L'anthracnose constitue pour plusieurs productions végétales une maladie importante qui engendre des pertes post récoltes considérables. Colletotrichum musae est le responsable de cette maladie chez la banane dessert. L'utilisation d'antimicrobiens naturels comme le système lactoperoxydase (LPS) représente une voie naturelle de lutte intéressante contre l'anthracnose. Dans cette étude, nous avons amélioré le LPS en ajoutant de l'iode dans le système existant ou en substituant le thiocyanate par de l'iode. La substitution du thiocyanate à l'iode a permis d'avoir un effet fongicide du LPS. De plus, d'autres substances comme la lactoferrine, le Bioxeda® et l'huile de Neem ont été étudiées pour leur effet antifongique. Les deux dernières substances ont donné une inhibition supérieure respectivement à 90% et à 40%., tandis qu'il n'y a eu aucun effet de la lactoferrine / Postharvest diseases are a major concern for several plant products, leading to considerable postharvest losses. Colletotrichum musae is responsible for anthracnose and is also involved in crown rot, the two main postharvest diseases of bananas. The use of natural anti-microbial agents such as the lactoperoxidase system (LPS) represents an interesting alternative to the use of fungicides for the control of postharvest diseases of bananas. This study consisted on optimization of the LPS by adding iodide or substituting the thiocyanate by iodide. Moreover, other substances like lactoferrin, Bioxeda® and Neem oil were analyzed for their antifungal effect. The last two compounds gave an inhibition higher than 90% and 40% respectively. No effect of lactoferrin was observed

<p>Kontrola zdravlja vimena krava je bitan element u procesu proizvodnje zdravstveno<br />bezbednog mleka, te se na farmama visokomlečnih krava, kroz program kontrole mastitisa,<br />redovno sprovode mere otkrivanja i prevencije bolesti vimena. Klinički pregled vimena<br />predstavlja osnovni metod koji pruţa korisne informacije o zdravstvenom statusu vimena<br />krava, ali nailazi na pote&scaron;koće u otkrivanju patolo&scaron;kih promena unutar parenhima i papile<br />vimena. U cilju otkrivanja promena u parenhimu vimena moţe se primeniti ultrazvučni<br />pregled koji omogućava vizualizaciju strukturnih promena vimena nastalih kao posledica<br />upalnih procesa i tako olak&scaron;ava dijagnostiku oboljenja.<br />Tokom poslednjih godina, javio se problem povećanja rezistencije bakterija na<br />antimikrobne lekove, &scaron;to oteţava lečenje bolesti, ali i ugroţava zdravlje ţivotinja i ljudi.<br />Najče&scaron;ći uzroci toga su nepravilna upotreba i eventualna zloupotreba antimikrobnih lekova.<br />Mnoga istraţivanja vr&scaron;ena su u in vitro i in vivo uslovima na primeni laktoferina samog ili u<br />kombinaciji sa antibioticima u terapiji i prevenciji mastitisa krava. Laktoferin, gvoţĎe<br />vezujući antimikrobni glikoprotein koji se nalazi u mleku i drugim sekretima, predstavlja<br />bitan deo sistema odbrane mlečne ţlezde.<br />Cilj istraţivanja u okviru ove disertacije je procena dijagnostičke mogućnosti<br />ultrazvučnog pregleda u detekciji subkliničkog mastitisa i poremećene sekrecije vimena,<br />kao i razmatranje opravdanosti primene laktoferina u prevenciji i lečenju mastitisa.<br />Kliničkim pregledom izvr&scaron;ena je procena op&scaron;teg zdravstvenog stanja krava, kao i<br />ispitivanje mlečne ţlezde adspekcijom i palpacijom. Za otkrivanje poremećene sekrecije<br />vimena i subkliničkih mastitisa kori&scaron;ćeni su brzi testovi, Kalifornija mastitis test i<br />Draminski test, kao i ultrazvučni pregled mlečne ţlezde krava. OdreĎivanje broja somatskih<br />ćelija u uzorcima mleka uraĎeno je metodom protočne citometrije. Za identifikaciju<br />uzročnika mastitisa kori&scaron;ćene su klasične mikrobiolo&scaron;ke metode. Krave sa pozitivnim<br />bakteriolo&scaron;kim nalazom podeljene su u dve ogledne grupe. Krave ogledne grupe I su<br />tretirane intramamarnom aplikacijom antibiotika, dok je kravama ogledne grupe II<br />aplikovana kombinacija antibiotika i laktoferina. OdreĎivanje koncentracije imunoglobulina<br />G u mlečnom serumu krava vr&scaron;eno je metodom radioimunodifuzije, a odreĎivanje<br />koncentracije laktoferina u mleku krava ELISA testom.<br />U istraţivanjima u okviru ove disertacije, najče&scaron;će izolovani major mastitis patogeni<br />bile su bakterije Staphylococcus aureus i Streptococcus agalactiae, a najče&scaron;će izolovani<br />minor mastitis patogeni Corynebacterium spp. i koagulaza negativne stafilokoke.<br />Ultrasonografija mlečne ţlezde krava pokazala se kao efikasna metoda u dijagnostici<br />poremećaja sekrecije vimena. Veće vrednosti koncentracije imunoglobulina G u mlečnom<br />serumu krava uočene su tokom perioda predzasu&scaron;enja i zasu&scaron;enja, u odnosu na period rane<br />laktacije. Najveći uticaj na porast koncentracije laktoferina u mleku krava su imali<br />bakterijski uzročnici mastitisa. Efikasnost antibiotske terapije primenjene tokom perioda<br />zasu&scaron;enja kod krava ogledne grupe I iznosila je 52,7%, dok je efikasnost primenjene terapije<br />sa goveĎim laktoferinom i antibiotikom kod krava ogledne grupe II iznosila 60%. Aplikacija<br />laktoferina tokom perioda zasu&scaron;enja doprinela je efikasnosti terapije intramamarnih<br />infekcija, ali nije imala uticaj na koncentraciju laktoferina u mleku tokom perioda rane<br />laktacije.<br />Dalja istraţivanja su neophodna kako bi se u potpunosti potvrdila efikasnost<br />intramamarne terapije sa goveĎim laktoferinom i antibiotikom u cilju eliminacije uzročnika<br />mastitisa i da se precizno odredi terapijska koncentracija laktoferina.</p> / <p>Control of udder health is an essential element in the process of safe milk<br />production, thus, through the mastitis control program, dairy farms regularly conduct<br />measures of detection and prevention of udder diseases. Clinical examination of the udder<br />is a basic method that provides useful information about the health status of cow udders,<br />but this method is limited in its ability to detect pathological changes in the udder<br />parenchyma and teat. In order to detect changes in the parenchyma of the udder, an<br />ultrasound can be applied which allows visualization of udder structural changes incurred<br />as a result of inflammatory processes, thus facilitating diagnosis of diseases.<br />Over the last few years, the problem of increasing resistance to antimicrobial drugs<br />has appeared, making it difficult to treat disease and also threaten the health of animals and<br />humans. The incorrect and widespread use of antimicrobial drugs are the most common<br />cause of this. Many studies were conducted in vitro and in vivo conditions on the use of<br />lactoferrin alone or in combination with antibiotics in the treatment and prevention of<br />mastitis. Lactoferrin, an iron-binding antimicrobial glycoprotein which is found in milk<br />and other secretions, represents an important part of the mammary gland defense system.<br />The aim of the research within this thesis was to evaluate the diagnostic<br />possibilities of udder ultrasonography in the detection of subclinical mastitis and udder<br />secretion disorders, as well as considering the feasibility of lactoferrin application in the<br />prevention and treatment of mastitis.<br />The general condition of the animals was evaluated by clinical examination, as<br />well as udder examination by adspection and palpation. The California mastitis test,<br />Draminski test and ultrasound examination of the cow&#39;s mammary glands were used for<br />detection of udder secretion disorders and subclinical mastitis. Somatic cell count in milk<br />samples was determined by flow cytometry method. Classical microbiological methods<br />were used for the isolation and identification of mastitis causative agents. Cows with<br />positive bacteriological findings were divided into two experimental groups. Cows in<br />experimental group I were treated with intramammary applications of antibiotics, while the<br />cows in experimental group II were treated with a combination of antibiotics and<br />lactoferrin. Immunoglobulin G concentration in bovine milk serum was determined by the<br />immunodiffusion method, while lactoferrin concentration in bovine milk was determined<br />using the ELISA test.<br />In this study, the most common isolated major mastitis pathogens were<br />Staphylococcus aureus and Streptococcus agalactiae, while Corynebacterium spp. and<br />coagulase-negatice staphilococci were the most commonly detected minor mastitis<br />pathogens. Ultrasonography of the bovine mammary gland proved to be an effective<br />method in the diagnosis of udder secretion disorders. Higher immunoglobulin G<br />concentrations were observed in milk serum from cows during pre-dry and dry period,<br />relative to early lactation period. The biggest influence on the increase in the concentration<br />of lactoferrin in the bovine milk had mastitis pathogens. The efficacy of antibiotic therapy<br />during the dry period in cows of experimental group I was 52.7%, while the efficacy of the<br />applied therapy with lactoferrina and antibiotics in cows of experimental group II was<br />60%. Application of lactoferrin during the dry period contributed to the effectiveness of<br />the treatment of intramammary infections, but had no influence on lactoferrin<br />concentration in the milk during the early lactation period.<br />Further studies are necessary to in order to fully confirm the efficacy of<br />intramammary therapy with bovine lactoferrin and antibiotic to eliminate the mastitis<br />pathogens and to determine the therapeutic concentration of lactoferrin.</p>

Bakgrund: Gingivit kännetecknas av svullet/lättblödande och ibland ömt tandkött. Gingivit är en inflammatoriskt orsakad reaktion som uppkommer i samband med närvaro av bakterier. För att motverka utvecklingen av gingivit rekommenderas munhygienvård regelbundet. Gingivit kan utvecklas till parodontit med skador på upphängningsapparaten och benstödet, orsaken till detta är idag dock ej helt klarlagd. Kliniska variabler (blödning vid sondering) är vedertagna diagnostiska metoderna för diagnostisk av gingivit idag. I pilotstudier har laktoferrinkoncentrationer setts vara förhöjda i stimulerad helsaliv och gingivalexsudat hos patienter med kronisk parodontit jämfört med patienter utan parodontit. Mål: Undersöka hur koncentrationen laktoferrin i ostimulerad helsaliv förändrades hos individer som utvecklade gingivit i en experimentell gingivitstudie och om C-reaktivt protein är en tillförlitlig markör för gingival inflammationsgrad.Material och metod: En experimentell gingivitstudie genomfördes med 13 försöksdeltagare där munhygienvanor kontrollerades och förändrades under 3 perioder á 2 veckor. Helsalivprov och mätning av CRP genomfördes. Resultat: Gingivalindex och CRP höjdes signifikant under försöksperioden Dag 0 till Dag 13(p=0,002 och p=0,027). Laktoferrin sågs först signifikant förhöjd under mitten av avslutningsperioden (Dag 20) (p=0,015). Laktoferrinkoncentrationen återgick inte till baselinevärdet under studien. Signifikant korrelation sågs mellan gingivalindex och laktoferrin från Dag 0 fram till Dag 13 (r=0,224, p=0,042). Utvecklingen av CRP sågs inte korrelera med någon variabel under försöket. Konklusion: Resultaten indikerar att utvecklingen av experimentell gingivit påverkar laktoferrinkoncentrationen över tid och kan därför vara en potentiell biomarkör för gingivit. De låga skillnader hos CRP visar att den inte är en tillförlitlig markör vid gingivit. / Background: Gingivitis is characterized by swollen and bleeding tissue, an inflammatory reaction with presence of pathological bacteria. To counteract the development of gingivitis, regular oral hygiene care is recommended. Gingivitis can progress into periodontitis which damages the periodontal tissue and bone. Clinical variables (bleeding on probing, plaque occurrence) are diagnostic methods for gingivitis. Elevated concentrations of lactoferrin in stimulated whole saliva and gingival crevicular fluid has been seen in patients with chronic periodontitis compared to patients without periodontitis.Objectives: Investigate the salivary concentration of lactoferrin in subjects enrolled in the experimental gingivitis model, the response of capillary CRP was also investigated.Methods: Thirteen individuals participated in a 14-day experimental gingivitis model and 10-day gingival healing period, measurements of whole saliva lactoferrin and CRP was performed. Results: Gingival index and CRP was increased statistical significant during the induction of gingivitis (p=0.002 and p=0,027). Lactoferrin increased during the experimental phase and reached a peak at day 20, which was statistical significant compared to baseline (p=0.015). A statistical significant correlation between gingival index and lactoferrin was seen from day 0 to day 13 (r=0.224, p=0.042). CRP did not match any correlation to any other measured variable from day 0 to day 24.Conclusion: These results indicate that gingivitis may have a systemic effect, which in this study has been expressed as increased levels of salivary lactoferrin. Lactoferrin may be a potential biomarker for gingivitis. Due to low changes in concentrations and because CRP is systemically affected it makes an unreliable biomarker for gingivitis.

To gain insight in the relationship between innate immune system and metabolic disease, we aimed to investigate the effects of lactoferrin in obesity-related metabolic disturbances. Circulating lactoferrin concentration was significantly decreased in subjects with altered glucose tolerance (AGT) and associated negatively with obesity-related metabolic disturbances. The SNPs-induced aminoacidic changes in lactoferrin N-terminus region were associated with a low atherogenic lipid profile. Lactoferrin production in neutrophils decreased significatively in aging, chronic low-grade inflammation and type 2 diabetes. In vitro, lactoferrin increased insulin signaling pathway, even under insulin resistance conditions and displayed dual effects on adipogenesis (antiadipogenic in 3T3-L1 and adipogenic in human adipocytes). In conclusion, lactoferrin might play a potential protective role against insulin resistance and obesity related metabolic disturbances. / Per aprofundir en la relació entre el sistema immunològic innat i els trastorns metabólics, s’investiga l’efecte de la lactoferrina en els desordres metabòlics associats a l’obesitat. Els nivells circulants de lactoferrina es trobaven significativament disminuits en subjetes amb la tolerància a la glucosa alterada (AGT), i aquests es correlacionaven negativament amb trastorns metabòlics associats a obesitat i resistència a la insulina. Els canvis aminoacídics en la seva regió N-Terminal s’associaven a un perfil lipídic menys aterogènic. La producció de lactoferrina es troba reduïda en condicions d’envelliment, inflamació crònica i diabetes tipus 2. In vitro, la lactoferrina incrementava la via de senyalització de la insulina, inclús en condicions de resistència a la insulina i presentava efectes dual en la adipogenesis (antiadipogenic en 3T3-L1 i adipogenic en adipòcits humans). En conclusió, la lactoferrina podria tenir un potencial efecte protector enfront les enfermetats metabòliques associades a obesitat i resistència a la insulina.

Various methods for extraction and analysis of high value minor proteins (lactoferrin, lactoperoxidase and immunoglobulins) directly from raw milk were explored. Extraction, purification and analysis of high-value minor proteins directly from milk without pre-treatment are major challenges for dairy industry, largely due to the complexity of milk and the presence of colloidal solids (casein micelles and milk fat globules). To overcome some of these challenges, this work focused on three main objectives: 1) characterization of cryogel monolith chromatography for purification of lactoferrin (LF) and lactoperoxidase (LP) directly from raw milk in single step, 2) identification and characterization of Protein A Mimetic affinity ligands for purification of immunoglobulins (Igs) from milk and 3) development and validation of a surface plasmon resonance method for simultaneous quantification of five whey proteins in multiple samples. Results portrayed the possibility of 40–50 column volumes of various milk samples (whole milk, skim milk and acid whey) to pass through a 5 mL cryogel monolith chromatography column at 525 cm hr⁻¹ without exceeding its pressure limits if the processing temperature is maintained around 35–37°C. Ideally, this should be the milk secretion temperature. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg mL⁻¹) was similar to that of the binding capacity (2.01 mg mL⁻¹) at equilibrium with 0.1 mg mL⁻¹ of lactoferrin in the feed samples. Lactoferrin and lactoperoxidase was selectively bound to the cryogel column with trivial leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of 85% and a purity of 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk, provided that the binding capacity can be increased. A Protein A Mimetic (PAM) hexapeptide (HWRGWV) peptide ligand that binds to the Fc portion of antibody molecules was explored for affinity purification of immunoglobulins from milk. The peptide has the ability to purify IgG from various milk and whey samples with a purity of greater than 85% in single step. More than 90% bound IgG was recovered with 0.2 M acetate buffer at pH 4.0 and total column regeneration was successfully achieved by 2.0 M guanidine-HCl. At 9.0 mg mL⁻¹ of IgG feed concentration, an equilibrium binding capacity of 21.7 mg mL⁻¹ and dynamic binding capacity of approximately 12.0 mg mL⁻¹ of resin was obtained. Recoveries and yields of IgG were significantly influenced by the feed IgG concentration. PAM hexamer ligand also contributed a significant amount of cross-reactivity with casein, glycomacropeptides and β-lactoglobulin proteins, however majority of these proteins were recovered in the regeneration step, except β-lactoglobulin, which co-eluted with IgG. Higher IgG concentration in feed vastly reduced the amount of cross-reactivity whilst increasing the recoveries and purities in the final product. PAM affinity ligands also showed interactions towards other classes of bovine immunoglobulins. These findings established the possibility of using PAM hexamer peptide as an alternative to conventional Protein A/G affinity chromatography for the isolation of Igs from milk in single step process. A surface plasmon resonance (SPR) method was developed for simultaneous, quantitative determination of commercially important whey proteins in raw and processed milk samples, whey fractions and various milk-derived products, with six samples per assay. Immobilized antibody stability and reproducibility of analyses were studied over time for 25 independent runs (n=300), giving a relative standard deviation (RSD) of <4%. Immobilized antibodies showed negligible non-specific interactions (<2–4 SPR response units (RU)) and no cross-reactivity towards other milk components (<1 RU). Regeneration of immobilised antibodies with glycine at pH 1.75 was determined to be optimal for maintaining the SPR response between samples. This method compared and validated well with reversed phase high performance liquid chromatography (RP-HPLC) and standard enzyme-linked immunosorbent assays (ELISA).

Coacervation between the milk proteins β-lactoglobulin (BLG) and Lactoferrin (LF) was studied as a model system for hetero-protein coacervation (HPC). Equilibria among BLG/LF complexes and the corresponding speciation were found to control coacervation, which can be quantitatively monitored by turbidimetry. Several methods were used to assess complexation as a function of LF : BLG (mol/mol) mixing ratio (r). Proton release, calculated from a shift in pH when LF is added to BLG, was used to identify regions of complexation. Dynamic light scattering (DLS) was used to determine regions of complexation by relating complex size to stoichiometry. Isothermal titration calorimetry (ITC) was used to measure enthalpies of binding upon addition of LF to BLG. These results are used to show that coacervation is related to speciation, with the LF(BLG2)2 complex as the coacervating species.

Les antibiotiques sont fréquemment utilisés dans l’alimentation de la volaille afin de prévenir certaines maladies, dont l’entérite nécrotique, ce qui occasionne l’émergence de souches bactériennes résistantes aux antibiotiques. Une alternative prometteuse est l’utilisation de peptides antimicrobiens (AMPs) comme suppléments alimentaires, tels les AMPs provenant des produits laitiers. L’objectif du projet était de développer une méthode de production d’extraits peptidiques à partir de coproduits de la transformation alimentaire (babeurre, lactoferrine, isolat de protéines de pois), afin de tester si ces extraits peptidiques possédaient une activité antimicrobienne sur les pathogènes spécifiques aviaires suivants : Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli et Staphylococcus aureus. Les protéines ont été mises en suspension dans l’eau (5% p/p) et hydrolysées par la pepsine, 6 heures, pH de 2.5. Les peptides furent récupérés par ultrafiltration (< 10 kDa), puis fractionnés selon leur charge nette : totaux, cationiques, anioniques et non liés. L’effet antimicrobien a été évalué surmicroplaques, par la survie bactérienne en présence de concentrations croissantes d’extraits peptidiques. Les extraits cationiques de babeurre ont démontré une efficacité à une concentration inférieure ou égale à 5 mg/mL; perte de 3 log pour Escherichia coli O78 :H80. En comparaison, la lactoferrine cationique a été efficace à une concentration inférieure ou égale à 0.6 mg/mL; perte de 6 log pour E. coli O78 :H80. Les extraits peptidiques du pois ont démontré une efficacité faible. Cette méthode s’avère prometteuse pour le développement d’une alternative ou d’un complément pour la réduction de l’utilisation des antibiotiques dans l’alimentation de la volaille. / Antibiotics are frequently used in poultry feed in order to prevent certain diseases, including necrotic enteritis, which causes the emergence of bacterial strains resistant to antibiotics. A promising alternative is the use of antimicrobial peptides (AMPs) as dietary supplements, such as AMPs from dairy products. The objective of this project was to develop a production method for the extraction peptides, from co-produced food processing (buttermilk, lactoferrin, pea protein isolates). These peptides were tested for the detection of antimicrobial activity on the following specific poultry pathogens; Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. Proteins were suspended in water (5% w/w) and pepsin hydrolyzed by pepsin for 6 hours at pH 2.5. Peptides were recovered by ultrafiltration (< 10 kDa) and fractionated based on the basis of their ionic charges: total, cationic, anionic and unbound peptides, to specifically target the fractions with antimicrobial activities. Bacterial survival was measured in contact with different peptides concentrations. Cationic buttermilk extracts were effective at a concentration less or equal to 5 mg / mL; loss of 3 log for Escherichia coli O78: H80, compared with lactoferrin which was effective at a concentration less than or equal to 0.6 mg / mL; loss of 6 log for E. coli O78: H80. The peptide extracts from pea showed low efficiency. The use of antimicrobial peptides, from buttermilk, lactoferrin and peas, is promising for the development of an alternative or a complement to reduce antimicrobial use.

Anaemia, which is caused by iron deficiency, is a global nutritional disorder of utmost concern. It has been assumed that meat, which contains haem as well as non-haem iron, enhances non-haem iron absorption due to the presence of the “meat factor”. In the experiment reported here, 24 semi-anaemic, 3-week-old piglets were utilised as human nutrition models to assess the effects of dietary lactoferrin, meat and LSF (Low Molecular Weight Sarcoplasmic Fraction) on iron bioavailability during a 4-week feeding period. The parameters that were used as measurements of iron bioavailability were changes in haematological indices, haemoglobin iron repletion efficiency, intestinal morphology and mineral balances. Non-significant (p > 0.05) dietary effects were observed for growth performance and for all the haematological and some histological parameters (small intestine villi height, crypt depth and mucosal thickness). Haemoglobin iron repletion efficiency was highest for the control group and was not significantly different between the other 3 diets. The superiority of the control diet in this respect was not consistent with previous trials and can not be explained. Results suggested that increased retention of calcium, magnesium, phosphorous and manganese tended to inhibit iron absorption. However, the LSF and meat diets significantly (p = 0.003) increased the number of goblet cells/100 µm suggesting that mucin secretion was favoured by these two diets. Additionally, all immunological parameters were significantly (p < 0.05) improved by the LSF diet. As such, the LSF diet can be a potential immunobooster feed ingredient for weanling piglets. Overall, the level of LSF in the LSF diet was insufficient to exert a desirable enhancement of iron bioavailability and betterment of 3 iron status of the semi-anaemic piglets relative to the control group. However, the diet containing LSF was as effective as the meat diet with respect to these characteristics.

Les cellules dendritiques présentes dans les muqueuses comme les cellules de Langerhans, sont capables de présenter le Virus d'Immunodéficience Humaine (VIH) aux lymphocytes T CD4+, aboutissant à l'infection par le VIH lors de contacts hétérosexuels non protégés. Le recrutement des cellules de Langerhans dans la muqueuse vaginale est assuré par la chimiokine Quimiocin C-C motif ligant 20 (CCL20) produite par les cellules épithéliales de la muqueuse. L'objectif de ce travail a été d’étudier la capacité du plasma séminal, de la salive et de la sécrétion vaginale à stimuler la production de CCL20 par les cellules de l’épithélium endocervical (cellules HEC-1A), et de corréler cette production avec la présence de la lactoferrine (LF) et de ses produits de clivage dans ces différents fluides biologiques. Des cellules HEC-1A ont été cultivées avec les plasmas séminaux, les salives et les sécrétions vaginales de patients séronégatifs et séropositifs pour le VIH, et la production de CCL20 a été mesurée dans les surnageants cellulaires en ELISA. Le dosage de la Lf par ELISA a été réalisé sur tous les échantillons. La présence d’un petide de clivage de la Lf (G-12-I) a été analysée en western blot. Les résultats obtenus montrent que les plasmas séminaux des patients séropositifs ont une plus grande capacité à stimuler la sécrétion de CCL20 que les patients séronégatifs pour le VIH. La concentration de la Lf dans le plasma séminal est corrélée à la stimulation de cette sécrétion par les cellules HEC-1A. Il semble que les échantillons ayant la plus grande capacité à stimuler la sécrétion de CCL20 possèdent une plus forte concentration en petide de clivage de la Lf (G-12-I), bien que ce résultat, obtenu en western blot. La salive est également capable de stimuler de la production de CCL20 par les cellules HEC-1A, mais cette stimulation n'est pas corrélée avec sa concentration en Lf. Nous concluons que la sécrétion de CCL20 par l’épithélium génital féminin induite par le plasma séminal est corrélée à la présence de la Lf dans ce fluide. Bien que les mécanismes puissent être différents, la salive peut également induire la production de CCL20 par les cellules épithéliales de la muqueuse génitale féminine / Dendritic cells, among them Langerhans cells, are recognized as presenters of Human Immunodeficiency Virus (HIV) to CD4 + T lymphocytes. The recruitment of Langerhans cells in the vaginal mucosa is modulated by Chemokine CC motif ligand 20 (CCL20) produced by epithelial cells of the female genital mucosa. The aim of this study was to evaluated the capacity of seminal plasma, saliva and vaginal secretions to induce the production of CCL20 by monostratified endocervical epithelium cells culture (HEC-1A), also, the relation of this production with the presence of lactoferrin and its cleavage products (G- 12-I) in seminal plasma and saliva. Cultures of HEC-1A cells were stimulated with seminal plasma, saliva and vaginal secretions of HIV-seronegative and HIV-seropositive participants, and the CCL20 production measured by enzyme linked immunosorbent assay (ELISA) in its cellular supernatant. ELISA for Lf was performed on all samples, and the peptide cleavage Lf (G-12-I) observed in the samples which more and less induced the CCL20 secretion on western blot. We observed that the seminal plasma of seropositive participants were responsible for a higher stimulation of CCL20 production by HEC-1A cells, when compared to the seminal plasma of HIV-seronegative participants. Lf concentration in seminal plasma was positively related to the CCL20 production by HEC-1A cells, the higher production of CCL20 was also more associated with Lf peptide cleavage Lf (G-12-I), on western blot. Saliva stimulated CCL20 production by HEC-1A cells, and such stimulation was not correlated to Lf concentration. In conclusion, the presence of Lf in seminal plasma positively related to the production of CCL20 by HEC-1A cells, and saliva may induce the production of CCL20 in the female genital mucosa / As células dentríticas, dentre elas as células de Langerhans, são capazes de apresentar o vírus da imunodeficiência humana (HIV) aos linfócitos T CD4+, cujo processo culmina com a infecção por esse vírus. O recrutamento das células de Langerhans na mucosa vaginal é modulada pela Quimiocin C-C motif ligant 20 (CCL20), produzida pelas células epiteliais da mucosa genital feminina. O objetivo desse trabalho foi estudar a capacidade do plasma seminal, da saliva e do conteúdo vaginal de induzir a produção de CCL20 pela cultura de células do epitélio monoestratificado endocervical (HEC-1A), relacionando tal produção com a presença da lactoferrina (Lf) e de seu produto de clivagem (G-12-I) nestes fluidos. Culturas de células HEC-1A foram estimuladas com plasmas seminais, salivas e conteúdos vaginais de participantes soronegativos e soropositivos para o HIV, visando avaliar a produção de CCL20, a qual foi mensurada utilizando-se o Enzyme Linked Immunosorbent Assay (ELISA). A dosagem de Lf (ELISA) foi realizada em todas as amostras estudadas e a presença do peptídeo de clivagem da Lf (G-12-I) observada nas amostras que mais e que menos induziram a secreção de CCL20 pelas células HEC-1A por Western blot. Verificou-se que os plasmas seminais de participantes soropositivos foram responsáveis por maior estimulação da produção de CCL20 pelas células HEC-1A, quando comparados aos plasmas seminais de participantes soronegativos para o HIV. A concentração de Lf no plasma seminal associou-se à indução na produção de CCL20 pelas células HEC-1A, assim como as amostras que mais induziram a produção de CCL20 apresentaram presenças mais evidentes do peptídeo de clivagem da Lf (G-12-I). A saliva foi responsável pela estimulação na produção de CCL20 pelas células HEC-1A, no entanto, tal estimulação não se associou à concentração de Lf salivar. Concluímos que a presença de Lf no plasma seminal esteve correlacionada à produção de CCL20 pelas células HEC-1A, e que a saliva pode induzir a produção da CCL20 pelas células epiteliais endocervicais

Die vorliegende Arbeit beschäftigt sich mit der Untersuchung und Beschreibung der Eigenschaften der synthetischen Dekapeptide Cetrorelix und Ozarelix durch analytische Methoden und computergestützte Modellierung. Diese Moleküle sind hydrophobe, aggregierende Antagonisten des Gonadotropin-Releasing-Hormons (GnRH). Zusätzlich wurden amyloidbildende Peptidstrukturen als Modelle für die Assoziationsprozesse in hydrophoben Peptiden untersucht und visualisiert. Die intrinsische Fluoreszenz der GnRH-Antagonisten und zusätzlich der Peptide Teverelix und D-Phe6-GnRH sowie von verkürzten Fragmenten des Cetrorelix wurde untersucht. Ein Strukturmodell für die Beschreibung der Aggregation der Dekapeptide wurde erarbeitet. Der Aufbau eines Rechenclusters durch das Einbinden der Computer am Lehrstuhl in ein Linux-System zur Verteilung von Rechenprozessen über das Netzwerk ermöglichte die Bereitstellung der notwendigen Leistung zur Realisierung der Berechnungen. Es wurden Werkzeuge zur Modellierung der solvatisierten Aggregate von Peptiden ohne eindeutige Vorzugsstruktur programmiert und in ein Docking-System für beliebige Moleküle eingebunden. Verwendet wurde das Kraftfeld MMFF94 mit einer Erweiterung durch ein Verfahren zur dynamischen Berechnung von Partialladungen in Molekülstrukturen. Solvatisierte Aggregate der Dekapeptide und von bekannten amyloidbildenden Strukturen wurden modelliert (Docking). Berechnet wurden als aggregierend beschriebene Sequenzen und entsprechende Vergleichsstrukturen des Calcitonins, des Insel-Amyloid-Polypeptides, des beta2-Mikroglobulins, des Amyloid-beta-Proteins, des Lactoferrins und weitere Modellpeptide. Die wesentlichen Wechselwirkungen während der Aggregation konnten schließlich anhand von Dynamik-Simulationen der faltblattartigen Dimere des Cetrorelix und Ozarelix beschrieben werden. So wurden die Prozesse der hydrophoben Assoziation und Stabilisierung durch Wasserstoffbrücken von Peptiden veranschaulicht und auf molekularer Ebene erfolgreich analysiert. Die Visualisierung der erhaltenen Modellierungsergebnisse erfolgt durch die Darstellung der Strukturen und Dynamik-Simulationen als interaktive 3D-Modelle in einem für diese Arbeit aufgebauten Internetauftritt. / This work discusses the analysis of the aggregation properties of the gonadotropin releasing hormone antagonists Cetrorelix, Teverelix, Ozarelix and of small amyloid forming model peptides by analytical fluorescence spectroscopy and molecular modelling. A high performance linux compute cluster was developed for calculation of molecular structures. Solvated aggregate clusters of peptides without defined secondary structure were modelled by molecular mechanics methods (forcefield mmff94) in combination with an advanced charge equilibration and docking technique. Molecular dynamics of solvated peptide dimers were implemented and the role of hydrophic association and hydrogen bond formation in hydrophobic peptide aggregates was explained. Finally, an aggregation model for the directed association of hydrophobic peptides is presented. The modelling results, 3d structures and dynamic simulations are visualized in an interactive web material.