O fluido folicular (FF) é um líquido extracelular complexo que se acumula no antro dos folículos ovarianos durante o seu desenvolvimento. É o meio essencial para o crescimento e a maturação das células ovarianas somáticas e germinativas e contém substâncias envolvidas na diferenciação celular, maturação do oócito, qualidade do gameta, ruptura da parede folicular e luteinização. O estudo de seus componentes é fundamental para um melhor entendimento dos mecanismos que envolvem a dinâmica folicular na espécie equina. O objetivo deste trabalho foi comparar o perfil proteico do maior folículo, e entre o maior e o segundo maior folículo, em diferentes momentos do desenvolvimento folicular. Para este estudo, quarenta ovários, oriundos de vinte éguas Crioulas, não gestantes e cíclicas, foram coletados durante a estação reprodutiva, em um abatedouro. Antes do abate, as éguas foram divididas em quarto grupos de acordo com o diâmetro folicular, ecotextura uterina (EU) e presença de corpo lúteo (CL): G 15 (emergência) (n = 3) folículos até 15 mm, EU ≥ 1, CL ≥ 20 mm; G 20 (divergência) (n = 9) folículos entre 20 e 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominância) (n = 4) folículos entre 30 e 35 mm, EU ≥ 2, CL ≤ 15 mm; G 40 (pré-ovulatória) (n = 4) folículos ≥ 40 mm, EU 2-3, CL ≤ 15 mm. Após o abate, os ovários foram coletados e o FF dos dois maiores folículos foi aspirado. A técnica de 2D-PAGE foi realizada, em duplicata, utilizando gel de acrilamida a 12%. Os géis foram corados com Comassie Brilliant Blue R-250, escaneados e analisados, utilizando o PDQuest software, para determinar a densidade óptica dos spots. A identificação proteica foi realizada através de espectometria de massa (MS). Um total de 43 spots foi observado. Sete spots, representando cinco proteínas (albumina, apolipoproteína A-1, gelsolina, transferrina e α-1-antiproteinase 2), apresentaram diferenças (P˂0,05) na expressão, no FF do maior folículo, nos diferentes grupos. Um spot, representado pela proteína POMZP3, demonstrou diferença (P=0,018) em sua expressão, entre o maior e o segundo maior folículo, nos diferentes grupos. E, por fim, um spot, identificado como a proteína α-1-antiproteinase 2, apresentou interação (P=0,047) entre o maior e o segundo maior folículo e as diferentes fases da foliculogênese. Os resultados deste trabalho demonstram que o perfil proteico do FF difere durante o desenvolvimento folicular e que, as maiores alterações, são observadas a partir da dominância. Além disso, provavelmente, algumas destas proteínas, bem como suas correlações, tenham grande importância nos eventos que ocorrem durante a foliculogênese. / The follicular fluid (FF) is a complex extracellular fluid that accumulates in the antrum follicles during the follicular development. It is the essential medium for the growth and maturation of somatic and germ ovarian cells and contains substances involved in cell differentiation, oocyte maturation, gamete quality, rupture of the follicle wall and luteinization. The study of its components is crucial for a better understanding of the mechanisms involved in follicular dynamics in mares. The objective of this study was to determine the protein profile of the largest follicle and among the largest and the second largest follicle at different stages of follicular development. In this study, 40 ovaries from 20 non pregnant Criollo cycling mares were collected during the breeding season in an abattoir. Before slaughter, the mares were divided into four groups according to follicular diameter, uterine ecotexture (UE) and the presence of corpus luteum (CL): G 15 (emergence) (n = 3), follicles up to 15mm, EU ≥ 1, CL ≥ 20 mm; G 20 (deviation) (n = 9), follicles between 20 and 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominance) (n = 4), follicles between 30 e 35 mm, EU ≥ 2, CL ≥15 mm; G 40 (ovulation) (n = 4), follicles ≥ 40 mm, EU 2-3, CL ≥ 15 mm. After slaughter, the ovaries were collected and the FF of the two largest follicles was aspirated. The technique of 2D-PAGE was performed in duplicate using 12% acrylamide gel. Gels were stained with Comassie Brilliant Blue R-250, scanned and analyzed using the PDQuest software to determine the optical density of the spots. Protein identification was performed by mass spectrometry (MS). A total of 43 spots was observed. Seven spots representing five proteins (albumin, apolipoprotein A-1, gelsolin, transferrin e α-1-Antitrypsin 2) showed differences (P˂0.05) in expression, the FF of the largest follicle in the different groups. One spot, represented by POMZP3 protein showed a difference (P=0.018) in expression between the largest and second largest follicle in the different groups. Finally, one spot, identified as the protein α-1-antitrypsin 2, showed interaction (P=0.047) between the largest and second largest follicle. The results of this study demonstrated that the protein profile of FF differs during follicular development and that the largest changes are observed from the dominance. Also probably some of these proteins, as well as their correlations, have great importance in the events that occur during folliculogenesis.

O fluido folicular (FF) é um líquido extracelular complexo que se acumula no antro dos folículos ovarianos durante o seu desenvolvimento. É o meio essencial para o crescimento e a maturação das células ovarianas somáticas e germinativas e contém substâncias envolvidas na diferenciação celular, maturação do oócito, qualidade do gameta, ruptura da parede folicular e luteinização. O estudo de seus componentes é fundamental para um melhor entendimento dos mecanismos que envolvem a dinâmica folicular na espécie equina. O objetivo deste trabalho foi comparar o perfil proteico do maior folículo, e entre o maior e o segundo maior folículo, em diferentes momentos do desenvolvimento folicular. Para este estudo, quarenta ovários, oriundos de vinte éguas Crioulas, não gestantes e cíclicas, foram coletados durante a estação reprodutiva, em um abatedouro. Antes do abate, as éguas foram divididas em quarto grupos de acordo com o diâmetro folicular, ecotextura uterina (EU) e presença de corpo lúteo (CL): G 15 (emergência) (n = 3) folículos até 15 mm, EU ≥ 1, CL ≥ 20 mm; G 20 (divergência) (n = 9) folículos entre 20 e 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominância) (n = 4) folículos entre 30 e 35 mm, EU ≥ 2, CL ≤ 15 mm; G 40 (pré-ovulatória) (n = 4) folículos ≥ 40 mm, EU 2-3, CL ≤ 15 mm. Após o abate, os ovários foram coletados e o FF dos dois maiores folículos foi aspirado. A técnica de 2D-PAGE foi realizada, em duplicata, utilizando gel de acrilamida a 12%. Os géis foram corados com Comassie Brilliant Blue R-250, escaneados e analisados, utilizando o PDQuest software, para determinar a densidade óptica dos spots. A identificação proteica foi realizada através de espectometria de massa (MS). Um total de 43 spots foi observado. Sete spots, representando cinco proteínas (albumina, apolipoproteína A-1, gelsolina, transferrina e α-1-antiproteinase 2), apresentaram diferenças (P˂0,05) na expressão, no FF do maior folículo, nos diferentes grupos. Um spot, representado pela proteína POMZP3, demonstrou diferença (P=0,018) em sua expressão, entre o maior e o segundo maior folículo, nos diferentes grupos. E, por fim, um spot, identificado como a proteína α-1-antiproteinase 2, apresentou interação (P=0,047) entre o maior e o segundo maior folículo e as diferentes fases da foliculogênese. Os resultados deste trabalho demonstram que o perfil proteico do FF difere durante o desenvolvimento folicular e que, as maiores alterações, são observadas a partir da dominância. Além disso, provavelmente, algumas destas proteínas, bem como suas correlações, tenham grande importância nos eventos que ocorrem durante a foliculogênese. / The follicular fluid (FF) is a complex extracellular fluid that accumulates in the antrum follicles during the follicular development. It is the essential medium for the growth and maturation of somatic and germ ovarian cells and contains substances involved in cell differentiation, oocyte maturation, gamete quality, rupture of the follicle wall and luteinization. The study of its components is crucial for a better understanding of the mechanisms involved in follicular dynamics in mares. The objective of this study was to determine the protein profile of the largest follicle and among the largest and the second largest follicle at different stages of follicular development. In this study, 40 ovaries from 20 non pregnant Criollo cycling mares were collected during the breeding season in an abattoir. Before slaughter, the mares were divided into four groups according to follicular diameter, uterine ecotexture (UE) and the presence of corpus luteum (CL): G 15 (emergence) (n = 3), follicles up to 15mm, EU ≥ 1, CL ≥ 20 mm; G 20 (deviation) (n = 9), follicles between 20 and 25 mm, EU 1-2, CL 15-20 mm; G 30 (dominance) (n = 4), follicles between 30 e 35 mm, EU ≥ 2, CL ≥15 mm; G 40 (ovulation) (n = 4), follicles ≥ 40 mm, EU 2-3, CL ≥ 15 mm. After slaughter, the ovaries were collected and the FF of the two largest follicles was aspirated. The technique of 2D-PAGE was performed in duplicate using 12% acrylamide gel. Gels were stained with Comassie Brilliant Blue R-250, scanned and analyzed using the PDQuest software to determine the optical density of the spots. Protein identification was performed by mass spectrometry (MS). A total of 43 spots was observed. Seven spots representing five proteins (albumin, apolipoprotein A-1, gelsolin, transferrin e α-1-Antitrypsin 2) showed differences (P˂0.05) in expression, the FF of the largest follicle in the different groups. One spot, represented by POMZP3 protein showed a difference (P=0.018) in expression between the largest and second largest follicle in the different groups. Finally, one spot, identified as the protein α-1-antitrypsin 2, showed interaction (P=0.047) between the largest and second largest follicle. The results of this study demonstrated that the protein profile of FF differs during follicular development and that the largest changes are observed from the dominance. Also probably some of these proteins, as well as their correlations, have great importance in the events that occur during folliculogenesis.

O saco vitelínico é uma das membranas embrionárias que desempenham um papel importante para a sobrevivência inicial do embrião em muitas espécies de mamíferos, além de produzir proteínas necessárias para o desenvolvimento do mesmo. Foram coletados 17 embriões bovinos, em diferentes períodos gestacionais afim de identificar as proteínas alfafetoproteína, alfa- 1 antitripsina e transferrina, presentes no saco vitelínico destes,para tanto realizou-se a técnica de Western Blot com eletroforese em gel de poliacrilamida, SDS-PAGE a 6%. Os géis, após a corrida, foram corados com Comassie blue, e as membranas de nitrocelulose, após a transferência, com Ponceau. Utilizaram-se os anticorpos monoclonal para alfafetoproteína anti-camundongo, monoclonal, receptpr de transferrin anti-camundongo IgG1, e policlonal para alfa- 1 antitripsina anti-coelho como anticorpos primário e conjugado para peroxidase e fosfatase como secundários. A revelação foi do tipo colorimétrica-fosfatase alcalina e por ECL. O saco vitelínico apresentou-se bem desenvolvido até os 50 dias de gestação, onde, a partir desse período o processo de involução está bem caracterizado Em algumas amostras do saco vitelínico detectamos a presença da alfafetoproteina, alfa-1 antitripsina e da transferrina, porém em algumas amostras as bandas estavam fracas, mostrando assim, que os anticorpos reagem com as proteínas bovinas. O fato de aparecerem bandas fracas pode estar relacionado a uma fraca reação cruzada por se tratar de um anticorpo não específico. / In many species of mammals, the yolk sac is one of the embrionary membranes that plays an important role in the embryo´s initial survival, as well as, in the manufacturing of the necessary proteins for its development. In order to identify the proteins: alfafetoprotein, alfa 1 - antitrypsin, and transferrin present in the cow´s embryo´s yolk sac, 17 bovine embryos were collected in different pregnancy periods. This procedure was performed by Western Blot Technique with a polyacrylamide gel electrophoresis, SDS-PAGE, at 6%. Gels following the electrophoresis, where tainted with Comassie blue, and the membranes of Nitrocellulose, following their transference (the proteins that were present in the gel go to the membrane), with Ponceau. Monoclonal Antibody mouse anti human α-fetoprotein, alphafetoprotein mouse monoclonal antibody, transferrin receptor mouse IgG1, and rabbit polyclonal to alpha 1 antitrypsin were used as primary antibodies, and Peroxidase labelled antimouse e Peroxidase labelled antirabbit e anti-mouse IgG- Alkaline Fosfatase as secundary ones. The membrane´s revelation was of the alcaline fosfatase colormetric type and by ECL. The yolk sac was presented well developed until the 50 days of gestation, where to break of this period the involution process well it is characterized. In some of the yolk sac samples we detected the presence of alfafetoprotein, alfa 1- antitrypsin, and transferrin, however, the bands in some specimens (samples) were weak, demonstrating that the antibodies react with the bovine proteins. The fact that weak bands appeared might be related to a weak cross reaction since we are dealing with a non specific antibody.

京都大学 / マギル大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第24203号 / 医博JD第1号 / 新制||医||JD1(附属図書館) / 京都大学大学院医学研究科京都大学マギル大学ゲノム医学国際連携専攻 / (主査)教授 稲垣 暢也, 教授 YOUSSEFIAN Shohab, 准教授 Majewski Jacek (マギル大学), 准教授 Gravel Simon (マギル大学), 教授 Gagneur Julien (ミュンヘン工科大学) / 学位規則第4条第1項該当 / Doctor of Philosophy in Human Genetics / Kyoto University / McGill University / DFAM

<p>Many elderly undergo cardiac surgery. The prevalence of malnutrition in elderly is high and increases with comorbidity. This thesis aims to clarify some aspects on performing surgery in elderly concerning nutritional status, nutritional treatment and age-related physiology.</p><p>Study I: 886 patients were assessed preoperatively by body mass index (BMI) and S-albumin and postoperatively for mortality and morbidity.. Low BMI increased the relative hazard for death and low S-albumin increased the risk for infection. BMI and S-albumin are useful in preoperative evaluations</p><p>Study II: we followed energy intake in 31 patients for five postoperative days. Scheduled and unscheduled surgery did not differ in preoperative resting energy expenditure (REE). REE increased by 10-12% postoperatively, more in unscheduled CABG. Nutritional supplementation increased total energy intake. All patients exhibited postoperative energy deficits, less prominent in the supplemented group. There were no differences in protein synthesis or muscle degradation. </p><p>Study III: in 16 patients, .we measured stress hormones and insulin resistance before surgery and for five postoperative days Patients were insulin resistant on the first two days. We saw no clearly adverse or beneficial effects of oral carbohydrate on insulin resistance or stress hormone response. </p><p>Study IV: 73 patients, with early enteral nutrition (EN), were observed until discharge or resumed oral nutrition. EN started within three days in most patients. In a minority, problems occurred (gastric residual volumes, tube dislocation, vomiting, diarrhoea, aspiration pneumonia). In the cardiothoracic ICU individually adjusted early EN is feasible. </p><p>Study V: in 16 patients, splanchnic blood flow (SBF) enhancing treatments (dopexamine (Dpx) or EN) were compared. Dpx increased systemic blood flow, but had only a transient effect on SBF. EN had no effect on systemic blood flow or SBF. Neither Dpx, EN or the combined treatment, exhibited any difference between groups on systemic or splanchnic VO₂ or oxygen extraction ratio. </p>

Many elderly undergo cardiac surgery. The prevalence of malnutrition in elderly is high and increases with comorbidity. This thesis aims to clarify some aspects on performing surgery in elderly concerning nutritional status, nutritional treatment and age-related physiology. Study I: 886 patients were assessed preoperatively by body mass index (BMI) and S-albumin and postoperatively for mortality and morbidity.. Low BMI increased the relative hazard for death and low S-albumin increased the risk for infection. BMI and S-albumin are useful in preoperative evaluations Study II: we followed energy intake in 31 patients for five postoperative days. Scheduled and unscheduled surgery did not differ in preoperative resting energy expenditure (REE). REE increased by 10-12% postoperatively, more in unscheduled CABG. Nutritional supplementation increased total energy intake. All patients exhibited postoperative energy deficits, less prominent in the supplemented group. There were no differences in protein synthesis or muscle degradation. Study III: in 16 patients, .we measured stress hormones and insulin resistance before surgery and for five postoperative days Patients were insulin resistant on the first two days. We saw no clearly adverse or beneficial effects of oral carbohydrate on insulin resistance or stress hormone response. Study IV: 73 patients, with early enteral nutrition (EN), were observed until discharge or resumed oral nutrition. EN started within three days in most patients. In a minority, problems occurred (gastric residual volumes, tube dislocation, vomiting, diarrhoea, aspiration pneumonia). In the cardiothoracic ICU individually adjusted early EN is feasible. Study V: in 16 patients, splanchnic blood flow (SBF) enhancing treatments (dopexamine (Dpx) or EN) were compared. Dpx increased systemic blood flow, but had only a transient effect on SBF. EN had no effect on systemic blood flow or SBF. Neither Dpx, EN or the combined treatment, exhibited any difference between groups on systemic or splanchnic VO2 or oxygen extraction ratio.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) show high prevalence and are economically important in equine athletes. RAO is considered a multifactorial disease due to genetic and environmental components in their patophysiology. The aim of this study was to determine the presence of polymorphism at exons 2, 3, 4 and 5 of the SPi2 gene and a possible association between them and ORA or IAD on 51 thoroughbred horses through single-strand conformational polymorphism (SSCP). Exons 2, 3 and 4 of the Spi2 gene showed no polymorphism. On exon 5, 3 alleles and 6 genotypes were identified. Frequency of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO but no association was found between any polymorphism and horses with RAO or IAD. / A obstrução recorrente das vias aéreas (ORA) e a doença inflamatória das vias aéreas (DIVA) são doenças de alta prevalência e economicamente importantes em cavalos atletas. A ORA é considerada uma enfermidade multifatorial por apresentar componentes ambientais e genéticos em sua fiosiopatologia. O presente trabalho teve por objetivo determinar a presença de polimorfismos nos éxons 2, 3, 4 e 5 do gene SPi2 e verificar uma possível associação destes com a ORA e/ou DIVA em 51 cavalos Puro Sangue de Corrida através da técnica de polimorfismo conformacional de fita simples (SSCP). Os éxons 2, 3 e 4 não apresentaram polimorfismo. No éxon 5 do gene Spi2 foram identificados três alelos e seis genótipos. Apesar do alelo A e o genótipo AA apresentarem freqüência (0,6388 e 0,3888, respectivamente) mais elevada nos animais com ORA, não houve associação entre os polimorfismos observados e ORA ou DIVA.

Indiana University-Purdue University Indianapolis (IUPUI) / Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt% and 36 wt%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.