O objetivo deste estudo foi avaliar o perfil proteico do plasma seminal equino utilizando eletroforese bidimensional de gel de acrilamida (2D-PAGE) e determinar se algumas das proteínas presentes estavam relacionadas com a congelabilidade do sêmen. O plasma seminal foi coletado de dez garanhões, de alta e baixa congelabilidade de sêmen, provenientes do Haras Estatal da Baixa Saxônia, na cidade de Celle, na Alemanha e rotineiramente utilizados em programas de inseminação artificial. Vinte e cinco bandas proteicas foram encontradas nos géis bidimensionais (12%) e sete delas foram identificadas por MALDI-MS. Das 25 proteínas encontradas nas amostras de plasma seminal dos garanhões, duas bandas proteicas apresentaram densidade óptica superior (P<0,05) nas amostras de garanhões de alta congelabilidade o sêmen: as bandas 5 (80-85 kDa, pI 7,54), que foi identificada como CRISP3 e a 45 (18,2 kDa, pI 5,0-5,2) identificada como HSP-2. Contrariamente a banda 7 (75,4 kDa, pI 6,9 – 7,4), identificada como lactoferrina, a 15 (26,7 kDa, pI 5,51) identificada como calicreína, a 25 (25 kDa, pI 7,54) como CRISP3 e a 35 (13,9 kDa, pI 3,8 – 4,2) que foi identificada como HSP-1, apresentaram valores de densidade óptica superior (P<0,05) nos reprodutores de baixa congelabilidade do sêmen. As proteínas foram identificadas através de espectometria de massa MALDI-MS. As evidências encontradas neste experimento mostram que existem diferenças no perfil proteico dos reprodutores de alta e baixa congelabilidade do sêmen, sugerindo as proteínas CRISP3 e a HSP-2 como possíveis marcadores da alta congelabilidade de sêmen de garanhões. / The objective of this study was to evaluate protein profile of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to find if any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions of high and low semen freezability routinely used in AI programs from the State Stud of Lower Saxony and Artificial Insemination Center. Twenty five protein spots were identified from the 2 D gel (12%), seven of which were present in all samples. Of the 25 proteins found in the research stallions, two spots showed superior relative content (P<0.05) in seminal plasma samples collected from stallions with high semen freezability: the spots 5 (80-85 kDa, pI 7.54) was identified as CRISP3 and 45 (18.2 kDa, pI 5.0-5.2) was identified as HSP-2. Conversely, the spot 7 (75.4 kDa, pI 6.9 – 7.4) was identified as lactoferrin, 15 (26.7 kDa, pI 5.51) was identified as kallikrein, 25 (25 kDa, pI 7.54) was identified as CRISP3 and 35 (13.9 kDa, pI 3.8 – 4.2) was identified as HSP-1, showed superior relative protein content (P<0.05) on seminal plasma samples from stallions with low semen freezability. The proteins were identified by MALDI-MS. There were differences in the seminal plasma protein profile between stallions with high and low semen freezability. It may be suggested that CRISP3 and HSP-2 may be considered possible seminal plasma markers of high semen freezability.

Le bénéfice de l’encapsulation des molécules bioactives a séduit les industries agroalimentaires depuis plusieurs décennies. Plus récemment des études ont montré la capacité de protéines alimentaires de charge opposée à s’assembler en microsphères par coacervation complexe. La compréhension des forces gouvernant le processus de coacervation entre protéines et l’influence exercée par la présence de bioactifs demeurent des prérequis pour l’utilisation des coacervats complexes comme agent d’encapsulation. Dans ce contexte, l’objectif de mon projet de thèse a été de comprendre le mécanisme de coacervation complexe entre la ¿-lactoglobuline (¿-LG) chargée négativement, et la lactoferrine (LF) chargée positivement, en absence et en présence de petits ligands. La LF a présenté une coacervation préférentielle avec le variant A de la¿¿-LG qui se distingue du variant B par la substitution de 2 acides aminés. Au niveau moléculaire, deux sites de fixation de la ¿-LG sur la LF ont été identifiés.En outre, par la mesure d’une part des coefficients de diffusion rotationnel et d’autre part de la cinétique de diffusion des entités moléculaires constituant les coacervats, il est suggéré que ces derniers sont formés à partir de -LG libre¿¿de pentamère, LF(-LG2)2, ainsi que des entités plus larges, (LF-LG2)n. Afin d’évaluer l’effet de la présence de petits ligands sur la coacervation complexe entre la -LG et la LF, des ligands modèles (ANS et acide folique) ont été utilisés. Dans les conditions expérimentales testées ces deux ligands n’ont pas d’affinité pour la -LG, mais après interact / Encapsulation of bioactives has been used by the food industries for decades and represents a great potential for the development of innovative products. Given their versatile functional properties, milk proteins in particular from whey have been used for encapsulation purposes using several encapsulation techniques. In parallel, recent studies showed the ability of oppositely charged food proteins to co-assemble into microspheres through complex coacervation. Understanding the driving forces governing heteroprotein coacervation process and how it is affected by the presence of ligands (bioactives) is a prerequisite to use heteroprotein coacervates as encapsulation device. In this context, the objective of my thesis work was to understand the mechanism of complex coacervation between -lactoglobulin (-LG) and lactoferrin (LF) in the absence and presence of small ligands. The conditions of optimal ¿-LG - LF coacervation were found at pH range 5.4-6 with a molar excess of ¿-LG. RemarkabAt molecular level, the presence of two binding sites on LF for -LG was evidenced. Moreover, the heterocomplexes such as pentamers LF(-LG2)2 and quite large complexes (LF-LG2)n were identified as the constituent molecular species of the coacervate phase. To evaluate the -LG - LF complex coacervation in the presence of small ligands, models of hydrophobic (ANS) and hydrophilic molecules (folic acid) were used. Although under the experimental conditions tested the small ligands did not interact with -LG, both interacted with LF inducing its self-association into nanoparticles. High relati

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The increase in obesity and overweight in many countries has led to an upsurge of interest in the development of reduced fat food products. However, the development of these products is challenging because of the many roles that fat droplets normally plays in these food products, including contributing to flavor, texture, appearance, and bioactivity. The goal of this research was to develop novel reduced-fat emulsions based on hetero-aggregation of oppositely charged food-grade colloidal particles or polymers. Initially, lactoferrin (LF) and β-lactoglobulin (β-Lg) were selected as emulsifiers to form protein-coated fat droplets (d43 ≈ 0.38 μm) with opposite charges at neutral pH: pKaβ-Lg ≈ 5 < pH 7 < pKaLF ≈ 8.5. Droplet aggregation occurred when these two emulsions were mixed together due to electrostatic attraction. The structural organization of the droplets in these mixed emulsions depended on the positive-to-negative particle ratio, particle concentration, pH, ionic strength, and temperature. The nature of the structures formed influenced the rheology, stability, and appearance of the mixed emulsions, which enabled some control over emulsion functionality. The largest microclusters were formed at particle ratios of 40% LF-coated and 60% β-Lg-coated fat droplets, which led to mixed emulsions with the highest apparent viscosity or gel strength. At low total particle concentrations (0.1%), there was a relatively large distance between microclusters and the mixed emulsions were fluid. At high particle concentrations (>20%), a three-dimensional network of aggregated droplets formed that led to gel-like or paste-like properties. The influence of environmental stresses on the physicochemical stability of the microclusters formed by hetero-aggregation was investigated: pH (2-9); ionic strength (0-400 mM NaCl); and temperature (30-90 ºC). Large microclusters were obtained at pH 7 (d43 ≈ 10 μm) with the absence of salt at room temperature. More acidic (< pH 6) or alkaline (> pH 8.5) solutions resulted in smaller aggregates by minimizing the electrostatic attraction between the protein-coated fat droplets. Microclusters dissociated upon addition of intermediate levels of salt, which was attributed to screening of attractive electrostatic interactions. Heating the microclusters above the thermal denaturation temperature of the proteins led to an increase in gel-strength, which was attributed to increased hydrophobic attraction. The influence of hetero-aggregation of lipid droplets on their potential biological fate was studied using a simulated gastrointestinal tract (GIT). Results showed that the mixed emulsions had high viscosity in the simulated oral environment but exhibited similar rheological properties and particle characteristics as single-protein emulsions in the simulated gastric and small intestinal tract regions. The mixed emulsions also had similar lipid digestion rates in the simulated small intestine as single-protein emulsions suggesting that they could be used as delivery systems for bioactive lipophilic compounds in reduced fat food products. The possibility of using more practical food ingredients to promote heteroaggregation system was also examined. Whey protein isolate (positive) and modified starch (negative) were selected as building blocks due to their opposite charges at pH 3.5. The largest aggregates and highest viscosities occurred at a particle ratio of 70% MS and 30% WPI, which was attributed to strong electrostatic attraction between the oppositely charged droplets. Particle aggregation and viscosity decreased when the pH was changed to reduce the electrostatic attraction between the droplets. Finally, the influence of interfacial properties on the chemical stability of bioactive components in emulsion-based delivery systems containing mixed proteins was studied. Lactoferrin (LF: pI ≈ 8) and β-lactoglobulin (β-Lg: pI ≈ 5) were selected to engineer the interfacial properties. Interfaces with different structures were formed: LF only; β-Lg only; LF-β-Lg (laminated); β-Lg -LF (laminated); β-Lg /LF (mixed). The influence of pH, ionic strength, and temperature on the physical stability of β-caroteneenriched emulsions was then investigated. LF- emulsions were stable to the pH change from 2 to 9 but the aggregation was occurred in intermediate pH for other emulsions. β- Lg- emulsions aggregated at low salt concentration (≥ 50mM NaCl), however other emulsions were stable (0 - 300mM NaCl). β-Lg /LF (mixed) emulsions were unstable to heating (≥ 60 ºC), but all other emulsions were stable (30 to 90 ºC). Color fading due to β-carotene degradation occurred relatively quickly in β-Lg-emulsions (37 ºC), but was considerably lower in all other emulsions, which was attributed to the ability of LF to bind iron or interact with β-carotene. Overall, this study shows that hetero-aggregation may be a viable method of creating novel structures and rheological properties that could be used in the food industry.

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Metallic flavors are of concern for many industries including food, health, and water. Metallic off-flavor, induced by ferrous sulfate solution (10mg/L), and its remediation using pre- and post-rinse treatments of water (control) or metal chelators, were studied. Metal chelators included lactoferrin (1 ?M), a natural metal-binding protein in milk and saliva, and EDTA (36 ?M), a synthetic chelator. Time-intensity (TI) evaluation (n=6, 4 female; age 40-70) of lingering metallic flavor indicated that metallic flavor decreased with a post-rinse adjuvant treatment of lactoferrin as indicated by a reduced maximum intensity and area under the curve compared to a pre-rinse treatment; EDTA and water post-rinses were equally effective for three of the TI parameters. Alterations in salivary components were studied in saliva collected (n=8; 5 female, age 40-70) after sipping a lactoferrin solution (1?M) followed with a ferrous sulfate sample (10 mg/ml) to stimulate metallic flavor, as compared to unstimulated whole saliva. Protein concentration, oral lipid oxidation as indicated by thiobarbituric acid reactive substances assay, and iron concentration were determined on individual saliva samples, with no significant differences found between treatments (p>0.05). Protein patterns were qualitatively characterized for each pre-rinse and metallic stimuli from four panelists by two-dimensional gel electrophoresis. A consistent pattern of regions containing major salivary components was observed. This research has shown that lactoferrin protein is a potential natural alternative to synthetic EDTA for reducing iron-induced metallic off-flavors. This study provides a foundation of method development to better understand salivary protein interaction with metals and flavor perception. / Master of Science in Life Sciences

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<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

The mare experiences a transient innate immune response to breeding, the resolution of which is crucial for optimal fertility. The majority of mares are able to modulate this inflammation in a timely fashion, but a subpopulation exists which fail to do so and are considered susceptible to persistent breeding-induced endometritis (PBIE). Seminal plasma has been shown to modulate aspects of this inflammation. Recently, two seminal plasma proteins have garnered interest for their immune modulating properties: cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin. These proteins have been found to alter the binding between sperm and neutrophils based on sperm viability in vitro, but minimal work has evaluated their effect on endometrial mRNA expression of cytokines and inflammation in response to breeding. Experiments were performed to analyze the expression of equine CRISP-3. Found to be primarily synthesized in the ampulla of the vas deferens and to a lesser extent in the vesicular gland, CRISP-3 expression was only seen in the postpubertal stallion. Due to the effect of sperm viability on protein function in vitro, varying sperm populations were analyzed for their effect on gene expression in the uterus. It was determined that viable sperm suppressed the gene expression of the inflammatory modulating cytokine interleukin-6 (IL-6) in comparison to dead sperm. Next, the effect of CRISP-3 and lactoferrin on endometrial gene expression in the normal and susceptible mare was investigated. Neither protein had a significant effect on the mRNA expression of inflammatory cytokines in the normal mares at six hours post-breeding. In contrast, lactoferrin was found to significantly suppress the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α in susceptible mares. Due to this, lactoferrin was further analyzed as an immunomodulant for the treatment of PBIE. Susceptible mares were infused with varying doses of lactoferrin at six hours post-breeding. Although not in a dose-dependent fashion, lactoferrin was found to decrease both fluid retention and neutrophil migration, in addition to suppressing the expression of the pro-inflammatory cytokine interferon gamma (IFNγ) and increasing the gene expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN). In conclusion, CRISP-3 expression occurs in secretory aspects of the male reproductive tract, and appears to be up regulated after sexual maturation. Viability of spermatozoa affects the immune response to breeding and should be taken into consideration for experimental design and interpretation of data. The seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on endometrial gene expression in normal mares, but lactoferrin suppresses the expression of TNF in susceptible mares. Finally, lactoferrin was found to function as a potent anti-inflammatory for the persistent inflammation seen in susceptible mares when administered post-breeding. This protein should be further investigated as a potential therapeutic for the treatment of persistent breeding-induced endometritis.