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Different models for the binding of the ligand tumour necrosis factor (TNF) to its receptors can be imagined. In a first model the homotrimeric ligand merges three single receptor molecules upon binding. Thereby their intracellular parts are brought into proximity, which enables signal initiation. Based on the discovery of a preassociation of the TNF receptors prior to ligand binding, a distinct model was proposed, in which the TNF trimer binds at once to several preassociated receptor molecules. A conformational change in the receptor molecules could then bring the intracellular receptor parts in proximity thereby initiating signalling. In the TNF derivative single chain TNF (scTNF) the three TNF monomers are linked by two Glycine-Serine linkers. The observed reversible dissociation of the TNF homotrimer in its monomers is thereby prevented. Furthermore it is possible to mutate single receptor binding sites in the trimer thereby generating TNF trimers with only one or only two active binding sites. With these molecules it is possible to draw conclusions on the binding mode of TNF. If TNF binds to several preaggregated receptor molecules at once, one would expect a strong reduction in affinity in saturation binding studies at 0°C. If TNF binds only to one single receptor or to only one receptor of a preformed aggregate, the omission of single binding sites should have no strong effects. Using this approach it was possible to show in this work that TNF receptor 2 (TNFR2) must exist in a preaggregated state prior to ligand binding, most likely as a receptor homotrimer. For TNF receptor 1 (TNFR1) the existence of preformed dimers can be assumed, because inactivation of one single binding site shows no significant effects on binding. Only when a second receptor binding site of scTNF was impaired, affinity was reduced. By using another mutation influencing receptor binding also with a scTNF with two impaired binding sites no effect on binding affinity was achieved. However, this could be the case because of a comparably weak impact of the used mutation on receptor binding in general. Alternatively, an aggregation of single receptor molecules or of receptor dimers/trimers by the ligand can be discussed. Hence the model of binding to already preaggregated receptors could be clearly proven, at least for TNFR2. Furthermore it was shown that there exist clear differences in ligand interaction between the two TNF receptors. This might be caused by the structural differences between the receptors (differences in stem- and transmembrane domains) and/or a localisation in different areas of the cell membrane (membrane microdomains). The partially detectable bioactivity of TNF trimers with only one binding site displaying full binding properties further gives a hint for the existence of higher aggregates of several ligand molecules, their bound receptors and intracellular signalling molecules. A signal initiation between two adjacent receptor molecules each bound to another TNF trimer could explain the observed residual activity. ; Für die Bindung des Tumor Nekrose Faktors an seine Rezeptoren sind unterschiedliche Modelle vorstellbar. Zum einen ist es naheliegend, dass der als Homotrimer vorliegende Ligand drei einzelne Rezeptormoleküle zusammenführt und dadurch die intrazellulären Rezeptorbereiche angenähert werden, was letztendlich die Signalinitiierung ermöglicht. Ausgehend von der Entdeckung, dass die TNF-Rezeptoren schon in Abwesenheit des Liganden voraggregiert auf der Membran vorliegen, wurde jedoch ein neues Modell vorgeschlagen, bei dem das TNF Trimer zugleich mehrere voraggregierte Rezeptormoleküle bindet. Eine Konformationsänderung in den Rezeptormolekülen könnte dann zur Signalinitiierung führen. In dem TNF Derivat single chain TNF (scTNF) sind die drei TNF Monomere über zwei Glycin-Serin Linker kovalent verknüpft. Die sonst beobachtbare reversible Dissoziation des TNF-Homotrimers in seine Monomere kann dadurch verhindert werden. Zudem ist es möglich einzelne Rezeptorbindestellen im TNF-Trimer zu mutieren und dadurch TNF-Derivate mit nur einer oder nur zwei voll bindungsfähigen Bindestellen herzustellen. Mit Hilfe dieser Moleküle ist es möglich Rückschlüsse auf die Art und Weise, mit der TNF an seine Rezeptoren bindet, zu ziehen. Bei einer gleichzeitigen Bindung an mehrere Rezeptoren würde in Gleichgewichtsbindungsstudien bei 0°C ein starker Abfall der Affinität zu erwarten sein. Bei der Bindung an einen einzelnen Rezeptor oder an ein Molekül eines Rezeptoraggregats, sollte ein Wegfall einzelner Bindungsstellen keinen großen Einfluß haben. Mit diesem experimentellen Ansatz war es in vorliegender Arbeit möglich zu zeigen, dass der TNF Rezeptor 2 (TNFR2) voraggregiert vorliegen muß, sehr wahrscheinlich als Homotrimer. Für TNFR1 wird aufgrund der erhaltenen Ergebnisse ein Vorliegen von Dimeren vermutet, da das Inaktivieren einer Bindungsstelle keinen Effekt zeigte. Erst das Mutieren einer weiteren Bindungsstelle führte zu einer Affinitätsabnahme. Bei der Verwendung einer weiteren, die Bindung beeinflussenden Mutation wurde jedoch auch mit einem scTNF mit zwei beeinflußten Bindungsstellen auf TNFR1 nur ein minimaler Effekt auf die Bindungsfähigkeit erzielt. Dies könnte man sich jedoch mit einer im Vergleich eher schwachen Auswirkung der verwendeten Mutation auf die Rezeptoraffinität erklären. Alternativ kann eine Aggregierung von einzelnen Rezeptormolekülen oder von Rezeptor Dimeren oder Trimeren durch den Liganden diskutiert werden. Somit konnte das Model der Bindung an schon voraggregierte Rezeptoren zumindest für TNFR2 eindeutig belegt werden. Es konnte aber auch gezeigt werden, dass deutliche Unterschiede zwischen beiden Rezeptoren vorhanden sind. Dies mag in der unterschiedlichen Struktur der Rezeptoren begründet sein (Unterschiede in Stiel- und Transmembrandomäne) und/oder in einer Lokalisation in verschiedenen Bereichen der Zellmembran (Mikrodomänen). Die teilweise noch nachweisbare Bioaktivität eines TNF Trimers mit nur einer voll bindefähigen Rezeptorbindestelle ist zudem ein Hinweis auf die Existenz größerer Aggregate bestehend aus mehreren Liganden, ihren gebundenen Rezeptoren und intrazellulären Signalproteinen. Eine Signalinitiation durch zwei benachbarte Rezeptormoleküle jeweils gebunden an ein anderes TNF-Trimer könnte die vorhandene Restaktivität erklären.

Unsere Lebensmittel haben eine zweifache, auch vom Gesetz vorgegebene Zweckbestimmung, d.h. sie dienen einerseits dem Genuss und andererseits der Ernährung. Aktuelle For-schungsstrategien in der Lebensmittelchemie auf diesen Gebieten lassen sich dahingehend zusammenfassen, dass im ersten Bereich die Suche nach wirksamen Aromastoffen und deren Vorläufern sowie auch zunehmend Probleme der Herkunftsanalytik im Vordergrund stehen. Auf dem Gebiet der Ernährung spiegeln die Schlagworte "Funktionalität" und "Bioaktivität" die derzeitigen Entwicklungen wider. Für alle Bereiche bilden instrumentell-analytische Techniken die Grundlage für Bewertungen. Im Rahmen der vorliegenden Arbeit haben wir daher das uns zur Verfügung stehende analytische Potential genutzt, in den oben genannten Gebieten einschlägige Beiträge zu leisten, d.h. die Suche nach wertgebenden Aromastoffen fortzuführen, Zuckerkonjugate zu untersuchen, die sowohl als Aromavorläufer als auch im Hinblick auf Bioaktivitätsstudien von Bedeutung sein können, und schließlich Methoden zur Herkunftsanalytik zu forcieren. Zu diesen Studien sind als attraktive und wirtschaftlich be-deutsame Rohwaren Rucola (Eruca sativa Mill.) und Kaffee (Coffea arabica) eingesetzt worden. Mit Hilfe der Kopplung aus S-selektiver Detektion und strukturselektiver massen-spektro-metrischer Analytik sind in Extrakten von Rucolablättern 22 schwefelhaltige Verbindungen strukturell charakterisiert worden. Darunter befanden sich neben Minorkomponenten wie 3-Sulfanyl-1-hexanol, Glucosinolatabbauprodukte wie 4-Methylthiobutylnitril, 4-Methylthiobutylthiocyanat und 4-Methylthiobutylisothiocyanat. Mit [1,3]-Thiazepan-2-thion ist eine bislang nicht bekannte Komponente erstmals beschrieben wor-den. Das Vorkommen von 3-Sulfanyl-1-hexanol, welches im Übrigen anhand von MDGC-MS-Analytik in nahezu racemischer Form (44:56 Prozent, R:S) in Rucola gefunden wurde, forderte dazu heraus, in einer Modellstudie eine allgemeine Methode zur Bestimmung der Absolutkonfiguration azyklischer 2- und 3-Sulfanyl-1-alkanole mittels der ECCD-Methode zu entwickeln. Dabei wurden in einer Ein-Schritt-Synthese die funktionellen Grup-pen der 2- und 3-Sulfanyl-1-alkanole (in der Studie handelte es sich um enantiomerenreine 2- und 3-Sulfanyl-1-hexanole) jeweils mit dem Chromophor 9-Anthroylflourid derivatisiert. Die ent-sprechenden CD-Splitkurven erlaubten eine eindeutige Zuordnung der Konfiguration. Die entwickelte Mikro-Maßstab-Methode wurde ebenfalls er-folgreich zur Bestimmung der Absolutkonfiguration von in der Studie als zusätzliche Modellverbindungen eingesetzten 1,2- und 1,3-Diolen angewendet. 3-Sulfanyl-1-hexanol wurde abschließend in einer Zwei-Schritt-Synthese mit 9- und 2-Anthroylflourid derivatisiert, auch diese Variation erlaubte eine eindeutige Zuordnung der Absolutkonfiguration. Mittels präparativer HPLC-Techniken wurden drei neue Flavonoidglykoside aus Rucolablättern isoliert. Deren Charakterisierung erfolgte mittels Tandemmassenspektrometrie sowie ein- und zweidimensionalen NMR-Messungen. Die Verbindungen sind als Quercetin-3,3',4'-tri-O-beta-D-glucopyranosid, Quercetin-3'-(6-sinapoyl-O-beta-D-gluco-py-ra-nosyl)-3,4'-di-O-beta-D-gluco-pyranosid und Quercetin-3-(2-sinapoyl-O-beta-D-gluco-pyranosyl)-3'-(6-sina-poyl-O-beta-D-glucopyranosyl)-4'-O-beta-D-gluco-py-rano-sid identifiziert worden. Diese Quercetin-Derivate zeigten sowohl hinsichtlich der Glucosilierung des Aglykons (an den Positionen 3, 3' und 4') als auch bezüglich der Acylierung ungewöhnliche Strukturen. Bei Rohkaffee hat man bislang keine Studien über das Vorkommen von Zuckerkonjugaten als Aromastoffvorläufer durchgeführt. Wir konnten diese Lücke anhand der Strukturaufklärung der nachfolgend aufgeführten Verbindungen schließen. Durch Kombination verschiedener präparativer Techniken (CCC, LC und HPLC) mit verschiedenen Methoden der Strukturaufklärung (HRGC-MS, HPLC-MS/MS, ein- und zweidimensionale NMR) sind aus Rohkaffee fünf Aromastoffprekursoren isoliert und identifiziert worden. Für die beiden aus Rohkaffee isolierten Linalyldisaccharide wurden als Strukturen 3(S)-Linalyl-3-O-beta-D-glucopyranosyl-(1-6)-alpha-L-arabinofuranosid (AK1) und 3(S)-Linalyl-3-O-beta-D-gluco-pyranosyl-(1-6)-beta-D-apiofuranosid ermittelt. Weiterhin sind erstmals drei neue Aromastoffprekursoren in Form von Zuckerestern, d.h. (3-Methylbutanoyl)-1-O-beta-D-glucopyranosyl-beta-D-apiofuranosid, (3-Methylbuta-noyl)-6-O-alpha-D-gluco-pyranosyl-(1-2)-beta-D-fructofuranosid und (3-Methyl-2-butenoyl)-O-beta-D-gluco-pyranosyl-beta-D-apio-furanosid in Rohkaffee charakterisiert worden. Die Kenntnis der Herkunft bestimmter Lebensmittel bzw. einzelner In-haltstoffe spielt für die Lebensmittelindustrie eine wichtige Rolle. Für die Zuordnung der geographischen Herkunft von Rohkaffee wurden in dieser Arbeit die 13C/12C-, 2H-/1H- und 18O/16O-Verhältnisse von extrahiertem Coffein mittels Elementaranalysator-Isotopen-massen-spektro-metrie (EA-IRMS) bestimmt. Zur Bewertung der Multielement-Messwerte wurden diese statistischen Berechnungen unterzogen. Zur Anwendung kamen die Lineare Diskrimi-nanzanalyse (LDA) und die Auswertung mittels sog. "Classification and Regression Trees" (CART). In der Anpassung wurde dabei eine Probe aus der Klasse 1 (Provenienz Afrika) der Klasse 2 (Provenienz Amerika) zugeordnet und eine Probe der Klasse 2 der Klasse 1 zugeordnet (Fehlerrate von 5,1 Prozent). Bei Kreuzvalidierung wurden drei der 39 Proben falsch zugeordnet (jeweils zwei der Klasse 1 und eine der Klasse 2), die Fehlerrate betrug hier somit 7,7 Prozent. Die Proben der Klasse 3 (synthetisches Coffein) ließen sich zu 100 Prozent von denjenigen natürlichen Ursprungs unterscheiden. ; Our foods exhibit two functions defined by legislation, too, i.e. to provide pleasure and nutri-tive effects. Current research strategies in food chemistry in these areas can be summarized as follows, i.e. that in the first-mentioned area the search for effective aroma substances and their precursors as well as, increasingly, also problems of authenticity assessments predominate. In the area of nutrition, the highlights "functionality" and "bioactivity" reflect the actual research trends. In all areas instrumental-analytical techniques form the fundament of evaluations. Thus, we used our analytical potential to provide new informations in the above-mentioned areas, i.e. to continue the search for valuable aroma compounds, to study sugar conjugates being important as flavour precursors and for bioactivity studies, as well as, finally, to strengthen methods for authenticity assessment. For these studies rocket salad (Eruca sativa Mill.) and green coffee (Arabica coffea), both known as attractive industrially important raw materials, were used. In flavour extracts of rocket salad leaves we characterized 22 sulphur-containing constituents by means of HRGC-MS/SCD analysis. This analytical tool allows parallel detection of com-pounds eluting from the GC column by mass spectrometry (to get structural information) and by chemiluminescence (to get selectively signals of sulphur-containing substances). Among the identified constituents we detected minor compounds such as 3-sulfanyl-1-hexanol, by-products of myrosinase hydrolysis such as 4-methylthiobutyl nitrile, 4-methylthio-butyl thiocyanate and 4-methylthiobutyl isothiocyanate as well as [1,3]-thiazepan-2-thion, the latter described for the first time. By means of multidimensional GC-MS (MDGC-MS) the enantiomeric ratio of 3-sulfanyl hexanol was determined as 44 per cent (R) to 56 per cent (S). In addition, a method for the determination of the absolute configuration of chiral 2- and 3-sulfanyl-1-alkanols (and 1,2 diols and 1,3 diols as model compounds) was developed. We demonstrated for the first time that the CD exciton chirality method can be extended to acyclic 2- and 3-sulfanyl-1-alkanols. The simple one-step derivatization using the 9-anthroate chromophore provided a general microscale method for the determination of the absolute configuration. Exciton coupling between the two 9-anthroate chromophores led to intense positive split CD curves for the (R)-configured 2-sulfanyl-1-alkanols and the (S)-configured 3-sulfanyl-1-alkanols as well as vice versa. The developed method was also useful for the stereochemical assignment of 1,2- and 1,3-diols. Furthermore, the application of the two step-derivatization using two different chromophores for both functional groups (2- and 9-anthroate) also resulted in mirror image split CD curves for both enantiomers, allowing the stereochemical assignment of 3-sulfanyl-1-alkanols. Three novel flavonoid glycosides were isolated of leaves of rocket salad by means of preparative techniques. Structural elucidation was performed using LC-MS/MS as well as one and two dimensional NMR experiments. The compounds were identified as quercetin 3',4'-tri-O-beta-D-gluco-py-ra-no-side, quercetin 3'-(6-sinapoyl-O-beta-D-gluco-pyra-no-syl)-3,4'-di-O-beta-D-gluco-py-ra-no-side and quercetin 3-(2-sinapoyl-O-beta-D-glucopyranosyl)-3'-(6-sinapoyl-O-beta-D-gluco-pyra-no-syl)-4'-O-beta-D-gluco-py-rano-side. These quercetin derivatives exhibited unusual structures as to the glucosylation of the aglycones (at positions 3, 3' and 4') and the type of acylation. Studies of bound flavour precursors in green coffee beans (Coffea arabica) have not been carried out to date. We were able to fill out this gap by means of structural analysis of the following compounds. By combination of different preparative analytical steps (CCC, LC and HPLC) with several analytical methods of structural elucidation (HRGC-MS, HPLC-MS/MS, one and two dimensional NMR experiments) five flavour precursors were isolated and identi-fied in green coffee beans. The two isolated linalool disaccharides were identified as 3(S)-linalyl-3-O-beta-D-gluco-py-ra-nosyl-(1-6)-alpha-L-arabinofuranoside and 3(S)-linalyl-3-O-beta-D-gluco-pyra-no-syl-(1-6)-beta-D-apiofuranoside. In addition, three novel flavour precursors were identified as sugar esters, i.e. (3-methyl-butanoyl)-1-O-beta-D-glucopyranosyl-beta-D-apiofuranoside, (3-methylbutanoyl)-6-O-alpha-D-gluco-pyranosyl-(1-2)-beta-D-fructo-furano-side and (3-Methyl-2-buteno-yl)-O-beta-D-gluco-pyranosyl-beta-D-apio-furanoside. The knowledge of the origin of food and its ingredients is actually one of the major topics for the food industry. For the assignment of the geographic origin of green coffee we determined the ratios of 13C/12C, 2H/1H and 18O/16O of extracted caffeine by means of elemental analysator/isotopic mass spectrometry (EA-IRMS). To check the suitability of the analytical data obtained by EA-IRMS we used statistical calculations, i.e. (i) linear discrimination analysis (LDA) and (ii) validation via classification and regression trees (CART). In the course of adap-tion one sample of class 1 (origin Africa) was assigned to class 2 (origin middle and south America) and vice versa, resulting in an error rate of 5.1 per cent. Using cross validation three of the 39 analyzed samples were assigned incorrectly resulting in an error rate of 7.7 per cent. Samples of class 3 (synthetic caffeine) were discriminated from that of natural origin at a rate of 100 per cent.

Article Details: Received: 2018-04-09 | Accepted: 2018-10-08 | Available online: 2018-09-31https://doi.org/10.15414/afz.2018.21.03.84-92This experiment was carried out at Ladoke Akintola University of Technology, Teaching and Research farm during the late and early planting seasons of 2011 and 2012 to determine the efficacy of Moringa oleifera(L) and Anacardium occidentales(L) extracts on major insect pests of pests of watermelon and their effects on fatty acid compound of watermelon. This experiment was arranged in a randomized complete block design and each treatment was replicated three times. Each of the plant extracts was applied at three different concentrations (5, 10 and 20%). Gas chromatography was used to determine the level of fatty acid composition.The results clearly showed that the applied plant extracts exhibited insecticidal action against the Aulocophora africana (Weise) and Dacus cucurbitae(Coquillet). 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(1991) The cultivated plants of the tropics and subtropics. Munchen: Verlag Josef Margraf, Weikersheim.SCHIPPERS, R.R. (2000) African indigenous vegetable. In: An overview of the cultivated species Chatthan, pp. 56–60.SELJASEN, R., and MEADOW, R. (2006) Effect of neem on oviposition and egg and larval development of Mamestra brassicae L: Dose response, residential activity. Repellent effect and systemic activity in cabbage plants. Crop Protection, vol. 25, pp. 238–345.SINGH, R.N, and SARATCHANDRA, B. (2005) The development of botanical products with special refrence to seri-ecosyetem. Caspain Journal Environmental Science, vol. 3, no. 1, pp. 1–8.SINGH, R.N, and SARATCHANDRA, B. (2002) An integrated Approach in the Pest Management in the sericulture. International Journal Industries Entomology, no. 5, pp. 141–151.SOFIDIYA, M.O. et al. (2006) Free-radical scavenging activity of some Nigerian medicinal plant extracts. 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Undernutrition is a public health problem that is the subject of a national policy whoseobjectives include prevention and treatment of this condition. Administration of parenteralnutrition (PN) involves metabolic complications, including hyperglycaemia as a major adverseeffect. Hyperglycemia increases the morbidity and mortality of patients receiving PN, either inhospital or at home. Therefore, insulin should be administered in conjunction with theadministration of PN. Addition of human insulin directly into PN bag is recommended only whenthe stability of the insulin on the one hand and the PN admixture on the other have beendemonstrated.The main objective of this work is to determine the stability of human insulin and the admixtureof ternary PN, mixed together.In order to achieve this objective, the following studies are being carried out : 1) evaluation ofthe physical stability of the ternary PN admixture 2) validation of an immunometric method forthe quantification of human insulin, in order to measure insulin over a 24-hour period afteraddition to an industrial ternary parenteral nutrition admixture supplemented with vitaminsand trace elements, then to study the impact of different factors (macronutritment composition,packaging, pH, etc.) on the evolution of the insulin concentration in this complex admixture 3)validation of a high performance liquid chromatography method with tandem massspectrometric detection to quantify insulin and detect glycated insulin in a mixture of binary PNduring 24-hour kinetics 4) study of the bioactivity of human insulin, using an animal testingprotocol to compare different admixtures administered by infusion (PN, PN withextemporaneous addition of human insulin and PN with insulin added 12 hours prior toadministration).Stability parameters of the PN emulsion show that the nutrient admixture is stable. A 35% lossin insulin content was observed during the 24-hour study on the ternary PN admixturesupplemented with vitamins and trace elements with a major decrease during the first 6 hours.This evolution is explained at 67% by the level of dextrose content of the admixture, the natureof the lipid emulsion, the absence of trace elements as well as the pH when taken intoconsideration with the dextrose content, the time and the amino acid content. The glycation ofinsulin, as demonstrated by the chromatographic method, explains this decrease in insulincontent in the presence of a pH close to neutrality. Total reversibility of insulin glycation isobserved when samples are diluted in an acidic medium. During the in vivo experiment, thehighest lagomorphic insulin concentration is lower when human insulin is addedextemporaneously to the PN, suggesting that in this condition, human insulin has a higherbioactivity, which is consistent with data in the literature on glycated insulin.In conclusion, addition of insulin to a ternary PN admixture has the physical stability to ensurethe integrity of the emulsion. On the other hand, the insulin stability study revealed theexistence of an insulin glycation phenomenon occurring mainly in the first 6 hours of contactwith the PN admixture. This glycated insulin showed reduced hypoglycemic activity. Furtherstudies are needed to precise glycation process and complete the chemical stability of the PNadmixture. ; La dénutrition est un problème de santé publique qui fait l'objet d'une politique nationalecomportant parmi les objectifs, la prévention et le traitement de cette affection. L'administrationde nutrition parentérale (NP) comporte parmi ses effets indésirables métaboliques majeurs,l'hyperglycémie. Celle-ci augmente la morbi-mortalité des patients recevant de la NP, que ce soità l'hôpital ou au domicile. L'administration d'insuline doit par conséquent accompagner celle dela NP. L'ajout direct d'insuline humaine dans la poche de NP n'est recommandé que lorsque lastabilité de l'insuline d'une part et du mélange de NP d'autre part ont été démontrées.L'objectif principal de ces travaux de thèse est de déterminer la stabilité de l'insuline humaine etdu mélange de NP ternaire lorsqu'ils sont mis en présence l'un de l'autre.Afin d'atteindre cet objectif, les études suivantes sont mises en oeuvre : 1) évaluation de lastabilité physique du mélange de NP ternaire 2) validation d'une méthode immunométrique dequantification de l'insuline humaine afin de doser l'insuline sur une durée de 24 heures aprèsajout dans un mélange industriel de nutrition parentérale ternaire supplémenté en vitamines etéléments-trace puis d'étudier l'impact de différents facteurs (composition en macronutritments,conditionnement, pH…) sur l'évolution de la concentration en insuline dans ce mélangecomplexe 3) validation d'une méthode de chromatographie liquide haute performance avecdétection par spectrométrie de masse en tandem afin de quantifier l'insuline et détecterl'insuline glyquée dans un mélange de NP binaire au cours d'une cinétique de 24 heures 4)étude de la bioactivité de l'insuline à l'aide d'un protocole d'expérimentation animale,permettant de comparer différents mélanges administrés par perfusion (NP seule, NP avec ajoutextemporané d'insuline et NP avec insuline ajoutée 12 heures avant l'administration).Les paramètres de stabilité de l'émulsion de NP témoignent d'une stabilité du mélange nutritif.Une perte de 35% de la teneur en insuline est observée au cours de l'étude de 24 heures sur lemélange de NP ternaire supplémenté en vitamines et éléments-trace avec une diminutionmajoritairement au cours des 6 premières heures. Cette évolution est expliquée à 67% par leniveau de teneur en glucose du mélange, la nature de l'émulsion lipidique, l'absence d'élémentstraceainsi que le pH lorsque celui-ci est pris en considération avec la teneur en glucose, le tempset la teneur en acides aminés. La glycation de l'insuline, mise en évidence à l'aide de la méthodechromatographique, explique cette diminution de la teneur en insuline en présence d'un pHproche de la neutralité. Une réversibilité totale de la glycation de l'insuline est observée lors dela dilution des échantillons dans un milieu acide. Au cours de l'expérimentation in vivo, laconcentration maximale de l'insuline endogène de lapin est plus faible lorsque l'insulinehumaine est ajoutée extemporanément dans la NP, ce qui suggère que dans cette condition,l'insuline humaine possède une bioactivité supérieure. Ce résultat est en accord avec lesdonnées de la littérature sur l'insuline glyquée.En conclusion, l'ajout d'insuline dans un mélange de NP ternaire présente une stabilité physiquepermettant l'intégrité de l'émulsion. En revanche, l'étude de stabilité de l'insuline a révélél'existence d'un phénomène de glycation de l'insuline survenant principalement dans les 6premières heures de contact avec le mélange de NP. Cette insuline glyquée a montré une activitéhypoglycémiante réduite. Une étude approfondie du mécanisme de glycation ainsi qu'uncomplément de l'étude de stabilité du mélange de NP sont nécessaires.

Article Details: Received: 2020-10-10 | Accepted: 2020-12-28 | Available online: 2021-06-30 https://doi.org/10.15414/afz.2021.24.02.124-128 A laboratory study was carried out to evaluate the efficacy of cashew nut shell extract in the control of cowpea bruchid, Callosobruchus maculatus (Fab.) under prevailing laboratory conditions. Fresh ethanolic and stored extract of cashew nut shell served as treatments which were compared with untreated control. Data collected on adult mortality, total number of emerged progeny (adults), number and weight of damaged seeds (seeds with holes) and undamaged seeds (seeds without holes) and percentage seed weight loss, and average number of seeds per 50 g in a container and the data were subjected to a two-way analysis of variance and significant different means were separated using Duncan`s Multiple Range test (DMRT) at 5% level of significance. The results revealed that treated plants generally performed better than the untreated. The different rates of treatment recorded significant differences (P <0.05) in causing adult mortality compared to the untreated control. The different rates of treatment also recorded significant differences (P <0.05) in emergence of F1 adults of each treatment compared to the control. It was also noted that the extract reduced or suppressed the weight loss and grain damage as a result of treatment with the extract compared to the untreated control. However, freshly extract of cashew nut shell recorded the highest adult mortality rate and lowest emergence while control had the lowest mortality rate and highest emergence of the insect. The rates of application were indicative of bioactive characteristics of the extract.Keywords: cowpea, Anacardium occidentale, Callosobruchus maculatus, botanicals, pest management ReferencesAbudulai, M. et al. (2016). Farmer participatory pest management evaluations and variety selection in diagnostic farmer field Fora in cowpea in Ghana. 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Insecticidal activity of alstoniaboonei de wild powder against cowpea bruchid, Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae] in stored cowpea seeds. International Journal of Biology, 4(2), 125–131. https://doi.org/10.5539/ijb.v4n2p125Khater, H. (2012) Prospects of botanical biopesticides in insect pest management. Journal of Applied Pharmaceutical Science, 2(5), 244–259.Kpoviessi, D.A. et al. (2017) Bioefficacy of powdery formulations based on kaolin powder and cashew (Anacardium occidentale L.) balms to control Callosobruchus maculatus F. (Coleoptera, Chrysomelideae: Bruchidae) in stored cowpea (Vigna unguiculata L.). International. Journal of Biological and Chemical Science, 11(4), 1424–1436.Nwosu, L. C. (2014). Evaluation of powder of weevil resistant maize as eco-friendly option in the management of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) infestation in stored maize. Journal of Entomological Research, 38, 245–250.Onekutu, A., Nwosu, L. & Nnolim, N. 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Esta tesis doctoral se ha realizado dentro del marco de un acuerdo de co-tutela entre la Universidad de Zaragoza (Universidad de origen), la Universidad de Calabria (Universidad anfitriona) y la Facultad de Ciencias y Tecnología de la Universidad NOVA de Lisboa (FCT NOVA) (Universidad anfitriona). El trabajo de investigación se ha llevado a cabo dentro del programa de Doctorado en Ingeniería de Membranas Erasmus Mundus (EUDIME), (FPA 2011-0014), financiado por la Unión Europea. La tesis se centró principalmente en el uso de la técnica de electrohilado para producir diferentes tipos de membranas que puedan ser utilizadas en distintas aplicaciones biomédicas. Se sintetizaron y produjeron nanopartículas orgánicas e inorgánicas para ser utilizadas como rellenos o como portadores (sistema de administración de fármacos), así como membranas nanofibrosas electrohiladas. Este trabajo se llevó a cabo en el Instituto de Nanociencia de Aragón (INA), específicamente en el grupo de Nanostructured Films and Particles (NFP) bajo la supervisión de la profesora Silvia Irusta y la Dra. Gracia Mendoza. Una parte importante de la caracterización físico-química se realizó en el INA. En la Universidad de Calabria se trabajó bajo la supervisión de la Dra. Loredana de Bartolo en el Instituto de Tecnología de Membranas (ITM). Allí se utilizaron técnicas específicas tanto para la caracterización como para estudiar diferentes señales biológicas producidas por las membranas sintetizadas, bajo la supervisión. Por otro lado, la movilidad llevada a cabo en la Facultad de Ciencias y Tecnología (FCT NOVA) de la Universidade NOVA (FCT NOVA) bajo la supervisión de la profesora Ana Isabel Aguiar-Ricardo, permitió realizar una caracterización completa de dos membranas asimétricas siguiendo diferentes Normas Internacionales que establecen diferentes ensayos a realizar en apósitos primarios utilizados en heridas. El desarrollo de nuevos scaffolds cargados con proteínas morfogenéticas o antibióticos es de gran interés en el campo de la ingeniería de tejidos óseos. Scaffolds electrohilados con una microporosidad mejorada puede ser beneficioso para mejorar la viabilidad celular debido a que una alta porosidad junto a la presencia de microporos puede proporcionar un entorno tridimensional (3D) que no solamente facilita la siembra y difusión celular sino también proporciona una mejor difusión de los nutrientes y residuos a través del scaffolds. La adición de cerámica de fosfato de calcio ha sido ampliamente investigada para fabricar scaffolds altamente porosos para la ingeniería de tejidos óseos debido a que presentan una composición muy similar al hueso, incluyendo excelentes propiedades de biocompatibilidad, osteoinductivas y osteoconductoras. Partículas cargadas con proteínas morfogenéticas de hueso distribuidas homogéneamente en el scaffolds podrían asegurar una liberación continua del factor de crecimiento proporcionando de esta forma las señales bioquímicas necesarias para la reparación y regeneración ósea. Los scaffolds cargados con antibióticos pueden proporcionar una liberación sostenida del fármaco en el sitio de interés, así como el mantenimiento de propiedades osteogénicas mejoradas para la regeneración exitosa del hueso. Evitando de esta forma que se alcancen niveles de toxicidad o niveles ineficaces en la zona de interés, así como la aparición de efectos secundarios indeseados en los pacientes que provocan un rechazo a los tratamientos prolongados de fármacos por vía sistemática (vía oral e intravenosa). Otra aplicación biomédica interesante de las membranas electrohiladas es la fabricación de apósitos inteligentes eficientes para el tratamiento de heridas. Para lograr una curación rápida de la herida es necesario desarrollar membranas apropiadas con poros interconectados capaces de prevenir la deshidratación rápida y la penetración de bacterias. Para mantener un ambiente húmedo en el lecho de la herida se necesita una alta capacidad de absorción y una adecuada transmisión de vapor de agua. Además, si la membrana electrohilada presenta propiedades bactericidas facilitará el proceso de curación. El objetivo principal de esta tesis fue el desarrollo mediante electrohilado de membranas fibrosas con las características apropiadas para ser utilizadas en la ingeniería de tejidos óseos o como apósito para heridas. En los Capítulos II al V se plantean una serie de objetivos específicos con el fin de cumplir el objetivo principal. Este documento de tesis se dividió en las siguientes secciones:  CAPÍTULO I, corresponde a la introducción general donde se describen los conceptos de biomateriales, scaffolds, ingeniería de tejidos y el objetivo principal de los sistemas de liberación de fármacos. Así como, la clasificación de los biomateriales y la ingeniería de tejidos según el origen de los materiales. Además se ponen de manifiesto todos los factores que deben tenerse en cuenta para desarrollar y aplicar adecuadamente los apósitos para heridas. Se mencionaron las diferentes técnicas utilizadas en la literatura haciendo énfasis en el uso de electrohilado y electropulverización para producir scaffolds o membranas para su uso en la ingeniería del tejido óseo y como apósitos para heridas.  CAPÍTULO II, se enfoca en el desarrollo y mejora de andamios 3D capaces de promover una eficiente regeneración ósea junto con la liberación de antibióticos dirigidos para prevenir la colonización de bacterias. El objetivo de este trabajo fue sintetizar y caracterizar un sistema de liberación de fármacos que consiste en nanofibras electrohiladas de policaprolactona (PCL) decoradas con partículas de poli (ácido láctico-coglicólico) (PLGA) cargadas con rifampicina (RFP). Este material debe promover la reparación ósea evitando el deterioro del scaffolds provocado por una infección. Se realizó la evaluación in vitro de la capacidad bactericida del material electrohilado sintetizado contra bacterias Gram positivas (Staphylococcus aureus) y Gram negativas (Escherichia coli), así como su citocompatibilidad en cultivos 3D con osteoblastos humanos. Estos resultados se enviaron a la Revista de farmacia "International Journal of Pharmaceuitics" para su publicación en formato de artículo y está bajo revisión.  CAPÍTULO III, se describe la síntesis y caracterización de membranas con estructura de núcleo-envoltura de PCL y acetato de polivinilo (PVAc) obtenidas por electrohilado. Las fibras se cargaron con nanopartículas de hidroxiapatita sintética (HAn) para aumentar la bioactividad de los materiales. Los scaffolds desarrollados se trataron con ablación láser para crear características topográficas deseadas a nivel micrométrico con el objetivo de favorecer la adhesión y crecimiento celular. Todas las membranas obtenidas presentaron una estructura de poros tridimensionalmente interconectados y el tratamiento con láser provocó un aumento en la viabilidad y densidad celular. Además, el aumento en la biocompatibilidad de los scaffolds sugiere que los microporos pequeños favorecen la adhesión y proliferación celular. Estos resultados fueron publicados en el artículo titulado "Laser-treated electrospun fibers loaded with nano-hydroxyapatite for bone tissue engineering". Javier Aragon, Nuria Navascues, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics 525,112–122, 2017. DOI:10.1016/j.ijpharm.2017.04.022.  CAPÍTULO IV, se refiere al desarrollo de un scaffold electrohilado compuesto por fibras con estructura de núcleo-cubierta de PCL o PCL/PVAc cargado con HAn sintética. Estas fibras se decoraron con partículas de PLGA cargadas con proteína morfogenética ósea 2 (BMP2) mediante el uso simultaneo de electrohilado coaxial y electropulverización. El objetivo de este trabajo fue evaluar las propiedades estructurales y físico-químicas así como el proceso de biodegradación de los nuevos scaffolds desarrollados y su capacidad para abordar las características arquitectónicas, bioquímicas y funcionales del tejido óseo. Para esto, se probó la bioactividad del scaffold mediante el cultivo de osteoblastos humanos sobre ellos y se monitoreo de la viabilidad celular durante 4 semanas. Se evaluó la actividad osteogénica in vitro de las células sembradas sobre los scaffolds determinando la actividad de la fosfatasa alcalina (ALP) y la expresión de osteocalcina (OCN) y osteopontina (OPN) como proteínas osteogénicas. Estos resultados fueron publicados en el artículo titulado "Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering". Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta and Gracia Mendoza. Journal of Colloid and Interface Science, 531 (2018) 126–137. DOI:10.1016/j.jcis.2018.07.029.  El CAPÍTULO V, describe la síntesis de un apósito antimicrobiano para heridas, con una resistencia mecánica adecuada que es capaz de absorber exudados y evitar la deshidratación rápida de una herida. Se prepararon membranas asimétricas de PCL/PVAc cargadas con carvacrol (CRV) mediante el uso simultáneo de electrohilado y electropulverización. Las membranas constan de dos capas; la primera es una capa de PCL electrohilado; la segunda, una lámina de PVAc que estaría en contacto con la piel liberando a su vez el compuesto antimicrobiano. Se demostró que el uso de diferentes disolventes pueden dar lugar a la obtención de diferentes morfologías de la capa PVAc-CRV. Los valores obtenidos de elongación máxima de las membranas antes de romperse son adecuados para ser utilizados como apósitos para heridas ya que están en el mismo rango reportado de elongaciones en la piel humana. Las membranas presentan una tasa óptima de Transmisión de vapor de agua (WVTR) con valores que se encuentran en el rango requerido para mantener un buen balance entre humedad y pérdida de agua en la herida. En la primera semana, se liberó más del 60 % del CRV cargado, mientras que después de tres semanas, las membranas liberaron entre el 85 y el 100 % del CRV cargado mediante la contribución de un proceso de difusión de tipo Fickiano y la relajación delas cadenas poliméricas. Las membranas sintetizadas son candidatas potenciales para ser utilizadas como apósitos para heridas. El manuscrito que resume estos resultados se envió a la revista "Materials Science and Engineering C" y está bajo revisión (MSEC_2018_3013).  CAPÍTULO VI, resume las conclusiones generales del trabajo de tesis.  APÉNDICE 1, describe las principales técnicas de caracterización y los métodos para evaluar diferentes propiedades en función de las posibles aplicaciones.  APÉNDICE 2, resume los artículos publicados y la participación en foros científicos durante el período de tesis. 1 The current Doctoral Thesis work has been performed under a co-supervision agreement between University of Zaragoza (Home University), University of Calabria (Host University) and Faculty of Sciences and Technology of the NOVA University of Lisbon (FCT NOVA) (Host University). This research has been carried out inside the Erasmus Mundus Doctorate in Membrane Engineering program (EUDIME), (FPA 2011-0014), funded by the European Union. This thesis focused mainly on the use of the electrospinning technique to produce different kind of membranes for biomedical applications. In particular, it described the synthesis and production of inorganic and organic nanoparticles to be used as fillers or as carriers (drug delivery system) as well as the production of electrospun nanofibrous membranes. This work was carried out within the Institute of Nanoscience of Aragon (INA), specifically in the Nanostructured Films and Particles (NFP) group under the supervision of the Professor Silvia Irusta and Dr Gracia Mendoza. Also an important part of the physico-chemical characterization was done at INA. The study of different biological signals and the use of specific techniques for membrane characterization were acquired at the University of Calabria under the supervision of Dr. Loredana De Bartolo in the Institute on Membrane Technology of the National Research Council of Italy (ITM-CNR). On the other hand, the mobility carried out at the Faculty of Sciences and Technology (FCT NOVA) of Universidade NOVA (FCT NOVA) under the supervision of Professor Ana Isabel Aguiar-Ricardo, allowed a total characterization of two asymmetric membranes following different International Standards to accomplish testing for primary wound dressing. The development of novel membranes loaded with morphogenetic proteins or antibiotic are of great interest in the field of bone tissue engineering. To promote the cellular viability and extracellular matrix production, electrospun membranes with enhanced porosity and micro-scale pores could be beneficial since increased porosity and pore size can provide a three-dimensional (3D) environment that not only facilitates cell seeding/diffusion but also provides better diffusion of nutrients and waste throughout the membranes. The addition of calcium phosphate ceramics has been extensively investigated to fabricate highly porous membranes to bone tissue engineering due to their close similar composition of bone, including excellent biocompatibility, osteoinductive and osteoconductive properties. A homogeneous distribution of the bone morphogenetic protein-loaded particles along the entire membrane could be ensuring a continuous release of the growth factor to provide the necessary biochemical cues for bone repair and regeneration. Antibiotic-loaded membranes may provide drug targeted and sustained release, avoiding the long-term oral and intravenous systematic multidrug administration, which implies toxic side effects, low delivery to the target site and low patient adherence to the treatment. Therefore, membranes loaded with antibiotics can overcome the drawbacks of the traditional therapy sustaining enhanced osteogenic properties for the successful regeneration of the bone. Another interesting biomedical application of electrospun membranes is the fabrication of efficient smart dressings for the treatment of wounds. A rapid wound healing requires developing appropriate membranes with interconnected pores that allow the oxygen diffusion and transport of metabolic waste, as well as an adequate pore size to prevent rapid dehydration and bacteria penetration. A high absorption capacity and adequate water vapor transmission will be necessary to keep a moist environment in the wound bed. Besides, if the electrospun membrane has some bactericidal properties will be better for the healing process. The main goal of this thesis was the development of fibrous membranes by electrospinning with the appropriate characteristics to be used in bone tissue engineering or as wound dressing materials. To achieve this target, several specific objectives were defined, which are described in Chapters II to V. The thesis was divided in the following sections: CHAPTER I, is an introduction where the concepts of biomaterials, scaffolds and tissue engineering and the main target of drug delivery systems are described. The chapter includes the classification of biomaterials according to the origin of the materials and tissue engineering is also described as well as all the factors that must be taken into account to develop and properly apply a wound dressing are discussed. Different kind of techniques used in the literature to produce scaffolds or membranes for bone tissue engineering and wound dressings are mentioned, focusing on the use of electrospinning and electrospray to produce them. CHAPTER II, focuses on the development of enhanced 3D membranes able to promote efficient bone regeneration together with targeted antibiotic release to prevent bacteria colonization. The aim of this work was to synthesize and characterize a drug delivery system consisting of polycaprolactone (PCL) electrospun nanofibers decorated with rifampicin (RFP) loaded into poly(lactic-coglicolic acid) (PLGA) particles. This material would promote bone repair avoiding the impairment of the membrane mediated by infection. The bactericidal ability of the synthesized electrospun material was assessed In vitro against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria, as well as its cytocompatibility in human osteoblasts 3D cultures. These results are included in the accepted article entitled "Composite scaffold obtained by electro-hydrodynamic technique for infection prevention and treatment in bone repair". Javier Aragon, Sergio Feoli, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics. CHAPTER III, describes the synthesis and characterization of core-shell membranes of PCL and polyvinyl acetate (PVAc) obtained by electrospinning. The fibers were loaded with synthetic hydroxyapatite nanoparticles (HAn) to increase the bioactivity of the materials. The prepared membranes were then treated by laser ablation to create desired microscale topographical features in order to favor cell adhesion and growth. All prepared membranes exhibited a three-dimensional network structure with interconnected pores; the laser treatment has modified the structural characteristics of the membrane causing an increase the cell viability and cell density. The materials biocompatibility is affected by the structural properties of the membranes, indeed smaller micropore sizes favor cell adhesion and proliferation. These results are published in the article entitled "Laser-treated electrospun fibers loaded with nano-hydroxyapatite for bone tissue engineering". Javier Aragon, Nuria Navascues, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics 525,112–122, 2017. DOI:10.1016/j.ijpharm.2017.04.022. CHAPTER IV, refers to the development of a composite electrospun membrane of PCL or PCL/PVAc core–shell fibers loaded with synthetic HAn. These fibers were decorated with bone morphogenetic protein 2 (BMP2) loaded in/into PLGA particles via simultaneous electrospraying and coaxial electrospinning. The aim of this study was to evaluate the structural and physico-chemical properties and biodegradation processes of the newly developed membranes assessing their ability to address the architectural, biochemical, and functional features of bone tissue. For this purpose, the membrane bioactivity was tested by culturing human osteoblasts on the membranes and by monitoring cell viability up to 4 weeks. The In vitro osteogenic activity of cells seeded onto the membranes was evaluated by assessing alkaline phosphatase (ALP) activity and the expression of osteogenic proteins osteocalcin (OCN) and osteopontin (OPN). These results are published in the article "Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering". Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta and Gracia Mendoza. Journal of Colloid and Interface Science, 531 (2018) 126–137. DOI:10.1016/j.jcis.2018.07.029. CHAPTER V, describes the synthesis of an antimicrobial wound dressing material, with appropriate mechanical resistance avoiding rapid dehydration and absorbing exudates. PCL/PVAc asymmetric membranes loaded with carvacrol (CRV) were prepared by electrospinning and electrospraying simultaneously. The membranes consist of two layers: the first is an electrospun PCL sheet, the second a PVAc sheet that would be in contact with the skin releasing the antimicrobial compound. The use of different solvents results in different morphologies for the PVAc-CRV layer. The membranes exhibit mechanical properties with strain to failure values that are in the range of human skin, being adequate to be deposited over a wound surface. The samples present Water Vapor Transmission (WVTR) values in the required range to keep good moisture balance with water loss from the wound at the optimal rate. In the first week, more than 60 % of the loaded CRV was released while after three weeks membranes released between 85 to 100 % of the loaded CRV through a Fickian diffusion and diffusion due to polymer relaxation. The synthesized membranes are potential candidates to be used for wound dressing applications. The manuscript summing up these results has been submitted to a scientific journal and is currently under review. GENERAL CONCLUSIONS, summarizes the conclusions of the thesis work. APPENDIX 1, describes the main characterization techniques and the methods to evaluate different properties according to the possible applications. APPENDIX 2, summarizes the articles published and the participation in scientific forums during the thesis period.