Academic literature on the topic 'Proteine allergeniche'

The general aim of the activities conducted in the framework of my PhD were to learn modern techniques for analyzing proteins by mass spectrometry (MS) and then to apply these techniques and approaches to the analysis of allergenic proteins contained in foods. My research activities were conducted at the Laboratory of Protein Chemistry of CRIBI, University of Padua, where previously I have conducted the research for my Thesis for the Doctor degree in Pharmaceutical Biotechnologies. During the fist year of my PhD I have concluded the Thesis project on the amyloid aggregation of -lactalbumin, a model protein utilized for investigating molecular aspects of protein amyloidogenesis. The results of this research were quite interesting and indeed they have been published in an international journal. During the first two years I acquired a solid knowledge on several aspects of the MS methodology and I was able to learn the theory and practice of several modern techniques and approaches in this ambit. The specific aim was to analyze allergenic proteins contained in complex matrices as foods and to this aim several proteins were extracted and purified from several food samples. The research has been focused mostly on the allergenic proteins from milk and eggs, known to cause widespread allergies. The proteins of interest were analyzed by using several chromatographic and electrophoretic techniques and also by means of HPLC connected to a tandem MS electrospray instrument. I was able to show that MS techniques can be used to identify allergenic proteins even when contained in very complex mixtures. Therefore, these MS techniques perhaps can be used as an alternative to the immunochemical methods nowadays in use for detecting allergens. I have also analyzed the chemical modifications that allergenic proteins suffer during several industrial treatments of foods, including heat treatment. During the third year of my PhD I spent a six months period at the Biochemistry Laboratory of the Imperial College in London, being involved in a project aimed to study in a large scale the proteins of the mosquito Anopheles gambiae. The MS analyses were focused on the proteins responsible of the mating behaviour of A. gambiae, hoping to identify a target for controlling the behaviour of this vector of the malaria disease. Summing up, besides the publication dealing with amyloid aggregates of beta- lactalbumin, this PhD Thesis is composed by a major part dealing with MS analysis of allergenic proteins and by a minor one dealing with MS analysis of proteins from A. gambiae.

MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes. Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data. Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays. Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins. In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked. Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample. The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research. Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis. The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation. Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).<br>Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).

2013 - 2014<br>The great interest of the research activity in food allergies could be attributed to the increase of allergic reactions all over the world not only in infants but even in adult age. As an alternative to the development of an allergen-free diet, many works have been focused on a novel approach for the treatment of allergens: instead of eliminating the allergens from the diet, the immunoresponse can be reduced or even eliminated by inducing some modifications of their molecular structure. In fact, changes in allergen conformation can modulate its identification by the specific antibody produced by immune system in allergic reactions. Structural modifications in allergens could be induced by conventional thermal treatments as well as by non-thermal technologies, namely High Hydrostatic Pressure (HHP), Pulsed Electric Fields (PEF), Pulsed Light (PL) and -radiations. Non-thermal technologies have been widely used in the last years for food preservation, having the advantage of increasing the shelf-life and freshness of the raw food products. These technologies are able to affect the food nutritional and organoleptic properties only slightly thanks to the use of a non-thermal stress to treat foodstuffs. Among them High Hydrostatic Pressure technology has been successfully used in food pasteurization, but also in processes involving the sol-gel transition such as the production of jams, jellies and dairy products. The ability of High Pressure to determine structural changes in foods was studied in order to assess if proteins unfolding and/or aggregation and gelation can be induced and if the treatment affects the functional properties and digestibility of proteins. These effects were studied on particular proteins, namely the allergens, for which unfolding and structural modification have been proven. However, the effectiveness of the High Pressure processing on the reduction of immunoresponse reduction was not clearly assessed so far. The objective of this PhD thesis was the study of the modifications induced by High Pressure Process on allergenic proteins and the possibility of obtaining hypoallergenic peptides by means of a combined High Hydrostatic Pressure hydrolysis. In particular, the effect of the HHP on the allergens structural modification was investigated in a wide range of operating conditions, including both gelling and ungelling conditions. Rheological behavior and functional properties of HHP processed allergens was also determined. [edited by Author]<br>XIII n.s.

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

Lipid transfer proteins (LTPs) are a class of low molecular weight hydrophobic conserved proteins comprising four intramolecular disulphide bonds making the structure very resistant to proteolysis and harsh food processing conditions. These proteins are identified as strong allergens sensitizing through the gut and share epitopes with LTPs from closely related species. Peach LTP, Pru p 3 is the primary sensitizer in the Mediterranean area being the most frequent food allergen. Wheat LTP, Tri a 14 is a relatively weak allergen with a very low prevalence. The study here compares the structural properties of these proteins and their resistance to various digestive and processing processes. Ligand binding experiments showed that Pru p 3 binds to ligands more strongly than Tri a 14. The gastroduodenal digestion of these LTPs revealed that both are stable to gastric digestion and while Pru p 3 is susceptible to duodenal digestion, Tri a 14 digestion is negligible. Ligand binding did not affect the digestibility of Pru p 3 but improved the duodenal digestibility of Tri a 14. The IgE binding studies using sera from peach allergic individuals confirmed that both Pru p 3 and its digestion fragments in the presence and absence of ligand were IgE reactive. Model processing conditions were employed to treat these LTPs. It was found that heat treatment destroys the secondary structure of Pru p 3 at 121°C and slightly affects that of Tri a 14. Heat treatment also increased the susceptibility of Pru p 3 to gastric digestion while Tri a 14 was less affected. The IgE binding studies showed that heat treatment of Pru p 3 appeared to reduce its IgE recognition while its digestion fragments lost all of their IgE reactivity. To investigate the effect of the food matrix on the digestibility of these LTPs, peach peel containing Pru p 3 and wheat flour containing Tri a 14 were digested under simulated conditions. It was found that they were resistant to proteolysis in their native matrices. Effect of heat treatment to the food matrix again confirmed that both of these proteins were more stable to heat in the matrix and were less digestible. In conclusion, this study shows that there are factors in food matrices which enhance structural stability of LTPs to both processing and digestion. Thus factors such as the effect of food matrix and effect of processing should be taken into account in assessing the allergenic risk posed by foods and not simply rely on data from purified proteins.

Orientadores: Maria Helena Andrade Santana, Ricardo de Lima Zollner<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica<br>Made available in DSpace on 2018-07-27T03:55:49Z (GMT). No. of bitstreams: 1 Victorino_IgorRicardodeSouza_M.pdf: 6760377 bytes, checksum: 05fc12e1f97a8b092dbd91b47b06441d (MD5) Previous issue date: 2000<br>Resumo: Os lipossomas têm sido utilizados como imunoadjuvantes antigênicos, em estudos que enfatizam a sua atuação no sistema imunológico. Extratos peptídicos ou protéicos de várias fontes foram encapsulados em lipossomas ou associados à sua superficie, e aplicados em imunoterapias e vacinas. Apesar disso, poucos são os estudos voltados para a performance do processo de preparação desses lipossomas, eficiência da associação proteína/lipídio e estabilidade das vesículas. Este trabalho trata da avaliação do encapsulamento das proteínas: Albumina de Soro Bovino (BSA), Mioglobina (Mio) e Cito cromo C (Cit C), simulando extratos alergênicos provenientes de fungos e ácaros, no que se refere à faixa de peso molecular das suas proteínas. Os lipossomas foram preparados com os lipídios L-a-distearoilfosfatidi1colina (DSPC) e Colesterol (Col) na razão molar 70:30 respectivamente. As proteínas foram encapsuladas individualmente e em forma de misturas de composições molares BSA:Mio:Cit C de 20:40:40, 40:20:40 e 40:40:20, respectivamente. Além disso, a mistura de proteínas de composição 40:20:40 (BSA:Mio:Cit C) foi também associada covalentemente à superficie das vesículas. Os lipossomas foram caracterizados pelo teor de fósforo e diâmetro médio. O desempenho do encapsulamento foi analisado através dos perfis e eficiências de encapsulamento das proteínas individuais e em misturas, além da estabilidade das vesículas em tensoativo penta etileno glicol mono-n-dodecil éter (C12Es), e de plasma humano. Para as misturas, analisou-se a exclusão de proteínas durante o encapsulamento e a associação à superficie dos lipossomas. Os resultados indicam que a eficiência de encapsulamento depende mais das interações proteína:lipídio que do tamanho das proteínas. As eficiências de encapsulamento variaram entre 0,03% e 3,55% para as proteínas individuais e entre 1,04% e 3,94% para as misturas. Não houve alteração na estabilidade dos lipossomas em C12Es com a presença das proteínas. Em plasma, essas vesículas permaneceram estáveis por aproximadamente 20 horas. Na associação à superficie dos lipossomas, predominou a presença de BSA em relação às outras proteínas<br>Abstract: Liposomes has been studied as antigenic immunoadjuvants with emphasis on their performance in the imunological system. Peptides or proteins of various sources were encapsulated in liposomes or associated to their surface, and applied in immunotherapy and vaccines. In spite of this, there are few studies about the performance of preparation process ofthese liposomes, efficacy ofthe association proteinllipid and the stability ofvesic1es. This work concems with the evaluation of the encapsulation of the proteins Bovine Serum Albumin (BSA), Myoglobin (Myo) and Cythochrome C (Cyt C), simulating allergen extracts from molds and mites, related to the range of molecular weight of their proteins. Liposomes were prepared with lipid L-a-disteraoylphosphatidylcholine (DSPC) and Cholesterol (Chol) at molar ratio 70:30 respectively. The proteins were encapsulated individualyand in mixtures with molar composition BSA:Myo:Cyt C of20:40:40, 40:20:40 and 40:40:20 respectively. Furthermore, the mixture 40:20:40 (BSA:Myo:Cyt C) was also covalent1y associated to the surface of vesic1es. Liposomes were characterized by their phosphate contents and mean diameter. The performance of the protein encapsulation was evaluated through the profiles and efficiency of the encapsulation and throught the stability of vesic1es in the presence of the surfactant penta ethylene glycol mono-n-dodecyl ether (C12ES) and human plasma. For the mixtures, the exclusion of proteins duringentrapment or association to the liposome surface was also evaluated. The experimental results indicate that the efficacy of encapsulation is more dependent of the interactions proteinllipid than the size of the proteins. The efficiencies of encapsulation changed between 0.03% and 3,55% for individual proteins and were between 1,04% and 3,94% for the mixtures. The presence ofproteins does not altered the stability of liposomes in C12ES . In human plasma the vesic1es remained with stability about 20 hours. For the mixtures, the presence ofthe BSA protein predominated in the vesicles<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior<br>This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10  8,36 TIU/mgP), chymotrypsin (25,36  0,86 IU/mgP), papain (65,419  0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 &#956;g/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract.<br>Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 &#956;g/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-30T15:53:39Z No. of bitstreams: 2 Dissertação - Moisés Queiroz Resende - 2014.pdf: 891007 bytes, checksum: 48a7b34dc669b45819e19f6d364dfdb5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)<br>Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-30T15:54:22Z (GMT) No. of bitstreams: 2 Dissertação - Moisés Queiroz Resende - 2014.pdf: 891007 bytes, checksum: 48a7b34dc669b45819e19f6d364dfdb5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)<br>Made available in DSpace on 2015-01-30T15:54:22Z (GMT). No. of bitstreams: 2 Dissertação - Moisés Queiroz Resende - 2014.pdf: 891007 bytes, checksum: 48a7b34dc669b45819e19f6d364dfdb5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-29<br>Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq<br>ABSTRACT: Two experiments were conduct to evaluate the effect of using soy protein concentrate (SPC) in pre-starter piglet diets over performance, intestinal histomofometria and blood parameters. In the first trial 72 piglets weaned at 21 days of age were used and 54 for the second trial. Both were conducted as a randomized blocks design with six replications and three piglets per unit. The first trial had four diets (0% SPC, 3% SPC, 6% SPC and 9% SPC), which evaluated different levels of SPC inclusion in diets, and the second trial, which evaluated the replacement of spray dried blood plasma (SDP) by SPC, had three diets (0.0%-SPC + 5.0-SDP, 2.5%-SPC + 2.5%-SDP and 5.0%-SPC + 0.0%-SDP). In both trials, diets and water were offered ad libitum in pre-starter l (21-32 days old) and pre-starter ll (33-42 days old) phases, but during the starter phase (43-66 days old) all piglets received a single diet. At 32 days of age, blood was collected from one animal per experimental unit and then these animals were euthanized to collect samples of the small intestine. Linear effect was observed In the first trial, over feed conversion (FC) as the level of CPS in diet was increased in the period between 21 and 32 days of age. Linear effect was also observed on the FC in the period of 33-42 days old, however in a reverse form from the previous period, wherein the increase of the SPC level in diets resulted in the increase of FC. In the total period of this trial (21-66 days old), it was found a quadratic effect of SPC on piglets FC. In the second trial, the replacement of SDP by CPS caused effect on performance variables, except for feed conversion (FC) during 21-32 days of piglets age. The average daily feed intake (ADFI) was higher for pigs fed 2.5%-SPC + 2.5%-SDP and the average daily gain (ADG) and the final body weight (FBW) of the piglets in this period was higher for 2.5%-SPC + 2.5%-SDP compared to 5.0%-SPC + 0.0%-SDP. In the period of 21-42 days old, it was observed best results of ADFI and FBW for piglets fed diet containing 2.5%-SPC + 2.5%-SDP compared to piglets fed 5.0%-SPC + 0.0%-SDP. The ADG showed better results for diets containing 0.0%-SPC + 5,0-SDP and 2.5%-SPC + 2.5%-SDP and FC was higher for the diet with 0.0%-SPC + 5.0%-SDP compared to the diet with 5.0%-SPC + 0.0%-SDP. During total period of the experiment (21-66 days old) ADG and ADFI was influenced by substitution of SDP for SPC, wherein the best results was presented by piglets fed 2.5%-SPC + 2.5%-SDP compared to those fed 5.0%-SPC + 0.0%-SDP. In both trials, no effect was observed upon histomorphometric variables (villus height, crypt depth and villus: crypt). Regarding the blood variables (leukocytes, eosinophils and lymphocytes), no effect of SPC at 32 days of age the piglets was observed in the first trial. But in the second trial, total leukocyte count was higher in animals fed 5.0%-SPC + 0.0%-SDP compared to ones fed 0.0%-SPC + 5.0%-SDP and lymphocyte count was lower in piglets receiving 0.0%-SPC + 5.0%-SDP. In the first trial, the use of SPC in diets of post-weaning piglets during pre-starter period (21-42 days old) did not influence performance from 21 to 66 days of life, or intestinal morphology and leukocyte, lymphocytes and eosinophils count in these animals. In the second, the combined use of SPC and SDP in the diets of pigs between 21 and 42 days of age reduces the activation of the immune system and improves productive performance.<br>Foram conduzidos dois experimentos com objetivo de avaliar o efeito da utilização de Concentrado proteico de soja (CPS) em dietas pré-inicias de leitões sobre o desempenho, histomofometria intestinal e alguns parâmetros sanguíneos. Foram utilizados 72 leitões machos castrados desmamados aos 21 dias de idade no primeiro experimento e 54 no segundo. Ambos foram delineados em blocos ao acaso com seis repetições e três leitões por unidade experimental, tendo o primeiro quatro tratamentos (0% CPS, 3% CPS, 6% CPS e 9% CPS), o qual avaliou diferentes níveis de inclusão de CPS nas dietas, e o segundo, para avaliar a substituição de plasma sanguíneo (SDP) por CPS, três (0,0%-CPS + 5,0-SDP, 2,5%-CPS + 2,5%-SDP e 5,0%-CPS + 0,0%-SDP). Em ambos as rações experimentais e água foram fornecidas à vontade nas fases pré-inicial l (21 a 32 dias de idade) e pré-inicial ll (33 a 42 dias de idade), sendo que na fase inicial (43 a 66 dias de idade) todos os leitões receberam uma única dieta. Aos 32 dias de idade foi coletado sangue de um animal por unidade experimental e em seguida estes animais foram eutanasiados para coleta de amostras de intestino delgado. No primeiro experimento, foi observado efeito linear decrescente sobre a conversão alimentar (CA) à medida que aumentou o nível de CPS na dieta no período entre 21 e 32 dias de idade. Efeito linear também foi observado sobre a CA no período entre 33 e 42 dias de idade dos leitões, entretanto de forma inversa ao período anterior, ou seja, à medida que se aumentou o nível de CPS na dieta a CA aumentou. Foi observado no período total (21 a 66 dias de idade) do primeiro experimento efeito quadrático do CPS sobre a CA dos leitões. No segundo experimento a substituição de SDP por CPS causou efeito sobre as variáveis de desempenho no período entre 21 e 32 dias de idade, exceto sobre a CA. O CDR foi superior para os leitões alimentados com 2,5%-CPS + 2,5%-SDP e o GDP e o peso final (PF) dos animais neste período foi superior com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. No período entre 21 e 42 dias de idade foi observado melhores resultados de CDR e PF para a dieta com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. O GDP apresentou melhores resultados para as dietas com 0,0%-CPS + 5,0-SDP e 2,5%-CPS + 2,5%-SDP e a CA foi superior para a dieta com 0,0%-CPS + 5,0%-SDP diante da dieta com 5,0%-CPS + 0,0%-SDP. No período total do experimento (21 a 66 dias de idade) foi observado efeito da substituição dos SDP por CPS sobre o GDP e o CDR, sendo que os animais alimentados com 2,5%-CPS + 2,5%-SDP apresentaram resultados superiores aos alimentados com 5,0%-CPS + 0,0%-SDP. Emambos os experimentos nenhum efeito foi observado sobre as variáveis histomorfométricas (altura de vilosidade, profundidade de cripta e relação vilo:cripta). Em relação às variáveis sanguíneas (leucócitos, linfócitos e eosinófilos) do primeiro experimento, não foi observado nenhum efeito do CPS aos 32 dias de idade dos leitões. No segundo experimento, a contagem total de leucócitos foi superior para os animais alimentados com 5,0%-CPS + 0,0%-SDP sobre os alimentados com 0,0%-CPS + 5,0%-SDP e a contagem de linfócitos foi menor nos leitões que receberam 0,0%-CPS + 5,0%-SDP. No primeiro experimento, a utilização de CPS em dieta de leitões pós-desmame no período préinicial (21 a 42 dias de idade) não influenciou o desempenho produtivo dos leitões no período entre 21 e 66 dias de vida, nem a histomorfometria intestinal e a contagem de leucócitos, linfócitos e eosinófilos desses animais. No segundo, a utilização conjunta de CPS e SDP nas rações reduz a ativação do sistema imune e melhora o desempenho produtivo.