12 and adjacent elbow helix 2 near the interface region of the inner leaflet, that have also been observed to interact with a cyclic peptide in a crystal structure of P-gp, were mutated to alanine. Y998, on TMH 12, was shown to interact with the cyclic peptide and is ideally located at the protein-lipid interface near a surface formed by elbow helix 2 and TMH 9 and was observed to have the largest effect on substrate accumulation. Accumulation of fluorescent substrates, relative to WT P-gp, was increased though not all substrates were affected similarly. No increase of accumulation was observed with rhodamine 123 while accumulation of BD-prazosin increased 65% relative to WT P-gp. It is to be expected that the large diversity of substrates recognized by P-gp would interact preferentially with carrying residues at the protein-lipid interface similar to observations of substrate binding at the substrate binding pocket. Variability in accumulation signifies that substrates do interact with P-gp at the lipid-protein interface and substrates interact differently at this interface similarly to substrate interaction at the substrate biding pocket.<br>

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+]e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+]e. It was also much slower in the presence of [Ca2+]e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797 , were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.

Flavonoid glycosides are required for a number of crucial roles in planta and have the potential for development in a variety of agricultural, medicinal, and biotechnological applications. A number of recent advancements have been made in characterizing glycosyltransferases, the enzymes that are responsible for the synthesis of these important molecules. In this review, glycosyltransferases are considered with regard to biochemical properties, expression patterns, levels of enzyme activity during development, and structure/function relationships. This is presented with historical context to highlight critical findings, particularly with regard to the innovative work that has come from research on citrus species. The plant glycosyltransferase crystal structures that have been solved over the past decade, either alone or in complex with sugar donor and/or acceptor molecules, are discussed. The application of results from these structures to inform current structure/function work as well as implications and goals for future crystallography and tertiary modeling studies are considered. A thorough understanding of the properties of glycosyltransferases will be a critical step in any future biotechnological application of these enzymes in areas such as crop improvement and custom design of enzymes to produce desired compounds for nutritional and/or medicinal usage.

Le projet de thèse à pour but de produire et caractériser un nouvel allergène de pollen d'ambroisie. Le projet est décliné en cinq axes: production d'une forme recombinante mature et en conformation native, caractérisation structurale, étude de la fonction enzymatique, étude de l'allergenicité et l'immunogénicité et évaluation du potentiel thérapeutique. / The goal of the thesis project is to product and caracterize a novel ragweed pollen allergen.The project have five axes: production of recombinant mature and native form,structurale caracterization, study of enzymatique function, study of allergenicity and immunogenicity and evaluation of therapeutic potential.

No description available.

β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Recent epidemiological studies have suggested that the development and progression of several chronic diseases may be initiated or augmented by oxidative stress. Reactive oxygen species and reactive nitrogen species react readily with and can damage nucleic acids, proteins, and lipids. While biological systems are equipped antioxidant defenses to cope with oxidative stress, oxidative damage may still occur when oxidative stress overwhelms antioxidant defenses. This damage, if left unchecked, may lead to a variety of degenerative diseases, including heart disease, Alzheimer's Disease, Parkinson's Disease and cancer. Several assays have been designed to describe the antioxidant activity of various phytochemicals, vitamins, and other compounds. The ORAC and TOSC assays have emerged as industry standards for measuring antioxidant activity due to their high reliability and sensitivity. Until recently, however, little has been done to assess the relative correlation between these two assays. Furthermore, no assay has been developed to measure changes in antioxidant activities of cells in response to oxidative stress. The current work investigates the correlation between measured antioxidant activities of samples in the both the ORAC and TOSC assays. Recent antioxidant research also focuses on relating chemical structure to antioxidant activity. Previous research in this area has included a broad range of chemical groups, but no study has attempted to formulate a structure-function framework that has applicability to compounds of any group. The current work uses amino acids as a simplest-case model for studying the relationships between chemical structure and antioxidant activity. One particular area of emerging research has centered around comparing organic and conventionally grown food products. The impetus of these investigations lies in claims made by organic supporting groups that these food products are generally more beneficial than their conventional counterparts. Despite the rapid rise in popularity of organic foods, there remains a dearth of research investigating these claims. The current work compares the antioxidant activities of organic and conventionally grown blueberries and apples.

<p dir="ltr">This thesis explores the intricate mechanisms governing paracellular permeability, a process vital for maintaining homeostasis and barrier integrity in epithelial and endothelial tissues. Central to this exploration are tight junctions (TJs), multi-protein complexes that regulate substance movement between cells. The thesis begins with a comprehensive literature review synthesizing current research on how bacterial and viral proteins can enhance paracellular permeability or modulate TJs, highlighting their significance in both scientific and medical fields.</p><p dir="ltr">A focal point of the thesis is the detailed study of the <i>Listeria</i> adhesion protein (LAP), an epithelial TJ modulator (TJM). LAP’s interaction with the epithelial receptor, Hsp60, facilitates the translocation of <i>Listeria monocytogenes </i>across the epithelial barrier, presenting a novel approach for drug delivery. Addressing the limitations of current TJMs, such as poor bioavailability and toxic side effects, the thesis provides an in-depth analysis of LAP as an alternative biologics’ delivery vehicle. Utilizing a multidisciplinary research approach, the study reveals the cryo-EM structure of LAP at 2.9 Å resolution, elucidating its multimeric formation and interaction with Hsp60 through electrostatic forces, as supported by computational modeling.</p><p dir="ltr">The functional efficacy of LAP is further established through experiments demonstrating its capability to transport drug analogs across epithelial cell monolayers comparably to established TJMs. Moreover, <i>in vivo</i> studies using a mouse oral gavage model show that LAP significantly enhances the blood levels of peptide drugs without triggering inflammatory responses or harming tight junction architecture. This thesis ultimately positions the LAP-Hsp60 complex as a promising TJM candidate, offering an innovative means for enhancing oral drug delivery and maintaining epithelial barrier integrity.</p>