Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of the present study was to determine the action of gestagens and estradiol benzoate (EB) on the mechanism of premature regression of the first corpus luteum (CL) after parturition, and to develop an efficient artificial insemination system for suckling beef cows. The effect of pre-exposure to gestagens and EB on the expression of the alpha (ERα) and beta (ERß) estrogen receptors and progesterone receptor (PR) in the endometrium and CL was evaluated after the first postpartum ovulation. Thirty days after calving, ten anestrous cows were divided into a control group (AN) and a treatment group (AN+MAP). On day 0, animals of the treatment group received 250 mg medroxyprogesterone (MAP) in a vaginal device for 7 days and 5 mg EB. In both groups, the follicular wave was monitored and ovulation was induced for the formation of a CL. Luteal and endometrial tissues were collected after 7 days. The samples were submitted to the analysis of gene expression by RT-PCR and endometrial and luteal histological evaluation. Preexposure to gestagen during the first postpartum estrous cycle resulted in a higher endometrial expression of the PR (AN+MAP group; P<0.05) and inhibition of ERß expression in endometrial tissue (P<0.05). The height of the endometrial glandular epithelium and the total number of large luteal cells were higher in the AN+MAP group than in the AN group. In order to promote follicular growth compatible with the ovulatory response in postpartum cows, we analyzed the follicular dynamics to determine the best time for the application of eCG and EB in combination with MAP. The cows received MAP for 7 days and EB on day 0 or 1, followed by eCG on day 6 (B0E6 or B1E6) or 7 (B0E7 or B1E7). In two groups, the animals were not injected with eCG but received EB on day 0 (B0) or 1 (B1). All cows received a GnRH agonist 45 h after removal of the vaginal device. Animals of the B0E6 group maintained a mean follicular diameter of 8 mm until eCG application and reached a mean diameter of 13 mm at the time of GnRH agonist injection (45 h after MAP removal), showing an ovulation rate of 80%. Covariance analysis showed a difference in this curve (P=0.0001) compared to the other groups, which reached a mean final follicular diameter of 7 to 12 mm and ovulation rate of 40%. Based on these results, we investigated an ovulation and estrus reinduction for a mixed system of estrus detection and fixed-time artificial insemination (ETAI) in postpartum suckling cows. A total of 553 animals was divided into the following groups: hormonal induction (HI; day 0 = EB and MAP for 7 days, day 6 = eCG, PGF2α and temporary weaning), early weaning at 60 days, and natural breeding. No difference in pregnancy rates (85%) was observed among groups. However, at 7 months of age, calves of the HI group had reached a 38 kg higher mean weight than those weaned at 60 days. In order to adjust the eCG dose to the HI system, doses of 300, 400 or 500 IU eCG were tested. No difference in the pregnancy rates (78.7%) was observed. In conclusion, MAP and EB regulate steroid receptors in the endometrium and CL, indicating an important role for these hormones in the mechanism of premature postpartum luteolysis. In addition, steroids participate in the postpartum restructuring of the endometrium and CL and, when applied 60 days after calving together with eCG administered one day before removal of the vaginal implant, increase the reproductive efficacy of beef cows. / O objetivo do presente estudo foi de elucidar a ação dos gestágenos e benzoato de estradiol (BE) no mecanismo de regressão prematura do primeiro corpo lúteo (CL) pós-parto, e desenvolver um sistema eficaz de inseminação artificial para vacas de corte amamentando. O efeito da pré-exposição a gestágenos e BE na expressão de receptor de estrógeno alfa (REα), beta (REβ) e receptor de progesterona (RP) no endométrio e CL foi avaliado após a primeira ovulação pós-parto. Dez vacas em anestro, 30 dias após o parto, foram divididas em grupo controle (AN) e tratamento (AN+MAP). No dia 0, os animais do grupo tratamento receberam 250 mg de medroxiprogesterona (MAP) em pessário vaginal por 7 dias e 5 mg de BE. Em ambos os grupos, a onda folicular foi acompanhada e a ovulação induzida para formação de um CL. Após 7 dias, foram obtidos tecidos luteal e endometrial. As amostras foram destinadas a expressão gênica (RT-PCR) e avaliação histológica endometrial e luteínica. A pré-exposição ao gestágeno no primeiro ciclo estral pós-parto resultou em uma maior expressão endometrial do RP (grupo AN+MAP; P<0,05) e um bloqueio da expressão dos REβ no tecido endometrial (P<0,05). No grupo AN+MAP, a altura do epitélio glandular endometrial e número total de células luteais grandes foram maiores do que no grupo AN. Visando promover o crescimento folicular compatível com resposta ovulatória, em vacas pós-parto, foi avaliada a dinâmica folicular, para determinar o melhor momento de aplicação do eCG e BE em associação ao MAP. As vacas receberam MAP por 7 dias e BE no Dia 0 ou 1, seguido de eCG no Dia 6 (B0E6 ou B1E6) ou 7 (B0E7 ou B1E7). Em dois grupos, os animais não foram injetados com eCG, mas com BE no Dia 0 (B0) ou no Dia 1 (B1). Todas as fêmeas receberam agonista de GnRH 45 h após remoção do implante vaginal. Os animais do grupo B0E6 mantiveram um diâmetro folicular médio de 8 mm até a aplicação do eCG e atingiram a média de 13 mm no momento do GnRH, com taxa de ovulação de 80%. Por análise de covariança, essa curva diferiu (P=0.0001) das demais, as quais atingiram média folicular final entre 8 e 12 mm e taxa de ovulação de 40%. A partir desses resultados, foi investigado um sistema de reindução de ovulação e estro e inseminação artificial em um processo misto de observação de estro e tempo fixo (IAETF) para vacas amamentando no período pós-parto. Um total de 553 animais foi dividido em indução hormonal (IH; Dia 0= BE e MAP por 7 dias, Dia 6= eCG, PGF2α e desmame temporário), desmame precoce aos 60 dias e monta natural. Não foi observada diferença nos índices de prenhez (85,0%) entre os grupos. No entanto, os terneiros do grupo IH, aos sete meses de idade, atingiram a média de 38 kg a mais do que os desmamados aos 60 dias. Com o objetivo de adequar a dose da gonadotrofina no sistema de IH, foram utilizadas 300, 400 ou 500 UI de eCG, não havendo diferença nos índices de prenhez (78,7%). Em conclusão, o MAP e BE regulam os receptores esteróides no endométrio e CL, evidenciando uma importante participação no mecanismo de luteólise prematura pós-parto. Além disso, esses esteróides participam da reestruturação pós-parto do endométrio e CL e, aplicados aos 60 dias pós-parto, associados ao eCG um dia antes da retirada do implante vaginal, resultam em maior eficácia reprodutiva em vacas de corte.

MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...

<p>U ovoj doktorskoj disertaciji&nbsp; razvijen&nbsp; je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada&nbsp; ER&alpha;, ER&beta; ili AR i kvantifikaciju&nbsp; njihovog&nbsp; afiniteta&nbsp; vezivanja sa mogućno&scaron;ću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana&nbsp; je primena optimizovanog biosenzora&nbsp; za&nbsp; procenu&nbsp; estrogenog&nbsp; potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma&nbsp; delovanja&nbsp; odabranih&nbsp; modifikovanih&nbsp; steroida&nbsp; ispitana&nbsp; je&nbsp; njihova antiproliferativna aktivnost prema ćelijskim&nbsp; linijama estrogen receptor pozitivnog kancera dojke&nbsp; (MCF-7) i kancera prostate (PC-3), dok su&nbsp; in silico metodom molekularnog&nbsp; dokinga&nbsp; predviđene&nbsp; energije&nbsp; i&nbsp; geometrije&nbsp; vezivanja&nbsp; ovih&nbsp; jedinjenja za ligand-vezujuće&nbsp; domene&nbsp; ER&alpha; i ER&beta;. Drugi deo ovog rada obuhvata razvoj testa za&nbsp; ispitivanje aktivnosti humanog enzima aromataze,&nbsp; heterologno eksprimiranog u ćelijama&nbsp; kvasca&nbsp; Saccharomyces cerevisiae&nbsp; i/ili&nbsp; bakterija Escherichia coli, u prisustvu&nbsp; ili&nbsp; odsustvu&nbsp; inhibitora.&nbsp; Interakcije modifikovanih&nbsp; steroida, odabranih na osnovu strukture,&nbsp; sa&nbsp; aromatazom&nbsp; ispitane&nbsp; su&nbsp; osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka&nbsp; triptofana&nbsp; iz&nbsp; aktivnog&nbsp; centra usled konformacione&nbsp; promene&nbsp; proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim&nbsp; visoke efikasnosti&nbsp; i&nbsp; bezbednosti&nbsp; po&nbsp; korisnika&nbsp; i&nbsp; okolinu, pokazali&nbsp; specifičnost&nbsp; i&nbsp; ekonomičnost&nbsp; u preliminarnom&nbsp; skriningu&nbsp; liganada&nbsp; steroidnih receptora&nbsp; i inhibitora aromataze. Jedinjenja&nbsp; kod kojih je detektovana&nbsp; značajna biolo&scaron;ka aktivnost mogu potencijalno poslužiti kao osnova za razvoj terapeutika u lečenju hormon-zavisnih bolesti i stanja, koja danas predstavljaju globalni zdravstveni problem.</p> / <p>In&nbsp; this&nbsp; doctoral&nbsp; dissertation,&nbsp; a&nbsp; fluorescent&nbsp; assay&nbsp; in&nbsp; yeast&nbsp; was&nbsp; developed&nbsp; for&nbsp; identification&nbsp; of&nbsp; potential&nbsp; natural or synthetic ligands of ER&alpha;, ER&beta; or AR and<br />quantification&nbsp; of&nbsp; their&nbsp; binding&nbsp; affinity,&nbsp; as&nbsp; well&nbsp; asevaluation&nbsp; of&nbsp; the&nbsp; estrogenic&nbsp; potential&nbsp; of&nbsp; synthetic steroids&nbsp; and&nbsp; selected&nbsp; plant&nbsp; extracts&nbsp; rich&nbsp; in phytoestrogen&nbsp; content.&nbsp; The&nbsp; assay&nbsp; could&nbsp; be&nbsp; used&nbsp; to&nbsp; screen&nbsp; libraries&nbsp; of&nbsp; modified&nbsp; steroids&nbsp; and xenoestrogens.&nbsp; In&nbsp; order&nbsp; to&nbsp; better&nbsp; understand&nbsp; the biomedical&nbsp; potential&nbsp; of&nbsp; selected&nbsp; modified&nbsp; steroids, results&nbsp; were&nbsp; compared&nbsp; to&nbsp; antiproliferative&nbsp; activity against&nbsp; estrogen&nbsp; receptor&nbsp; positive&nbsp; breast&nbsp; cancer (MCF-7)&nbsp; and&nbsp; prostate&nbsp; cancer&nbsp; (PC-3)&nbsp; cell&nbsp; lines. Binding&nbsp; energies&nbsp; and&nbsp; the&nbsp; geometry&nbsp; of&nbsp; binding&nbsp; of these&nbsp; compounds&nbsp; for&nbsp; ER&alpha;&nbsp; and&nbsp; ER&beta;&nbsp; ligand&nbsp; binding domains&nbsp; were&nbsp; predicted&nbsp; in&nbsp; silico&nbsp; by&nbsp; molecular&nbsp; docking&nbsp; methods.&nbsp; The&nbsp; second&nbsp; part&nbsp; of&nbsp; this&nbsp; study includes development&nbsp; of&nbsp; an&nbsp; assay&nbsp; for&nbsp; study&nbsp; of&nbsp; aromatase&nbsp; activity&nbsp; in&nbsp; the&nbsp; presence&nbsp; or&nbsp; absence&nbsp; of inhibitors&nbsp; by&nbsp; heterologous&nbsp; expression&nbsp; of&nbsp; human aromatase&nbsp; in&nbsp; Saccharomyces&nbsp; cerevisiae&nbsp; and/or Escherichia&nbsp; coli&nbsp; cells,&nbsp; as&nbsp; model-organisms. Furthermore, interactions between modified steroids, selected&nbsp; according&nbsp; to&nbsp; their&nbsp; structure,&nbsp; and&nbsp; aromatase were&nbsp; tested&nbsp; using&nbsp; sensitive&nbsp; spectroscopic&nbsp; methods based on ligand-induced changes&nbsp; in&nbsp; the&nbsp; spin state of Fe&nbsp; from&nbsp; the&nbsp; heme&nbsp; group&nbsp; or&nbsp; changes&nbsp; in&nbsp; the fluorescence&nbsp; of&nbsp; a&nbsp; tryptophan&nbsp; residue&nbsp; in&nbsp; the&nbsp; active site.&nbsp; The&nbsp; non-radioactive&nbsp; in&nbsp; vitro&nbsp; assays&nbsp; developed&nbsp; here, besides high efficiency, user and environmental safety,&nbsp; also&nbsp; have&nbsp; greater&nbsp; specificity&nbsp; and&nbsp; are&nbsp; more cost-effective&nbsp; for&nbsp; preliminary&nbsp; screening&nbsp; of&nbsp; steroid receptor&nbsp; ligands&nbsp; and&nbsp; aromatase&nbsp; inhibitors. Additionally,&nbsp; compounds&nbsp; identified&nbsp; to&nbsp; express significant biological activity can serve as a basis for the&nbsp; development&nbsp; of&nbsp; potential&nbsp; therapeutics&nbsp; in&nbsp; the treatment&nbsp; of&nbsp; hormone-dependent&nbsp; diseases&nbsp; and conditions, a global health issue today.</p>