Gelsolin from horse plasma has been isolated in good and reproducible yields. It has an absorption coefficient of 1.4 ml/(mg cm) and is similar in amino acid composition to other plasma gelsolins. It migrates as a single polypeptide chain in polyacrylamide gel electrophoresis with an apparent molecular mass of 90 kDa. Hydrodynamic calculations suggest that gelsolin is a globular protein of 75 kDa. Thermal and chemical denaturation profiles were obtained for gelsolin by measuring its intrinsic fluorescence and ellipticity values. The melting temperature values obtained were Tm ≈ 46 °C in the absence of calcium and Tm ≈ 54 °C in the presence of the divalent cation. The mid-point of the transition in the guanidine hydrochloride-induced unfolding of gelsolin was found to be at ≈ 1.5 M denaturant. Gelsolin is able to nucleate actin polymerization and sever actin filaments. These activities are manifested by the abolition of the lag phase in the time course of actin polymerization and the decreased steady state viscosity of actin solutions polymerized in the presence of gelsolin. Gelsolin interacts with actin labelled with the fluorescent probe 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) to produce a 2:1 actin:gelsolin complex in the presence of calcium. Upon chelation of the divalent cation from the 2:1 complex, one actin molecule is released producing a 1:1 EGTA-resistant complex . The fluorescence of 2-(N-methylanilino)naphthalene-6-sulphonate (MANS), a fluorescent probe that is sensitive to the polarity of its environment, is both enhanced and blue shifted upon binding to gelsolin. These results are indicative of the binding of MANS to hydrophobic regions in gelsolin. Gelsolin binds 2.5 ± 0.9 molecules of MANS with a dissociation constant of 0.24 ± 0.13 μM. Gelsolin can be labelled in the absence of divalent cations with the sulfhydryl-specific probe N-(l-pyrenyl)iodoacetamide (PIA) without altering its structural integrity. The labelled protein presents excimer-like pyrene emission indicative of the proximity of the labelled cysteines in the three dimensional structure of the protein. The spectroscopic characteristics of the labelled protein indicate that the excimer emission arises from interactions that can be traced to the ground state of the pyrene molecules. This is in contrast to the well studied excimer emission that arises from molecules that repel each other in the ground state. / Science, Faculty of / Chemistry, Department of / Graduate

Thesis (doctoral)--Rheinische Friedrich-Wilhelms-Universität Bonn, 2005.

Gelsolin was labelled with the sulphydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labelling using non-denaturing conditions was 1.9 ± 0.5 acrylodans per gelsolin molecule. Circular dichroism and viscosity studies showed no significant effect on gelsolin structure and function on incorporation of the label. Circular dichroism studies did not detect Ca²⁺ effects on aerylodan-labelled gelsolin, but fluorescence studies detected subtle changes in the protein. The presence of Ca²⁺ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I⁻ and a decrease in fluorescence polarisation of the acrylodan-labelled gelsolin. These indicate an increased degree of exposure of the fluorescent label to the solvent environment on interaction of gelsolin with Ca²⁺. Actin binding to gelsolin was evident from a decrease in fluorescence intensity, an increase in sensitivity to quenching and an increase in fluorescence polarisation. Actin binding increases the exposure of the acrylodan label to solvent, as does Ca²⁺ binding. / Science, Faculty of / Chemistry, Department of / Graduate

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 3 uppsatser.

Neuroprotektion und Verbesserung der zerebralen Durchblutung stellen die beiden zentralen therapeutischen Ansätze für den ischämischen Schlaganfall dar. In dieser Arbeit stellen wir ein neues experimentelles Konzept vor, welches das neuronale und endotheliale Zytoskelett als therapeutisches Target zur Schlaganfallbehandlung identifiziert. Ungebremster intrazellulärer Kalziumeinstrom durch aktivierte N-methyl-D-aspartat (NMDA)-Rezeptoren und spannungsabhängige Ca++-(VDCC)-Kanäle ist ein entscheidender Trigger für den Zelltod nach Schlaganfall. Die Aktivität dieser Kanäle wiederum wird dynamisch durch Veränderungen des Aktin-Zytoskeletts modifiziert, welches u.a. durch das aktindegradierende Protein Gelsolin vermittelt wird. Neurone, denen Gelsolin fehlt (Gelsolin-Null), zeigen einen vermehrten Zelltod und erhöhten Ca++-Einstrom nach Sauerstoff/Glukose Deprivation und weiterhin erhöhte zytosolische Ca++-Spiegel in den Nervenendigungen nach Depolarisation in vitro. Nach transienter zerebraler Ischämie wiesen Gelsolin-Null Mäuse deutlich größere Infarkte im Vergleich zu den Kontrollen auf. Eine akute Behandlung mit Cytochalasin D, einem Pilztoxin, das spezifisch Aktinfilamente depolymerisiert, reduzierte das Schlaganfallvolumen in Gelsolin-Null und Wildtypmäusen auf das gleiche Volumen. Gelsolinaktivierung und Aktindepolymerisierung schützen somit vor Exzitotoxizität und Zelltod nach zerebraler Ischämie. Ein entscheidender Regulator des zerebralen Blutflusses ist die endotheliale NO Synthase (eNOS). Mäuse, denen dieses Enzym fehlt (eNOS-Null Mäuse), entwickeln grössere Infarkte nach fokaler zerebraler Ischämie. In unserer Arbeit zeigen wir, daß das G-Protein Rho Veränderungen im Zytosklett von Endothelzellen bewirkt, die zur eNOS Herunterregulierung führen. Die Behandlung von Mäusen mit Rho Inhibitoren, wie Statinen (HMG-CoA Reduktasehemmern), C3 Transferase von C. botulinum, oder aber mit dem (oben genannten) aktindepolymerisierenden Toxin Cytochalasin D führten zu einer höheren eNOS Aktivität, vermehrtem zerebralem Blutfluß und kleineren Infarkten nach zerebraler Ischämie. Diese neuroprotektiven Effekte konnten nicht in eNOS-Null Mäusen erzielt werden, was dieses Enzym als den entscheidenden Effektor dieses prophylaktischen Ansatzes identifiziert. Zusammenfassend stellen sowohl das neuronale Zytoskelett (durch Schutz vor Ca++-Influx) als auch das endotheliale Zytoskelett (über einen eNOS-abhängigen Mechanimus) ein neurartiges Target zur Schlaganfalltherapie dar. Diese ermöglichen neuartige Behandlungsstrategien sowohl in der Akutphase als auch zur Prophylaxe. Insbesondere die Tatsache, daß mit den Statinen bereits zugelassene spezifische Therapeutika zur Verfügung stehen, unterstreicht die unmittelbare klinische Relevanz der Untersuchungen. / Neuroprotection and reperfusion are the basic therapeutic concepts for the treatment of ischemic stroke. Here, we present a novel strategy which identifies both the neuronal and endothelial cytoskeleton as potential therapeutic targets. Increased Ca++ influx through activated NMDA receptors and voltage-dependent Ca++ channels (VDCC) is a major determinant of cell injury after brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur in part through the actions of the actin filament-severing protein gelsolin. We show that gelsolin null neurons have enhanced cell death and rapid, sustained elevation of Ca++ levels after glucose/oxygen deprivation as well as augmented cytosolic Ca++ levels in nerve terminals after depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin null mice after reversible middle cerebral artery occlusion compared to controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin null and control mice to the same final volume. Hence, enhancement or mimickry of gelsolin activity may be neuroprotective during stroke. Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-/- "knockout") mice develop larger cerebral infarcts following middle cerebral artery (MCA) occlusion. We report that the small G-protein rho mediates disruption of the endothelial actin cytoskeleton that leads to upregulation of eNOS. Mice treated with Rho inhibitors like statins (HMG-CoA reductase inhibitors), C3 transferase from C. botulinum, or with the actin cytoskeleton disruptor cytochalasin have higher eNOS expression and activity, increased cerebral blood flow and smaller ischemic lesions following MCA occlusion. No neuroprotection was observed with these agents in eNOS-/- mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke. Both the neuronal and the endothelial actin cytoskeleton were identified as novel therapeutic targets following cerebral ischemia stroke. This may have implications for the treatment and prophylaxis of ischemic stroke in man.

Ovarian cancer (OVCA) is the most lethal gynecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as targeted therapies and immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long term therapeutic success. Therefore, there is an urgent need to understand the cellular and molecular mechanisms of immune dysregulation in chemoresistant ovarian cancer in order to harness the host’s immune system to improve cancer survival. Early diagnosis and residual disease are key determinants of favorable survival in OVCA; however, CA125 which is the conventional marker is not reliable and has modest diagnostic accuracy. There is therefore an urgent need to discover reliable biomarkers to optimize individualized treatment and diagnostic recommendations. Plasma gelsolin (pGSN; an actin binding protein) is the secreted isoform of the gelsolin (GSN) gene implicated in inflammatory disorders, colon cancer and prostate cancer. Increased expression of total GSN is associated with poor survival of patients with gynecological cancers. As to whether this is due to pGSN is yet to be investigated. Increased expression of pGSN is significantly associated with the down-regulation of immune cell markers; however, the exact mechanism has not been explored. If and how pGSN is involved in the cellular and molecular mechanisms of OVCA remains to be determined. In our current research, we have demonstrated that pGSN is involved in the regulation of immune cells, early diagnosis, tumor recurrence and chemoresistance in OVCA, using standard in vitro techniques and human clinical samples (North America, Asia and public datasets). We have shown that pGSN is highly expressed and secreted in chemoresistant OVCA cells than their chemosensitive counterparts. pGSN, secreted and transported via exosomes, upregulated HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as conferred cisplatin resistance in otherwise chemosensitive OVCA cells. pGSN also induced the OVCA expression of the antioxidant and tumor growth promoter, glutathione (GSH), by activating Nuclear factor erythroid 2-related factor 2 (NRF2), a response that attenuated cisplatin (CDDP)-induced apoptosis. In human tumor tissues, increased pGSN mRNA and protein expressions were significantly associated with advanced tumor stage, suboptimal residual disease, tumor recurrence, chemoresistance and poor survival regardless of patients’ ethnic background and histologic subtypes. Increased Infiltration of CD8+ T cells was significantly associated with favorable patient survival; however, increased pGSN hindered the survival impact of these infiltrated CD8+ T cells. Further investigation revealed that pGSN induced CD8+ T cell death via caspase-3 activation, an action that resulted in decreased IFNγ levels. Increased epithelial pGSN expression was significantly associated with reduced survival benefits of infiltrated M1 macrophages, through caspase-3-dependent apoptosis as well as reduced production of TNFα and iNOS. The clinical application of circulatory pGSN as a biomarker for early detection and patients’ survival was investigated. Pre-operative circulating pGSN presented as a favorable and independent biomarker for early disease detection and residual disease prediction compared with CA125. The test accuracy of pGSN was significantly enhanced when combined with CA125 in multianalyte index assay. The findings suggest that pGSN is a potential target for chemoresistant OVCA and presents as a diagnostic marker for early stage disease and surgical outcomes, interventions that could maximize the therapeutic success of immunotherapies.

Ovarian cancer (OVCA) is the most lethal gynecologic cancer. Chemoresistance remains a major hurdle to successful therapy and patient survival. The secreted isoform of the actin-associated protein, gelsolin (plasma gelsolin; pGSN), is highly expressed in chemoresistant than chemosensitive OVCA cells, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. DNA methylation plays a key role in the regulation of genes expression and contributing to cancer development and chemoresistance with the help of DNA methyltransferases (DNMTs) or Ten eleven translocation (TETs) enzymes. TET1 is the most studied isoform of TETs family and primarily responsible for 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) oxidation to initiate demethylation and increase in the expression of methylated genes. Whether pGSN expression in OVCA cells is regulated by DNA methylation and TET1 regulates the differential pGSN expression between chemosensitive and resistant OVCA cells is not known. In this study, we hypothesized pGSN overexpression in chemoresistant OVCA cells is due to the hypomethylation at its promoter region by TET1. Our objective was to investigate whether DNA methylation and specifically TET1 plays a role in the regulation of differential pGSN expression and chemosensitivity in OVCA. Chemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance and protein contents by qPCR and Western blotting respectively. Cisplatin-induced chemoresponsiveness was morphologically assessed by Hoechst staining (apoptosis). Infinium HumanMethylation450 BeadChip assay was used for global methylation analysis of twelve (12) different OVCA cells and to investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance. DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cell using specific inhibitors. Gain-and-loss-of-function assays were carried to identify the relationship between TET1 and pGSN in OVCA chemoresponsiveness. Differential protein and mRNA expressions of pGSN and TET1 were observed between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. Global methylation analysis revealed hypomethylation in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreases their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN and positively related to chemoresponsiveness of OVCA cells. This project does not only broaden our knowledge about the mechanistic insights into the epigenetic regulation of pGSN in OVCA chemoresistance, but it also reveals a new potential target to re-sensitize chemotherapy resistant OVCA cells. This may provide a future strategy to improve overall OVCA patient survival.

In unseren Studien haben wir uns unter Verwendung von in vitro und in vivo Modellen des neuronalen Zelltodes mit verschiedenen neuroprotektiven Mechanismen beschäftigt. Uns interessierten Faktoren aus drei verschiedenen Komplexen, die an zellulärer Vitalität teilhaben. Im Mittelpunkt unserer Untersuchung stand ein antioxidatives Enzym und dessen Rolle bezüglich des neuronalen Zelltodes - die NAD(P)H-Quinonoxidoreduktase1. In den Sudien haben wir die Aktivität, Expression und die Lokalisation dieses Enzyms nach neuronalem Schaden untersucht. Wir konnten zeigen, dass die Quinonoxidoreduktase den neuronalen Schaden exazerbieren kann. Ein anderer Schwerpunkt unserer Arbeit bildete das Steroidhormon 17-beta-Estradiol und sein neuroprotektiver Einfluss auf neurodegenerative Prozesse. Wir deckten verschiedene Expressionen der beiden Estradiolrezeptoren alpha und beta in kortikalen, septalen und hippokampalen Nervenzellen auf und das daraus folgende unterschiedliche neuroprotektive Potenzial des Hormons. Im dritten Teil der Studien konnten wir den Einfluss des zytoskelettmodulierenden Proteins Gelsolin auf die Progression des neuronalen Zelltodes nach zellulärem Schaden charakterisieren. / In our studies we characterized different neuroprotective mechanism by in vitro and in vivo models of neuronal cell death. We were interested in factors of three different complexes wich play a role in cellulare vitality. In the centre of our studies there was an antioxidative enzyme and his role in the neuronal cell death-the NAD(P)H:quinone oxidoreductase1. We have investigated in the activity, expression and localisation of this enzyme after neuronal damage. We could show that this enzyme can exacerbate neuronal cell death. Another point of our work were the steroid hormon 17-beta-Estradiol and the neuroprotective character in neurodegeneration. We discovered different expression of the two estradiol- receptors alpha and beta in brain caused in different protective potential to cortical, septal and hippocampal structures by the hormon. The cytoskeletal structures modulate by the protein gelsolin was the third part of the studies. We could show that the modulation of the cytoskeleton were involved in the progression of neuronal cell death after cellulare damage.

A região anterior do intestino médio de Lepidoptera apresenta uma secreção microapócrina de enzimas digestivas com vesículas migrando pelo interior das microvilosidades. Essas vesículas brotam das microvilosidades intestinais como vesículas de membrana dupla e são descarregadas dentro do lúmen. O objetivo desse trabalho foi identificar as proteínas secretadas e aquelas envolvidas na maquinaria secretória microapócrina em Spodoptera frugiperda. Para isso, vesículas microapócrinas foram preparadas e usadas para a produção de anticorpo policlonal. Esse anticorpo foi utilizado para varrer uma biblioteca de expressão de cDNA do intestino médio de S. frugiperda. Também obtivemos um transcriptoma por pirosequenciamento de uma biblioteca de cDNA proveniente dos transcritos do intestino médio do mesmo inseto. Os clones positivos da varredura foram sequenciados, montados e submetidos a um BLASTN contra as sequências obtidas pelo pirosequenciamento, o que resultou na extensão dessas sequências. Usamos ainda as sequências geradas pelo pirosequenciamento para reanalisar sequências de proteínas presentes nas membranas microvilares, que tinham sido obtidas anteriormente em nosso laboratório (Ferreira et al., 2007). A reanálise das sequências de proteínas microvilares gerou 66 proteínas preditas. Dessas, 18 foram consideradas contaminantes de outros compartimentos celulares e 48 associadas às membranas microvilares. A análise das sequências obtidas das vesículas microapócrinas gerou 50 proteínas preditas que podem ser secretadas por essa rota. As sequências encontradas tanto em membrana microvilar quanto em vesículas microapócrinas podem ser classificadas em 8 grupos, de acordo com sua função: (1) enzimas digestivas; (2) proteínas da membrana peritrófica; (3) envolvidas com proteção; (4) transportadores; (5) receptores; (6) proteínas da maquinaria secretória; (7) proteínas de citoesqueleto; (8) com função desconhecida nesse local. Em ambas as preparações existem uma predominância de sequências de enzimas digestivas. Nas membranas microvilares a maioria das sequências são aminopeptidases, enquanto nas vesículas microapócrinas a maioria são lipases. Os cDNAs correspondentes as proteínas que poderiam estar envolvidas na maquinaria secretória foram clonados e sequenciados. São elas: fimbrina, cofilina, gelsolina-1 e miosina I. RT-PCRs semi-quantitativas dessas proteínas em diferentes tecidos do inseto (intestino, túbulos de Malpighi, corpo gorduroso e carcaça) mostraram que somente gelsolina-1 está presente exclusivamente no intestino. Os domínios G1-G3 da gelsolina característica do intestino (gelsolina-1) foram expressos e usados para produzir anticorpos em coelhos. Esses anticorpos reconhecem a proteína recombinante e uma proteína presente no epitélio intestinal com massa molecular compatível com a massa predita para gelsolina-1. Foi possível diminuir a expressão de gelsolina-1 utilizando RNA interferente. / Lepidoptera anterior midgut presents a microapocrine secretion of digestive enzymes with secretory vesicles migrating inside the microvilli. These vesicles bud from the midgut microvilli as double membrane vesicles and are discharged into the lumen. The aim of this work was to identify the proteins secreted and those involved in the microapocrine secretory machinery in Spodoptera frugiperda larvae. For this, microapocrine vesicles were prepared and used for polyclonal antibody production. This antibody was used to screen a cDNA expression library of S. frugiperda midgut. We also obtained a transcriptome by pyrosequencing a cDNA library derived from transcripts of the midgut. Positive clones from the screening were sequenced, assembled and N-blasted against S. frugiperda sequences obtained by pyrosequencing. This procedure led to the extension of the sequences previously obtained. We also used the sequences generated by pyrosequencing to reanalyze the sequences of microvillar membrane proteins obtained previously by Ferreira et al. (2007). This reanalysis generated 66 predicted proteins that are present in the microvillar membranes. Eighteen were considered to be contaminants from other compartments and 48 associated with the microvillar membranes. Analysing the sequences from microapocrine vesicles we found 50 predicted proteins that should be secreted by microapocrine vesicles. The sequences found in both microvillar membrane and microapocrine vesicles may be classified into 8 groups, according to their function: (1) digestive enzymes; (2) peritrophic membrane proteins; (3) protection; (4) transporters; (5) receptors; (6) secretory machinery; (7) cytoskeleton; (8) with unknown function. In both preparations there is a predominance of sequences of digestive enzymes. In microvillar membranes, there is a remarkable amount of aminopeptidases, while in microapocrine vesicles this is true for lipases. cDNAs coding for proteins that could be involved in the microapocrine secretory machinery were cloned and sequenced. They are: fimbrin, cofilin, gelsolin-1 and myosin I. Using RT-PCR, we showed that mRNAs coding for gelsolin-1 and myosin I are present only in the intestinal tissue. The mRNAs coding for other proteins were found in all tissues. The domains G1-G3 from gelsolin specific from intestinal midgut (gelsolin 1) were expressed and used to raise antibodies in rabbit. These antibodies were able to recognize the recombinant protein and a protein from the midgut epithelium that has a molecular weight similar to the one predicted from gelsolin-1 sequence. We succeed in decreasing the expression of gelsolin-1 by using interfering RNA

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.