Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-04-25T14:31:12Z No. of bitstreams: 1 ALLYSSON FORTUNATO DE ABRANTES - DISSERTAÇÃO PPGSA 2015..pdf: 1275565 bytes, checksum: 5e0019b5b460441a322e5e237b8eac1c (MD5) / Made available in DSpace on 2018-04-25T14:31:12Z (GMT). No. of bitstreams: 1 ALLYSSON FORTUNATO DE ABRANTES - DISSERTAÇÃO PPGSA 2015..pdf: 1275565 bytes, checksum: 5e0019b5b460441a322e5e237b8eac1c (MD5) Previous issue date: 2015 / O veneno da abelha, a apitoxina, consiste de uma mistura complexa de compostos nitrogenados, constando de uma maior parte proteica(Melitina) e menor fração por: Apamina, adolapina, fosfolipase A2, hialuronidase e peptídeos MCD. Trata-se de um líquido transparente, com odor característico, amargo e que apresenta pH básico (4,5 a 5,5), usado pelas abelhas como elemento de defesa e que seca facilmente em temperatura ambiente. A Melitina é o componente predominante no veneno apresentando, aproximadamente, 50 % da matéria seca, trata-se de uma proteína de elevado potencial anti-inflamatório, sendo considerada o principal agente da apitoxina na terapia da artrite reumática. Junto com a apamina, a melitina estimula os sistemas adrenal e pituitário a produzirem cortisol e outros esteroides naturais, que tem relevante papel na terapia da artrite, bem como na indústria cosmecêutica. A apitoxina foi coletada no apiário El-Shaday, Assentamento Rosário, Agrovila Canudos do município de Ceará-Mirim/RN, a coleta da amostra se deu utilizando o método de choques elétricos através de eletrodos de aço inox conectados a uma bateria e lâminas de vidro posicionado no alvado da colmeia que retêm a apitoxina após a picada da abelha. O apiário produz dois tipos de apitoxina, classificada como tipo 01, pura, e tipo 02, beneficiada. Foram realizados testes de citotoxicidade, quantificação proteica e eletroforese, em ambas as amostras, a fim de avaliá-las e comparar os dois tipos de apitoxina.Na apitoxina 01 foi realizada a concentração inibitória mínima. As amostras também foram incorporadas em duas bases dermatológicas, Gel carbopol® e Emulsão aniônica para que fossem avaliadas a estabilidade Preliminar e Acelerada das formulações.A apitoxina tipo 01, apresentou 77,83% de proteínas, uma concentração inibitória mínima a partir de 1,0% e toxicidade de 962,60 µg/mL; Os testes na apitoxina 02 apresentaram um teor proteico de 51,87% e uma citotoxicidade 154,82 µg/mL resultados possivelmente afetados pela contaminação ainda presente neste tipo de amostra após o beneficiamento. A eletroforese demonstrou que a apitoxina apresenta o mesmo perfil proteico, independente do processo de extração. Porém, a apitoxina tipo 02, aponta a diferença de uma unidade que não se revelou neste produto. As formulações mantiveram-se estáveis durante todo período de avaliação de sua estabilidade, 90 dias, onde foram avaliados pH, viscosidade, caracteres organolépticos, espalhabilidade, nas três diferentes condições de armazenamento: temperatura ambiente, 5 °C e 40 °C, exceção feita à emulsão aniônica conservada à 40 °C em que perdeu estabilidade a partir do trigésimo dia. / The bee venom, the apitoxin, is a complex mixture of nitrogenous compounds, having largely represented by melittin protein and a smaller fraction consisting of: Apamin, adolapina, phospholipase A2, hyaluronidase and peptides MCD. It is a transparent liquid, with characteristic odour, bitter and that presents basic pH (4.5 to 5.5), used by bees as a defense and that dry easily at room temperature. The Melittin is the predominant component in poison showing approximately 50% of the dry matter, it is a high protein anti-inflammatory potential, being considered the main agent of apitoxin on rheumatic arthritis therapy. Along with the apamin, melittin stimulates the adrenal and pituitary systems to produce cortisol and other steroids, which has important role in arthritis therapy, as well as in the cosmeceutical industry. The apitoxin was collected in the Apiary El-Shaday, Settlement, Agrovila Straws of municipality of Ceara – Mirin/ RN. sample collection occurred using the method of electric shocks through electrodes of stainless steel connected to a battery and glass blades positioned at the hive entrance of the hive that retain the apitoxin after bee sting. The Apiary produces two types of apitoxin, classified as type 01, pure, and type 02 milled. Cytotoxicity tests were performed, and protein quantification in both samples in order to evaluate them and compare the two types of apitoxin, on 01 was still held the apitoxin minimum inhibitory concentration. The samples were also incorporated into two bases, carbopol Gel ® skin and anionic emulsion for primary and Accelerated stability evaluated formulations. The apitoxin type 01, 77.83% protein, introduced a minimum inhibitory concentration from 1% and toxicity of 962.60 µ g/mL; Tests on apitoxin 02 presented a protein content of 51.87% and a cytotoxicity 154.82 µ/mL results possibly affected by the contamination still present in this sample type after the processing. Electrophoresis showed that apitoxin features the same protein profile, regardless of the extraction process. However, the apitoxin 02 type, presents the difference in a unit that didn't report revealed in this product. The wording remained stable throughout assessment period of its stability, 90 days, where they were evaluated pH, viscosity, organoleptic characters, spreadability, in three different storage conditions: room temperature 5° C and 40° C, except the anionic emulsion stored at 40° C that lost stability from the 30th day.

Protéines sont largement utilisés dans la formulation dans le domaine pharmaceutique et de jouer un rôle important dans les fonctions biologiques. Il est bien connu que l'adsorption de protéines sur la surface solide est toujours observé pour un stockage à long terme, ce qui entraînera une réduction de la dose de substance active ou une perte de l'activité biologique. Dans certains cas, une courte période de contact avec la surface est suffisante pour modifier fortement la conformation des protéines : par exemple, l'insuline pertes 52% de son activité biologique après 5 minutes de contact avec la surface de verre, ainsi qu'une perte de 30% d’activité biologique du cétrorélix est observé après 2 heures de contact. Parmi tous les paramètres, la dénaturation des protéines est fortement liée à sa stabilité des propriétés de surface. La compréhension de l'adsorption de protéines est devenue une question cruciale dans l'industrie pharmaceutique.Pour mieux comprendre le comportement des protéines à la surface, la quantification des protéines adsorbées et sa conformation devrait être étudiée. L'objectif de notre recherche sera de comprendre les comportements des protéines sur différents surfaces de seringue pré - remplie classique.Le principal objectif de ce projet est de comprendre le comportement de plusieurs modèles de protéines comme la sérum d’albumine bovine (BSA), le lysozyme (LSZ) et la myoglobine (MGB) en contact avec des surfaces de seringues pré-remplie comme le verre et l’élastomère. Nous proposons d'utiliser la chromatographie liquide à haute performance (HPLC) pour la quantification de protéine adsorbée sur une surface plane en déterminant la déplétion des protéines en solution. La réflexion totale atténuée infrarouge à transformée de fourier (FTIR-ATR) spectroscopie est utilisée de suivre les changements structurels des protéines adsorbées sur des surfaces solides. [...] / Proteins are widely used in formulation in the pharmaceutical field and play a major role in biological functions. It is well known that protein adsorption on solid surface is always observed for a long-term storage, which will result in a reduced dose of active compound or a loss of biological activity. In some cases, only short time of contact are sufficient to drastically modify the protein conformation: for instance, insulin losses 52% of its biological activity after 5 minutes contacting with glass surface, as well as a loss of 30% of cetrorelix is observed after 2 hours. Among all parameters, the time frame of the denaturation process is strongly related to the protein stability and surface properties. The understanding of protein adsorption has therefore become a crucial issue in the pharmaceutical industry.To gain a better understanding of proteins’ behavior on the surface, adsorbed protein quantification and its conformation should be studied. The objective of our research in a first will be to understand proteins’ behaviors on various surfaces which composed a classical prefilled syringe.The main goal of this PhD project is to understand the behaviors of several model proteins like bovine serum albumin (BSA), lysozyme (LSZ) and myoglobin (MGB) in contact with the surfaces of prefilled syringes such as glass and elastomer. We propose to use the high performance liquid chromatography (HPLC) to quantify the amount of protein adsorbed on a flat surface by determining the depletion of the proteins in solution. Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy was as well as employed to follow the structural changes of adsorbed BSA on solid surface. [...]

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Der Sprengstoff PETN ist äußerst schwer zu detektieren. Ein verbesserter anti-PETN-Antikörper wurde durch Anwendung des Bioisosterie-Konzepts entwickelt. Diese polyklonalen IgGs sind sehr selektiv und sensitiv. Die Nachweisgrenze des ELISAs beträgt 0,15 µg/L. Der Messbereich des Immunoassays liegt zwischen 1 und 1000 µg/L. Die Antikörper sind recht pH-stabil als auch robust gegen Lösungsmittelzusätze. Für die Umweltanalytik von TNT wurde eine HPLC-kompatible Affinitätssäule mit porösem Glas als Trägermaterial hergestellt. Um die anti-TNT-Antikörper selektiv aus den TNT-Seren zu isolieren, wurde eine Trennung an einer Dinitrophenyl-Affinitätssäule durchgeführt. Zur Optimierung der Kopplungsmethode wurden orangefarbene Dabsyl-Proteine synthetisiert und auf der Oberfläche gebunden. Die Färbung wurde als Indikator für die Ligandendichte verwendet. Wegen der hohen Affinitätskonstanten der anti-TNT-IgGs lässt sich TNT nicht reversibel von der TNT-Affinitätssäule eluieren. Daher wurde eine neuartige Elutionsmethode entwickelt, die thermische Online-Elution. Die maximale Kapazität einer TNT- Affinitätssäule betrug 650 ng TNT bzw. 10 µg/mL Säulenvolumen. Um die Ligandendichte der TNT-Affinitätssäulen zu bestimmen, wurde ein neues Verfahren entwickelt, da die spektroskopischen Proteinbestimmungsmethoden nicht geeignet waren. Zur Proteinbestimmung wurde eine HPLC-Trennung der Aminosäuren Tyr und Phe ohne vorherige Derivatisierung entwickelt. Die Proteinhydrolysezeit wurde durch Einsatz einer Mikrowelle von 22 h auf 30 min verkürzt. Zur internen Kalibrierung wurden HTyr und FPhe verwendet. Die Nachweisgrenze bei 215 nm ist sowohl für Tyr als auch für Phe 0,05 µM (~ 10 µg/L). Dieses neue Verfahren, das als Aromatische Aminosäureanalyse (AAAA) bezeichnet werden kann, wurde zur Proteinbestimmung von homogenen Proben mit NIST-BSA validiert, wobei die Nachweisgrenze für Proteine 16 mg/L (~ 300 ng BSA) ist. Die relative Standardabweichung incl. der Hydrolysestufe beträgt 5%. / The explosive Pentaerythritol tetranitrate (PETN) is extremely difficult to detect. An improved antibody against PETN was developed by using the bioisosteric concept. These polyclonal antibodies are highly selective and sensitive. The limit of detection (LOD) of the ELISA was determined to be 0.15 µg/L. The dynamic range of the assay was found to be between 1 and 1000 µg/L. The antibodies are sufficiently pH-stable and resistant to solvent additives. An HPLC-compatible TNT-affinity column with porous glass as support material was prepared for the environmental analysis. In order to isolate the anti-TNT antibodies of the TNT sera a separation was carried out on a dinitrophenyl-affinity column. To optimize the immobilization method, orange-coloured dabsyl proteins were synthesized and bound to the surface. The colour intensity was found to be an indicator for the immobilization rate. In consequence of the high affinity constants of the anti-TNT antibodies, TNT can''t elute by a typical acidic elution step. Therefore, a novel separation approach, the thermal online-elution was developed. The maximum capacity of an affinity column was 650 ng TNT or 10 µg/mL of column volume. To quantify the immobilization rate of proteins, a new method has been developed, because the usual protein determination methods were unsuitable. Therefore an HPLC separation method of Tyr and Phe was developed without prior derivatization. Two internal standard compounds, HTyr and FPhe, were used for calibration. The LOD was estimated to be 0.05 µM (~ 10 µg/L) for Tyr and Phe at 215 nm. The protein hydrolysis time was reduced from 22 h to 30 min using microwave technique. This procedure, that was termed aromatic amino acid analysis (AAAA), has been validated for protein determination of homogeneous samples with NIST-BSA. The LOD for proteins was calculated to be below 16 mg/L (~ 300 ng BSA absolute). The relative standard deviation, including the hydrolysis step, is 5%.