71 |
The role of transforming growth factor-beta 1 in steroidogenesis, cell proliferation, and apoptosis in cultured bovine granulosa cellsZheng, Xiaofeng January 2008 (has links)
Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal
|
72 |
Elucidation of 17β-Estradiol (E2) Role in the Regulation of Corpus Luteum Function in Mammals : Analysis of IGFBP5 Expression during Ea-mediated ActionsTripathy, Sudeshna January 2014 (has links) (PDF)
Corpus luteum is a transient endocrine structure formed from the ruptured ovarian follicle. Its main function is to secrete P4, a pro-gestational hormone, essential for establishment and maintenance of pregnancy in mammals. The modulators of CL structure and function are classified as trophic and lytic factors. The luteotrophic factors include pituitary hormones, growth factors, intra luteal factors and cytokines, while luteolytic factors include PGF2α and oxytocin. The interplay between luteotrophic and luteolytic factors regulates luteal steroidogenesis. The precise timing of expression of various enzymes/proteins required for synthesis and metabolism of P4 constitutes an important process in the overall regulation of CL function. The three hormones LH/CG, E2 and PRL are regarded as luteotrophic factors crucial for control of CL function in mammals. Depending on species, either individually or all three hormones in the form of luteotrophic complex have been shown to participate in the regulation of CL function. In addition to the well-established endocrine role of E2, its secretion is the hallmark of the ovulating follicle, has an important role in the intraovarian growth, differentiation and survival of cells.
Chapter I provides a comprehensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in bovines and rodents. In Chapter II, studies have been carried out to examine 20α-HSD expression and its activity in the CL of buffalo cow. During induced and spontaneous luteolysis, rapid fall in circulating P4 is one of the early signs of initiation of luteolytic process in several species. In rodents, it is well recognized that during luteolysis, P4 is catabolized into inactive metabolite, 20α-OHP by the reaction of 20α-HSD enzyme during luteolysis. Experiments were carried out to determine 20α-HSD expression and activity throughout the luteal phase and during induced luteolysis in the buffalo cow. Circulating P4 concentration declined rapidly in response to PGF2α treatment, but HPLC analysis of serum samples did not reveal changes in circulating 20α-OHP levels in buffalo cows. In contrast, pseudo pregnant rats receiving PGF2α treatment showed higher 20α-OHP levels at 24 h post treatment. qPCR expression of 20α-HSD in CL during different stages of luteal phase and PGF2α-treated buffalo cows was carried out and higher expression of 20α-HSD was observed at 3 and 18 h post treatment, but its activity was not altered post PGF2α treatment at other time points examined. The expression of the transcription factor Nurr77 which is involved in increased expression of 20α-HSD increased several fold 3 h post PGF2α treatment similar to the observation in PGF2α-treated pseudo pregnant rats. The results suggested that the synthesis rather than catabolism of P4 appears to be primarily
affected by PGF2α treatment in buffalo cows in contrast to increased metabolism of P4 as seen in rodents.
In bovines, to date no luteotropic actions for E2 has been demonstrated and whether E2 has direct effect on CL function has also not been reported. Expression of CYP19A1 gene that encodes aromatase enzyme although gets down regulated post ovulation but its expression recovers in the CL and also E2 biosynthesis has been reported in the bovine CL. Recently it was observed that CYP19A1 expression was consistently down regulated following administration of luteolytic dose of PGF2α. Experiments were conducted to examine the expression of ERα and ERβ in the CL throughout the buffalo estrous cycle as well as examined the luteal E2 levels post PGF2α treatment. The results indicated that ER expression was detectable during different stages of CL and that circulating and luteal E2 levels declined post PGF2α treatment. It was hypothesized that decrease in luteal E2 levels leads to down regulation of ER signaling and changes in expression of E2 responsive genes in the CL. To test the hypothesis, 89 genes which were regarded as E2 responsive genes were selected and the previously published global gene expression data of the buffalo CL was mined for E2 responsive genes. It was observed that 57 of 89 genes regarded as E2 responsive genes were found to be differentially expressed. Since non pregnant buffalo CL is not regarded as major site of E2 production, to validate the authenticity of differentially E2 expressed genes post PGF2α, CL of another species, the macaque, which is known to secrete abundant E2 was included for the analysis. Incidentally, the global gene expression data for the PGF2 α treated macaques (in which CYP19A1 gene expression also gets down regulated) has previously been reported from the laboratory. Here again, it was observed that nearly 79 of 89 genes were identified to be differentially expressed. To further determine the consequences of decreased ER signaling, molecules associated with survival and apoptosis were examined. The results indicated decreased expression (both mRNA and protein levels) of Akt, Bax and Bcl-2 genes. The results suggested an important role for E2 on CL function in the buffalo cow.
In Chapter III, several experiments were conducted in another model system, pregnant rat, in which aromatase expression and therefore E2 production is high in the CL. Experiments were conducted to examine the effects of E2 inhibition and E2 replacement on the expression of genes. For this purpose, pregnant rats were treated with a specific aromatase inhibitor on day 12-15 of pregnancy. Together with AI, exogenous E2 was administered to another group of pregnant rats. The CL collected from different groups of rats on day 16 of pregnancy was subjected to microarray analysis. The analysis post
validation of microarray data has shown that clusters of genes could be segregated into various pathways involving luteal steroidogenesis, immune system, various growth factors and apoptotic processes, all directed towards the regulation of CL function. The involvement of E2 in luteal cell proliferation and lipid deposition well corroborated with protein levels for cyclin D1 and ki67 and the results of oil red O staining, respectively. There have been reports implicating PI3K/Akt signaling in cyclin D1 accumulation, but mechanism of action does not appear to involve transcriptional activation of cyclin D1. The results of the present study indicate a decrease in cyclin D1 protein levels due to inhibition of PI3K/Akt signaling by AI treatment which is prevented upon administration of E2 during AI treatment. The findings provide a comprehensive overview for the mechanisms associated with the cell survival, progression, etc. The bioinformatics approach provided complete landscape of functional changes affected by the upstream regulators of genes associated with survival and apoptosis. Also, the findings further strengthen the hypothesis of involvement of E2 in the regulation of CL function by way of activation of Akt, the primary mediator of PI3K signaling in the regulation of cellular component that affect cell survival.
In the present study, IGFBP5 which was up regulated during luteal inhibition of E2 with AI treatment was selected for further studies. Although IGFBP5 is known to be associated with follicular atresia in the rat ovary, there is limited data for the involvement of IGFBP5 in either a growth stimulatory or inhibitory action on ovarian cells. Based on present findings, a causal link between reduced ERα transcriptional activities resulting in inhibition of Akt/PKB in the presence of IGFBP5 expression could be proposed. Further, the cellular hypertrophy mediated by E2 has been speculated due to increased proliferation of vascular endothelial cells, blood supply and thus nutrients. E2, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat CL of pregnancy. In CL, the prominent IGFBP5 mRNA expression in different types of luteal cells has not been reported. The mRNA expression for IGFBP5 across the two types of luteal cells showed higher expression in SLC. Hence, in the present study, it has been speculated that prevention of conversion of SLC to LLC due to lack of E2 biosynthesis in presence of AI might be acting as a source for the increased IGFBP5 levels during mid pregnancy in rat CL and brings about changes associated with lack of E2.
Various receptor studies on rat CL have demonstrated the lack of progesterone receptor (PR) mRNA expression in the rat CL negating its involvement as an autocrine/paracrine regulator of CL function through an intracellular receptor, but the
involvement of non-PR involvement in mediating such mechanism further strengthens the role of ERs. The luteotrophic complex formation in pregnant rat principally by PRL and E2 has been discussed at length in Chapter III. PRL appears to maintain luteal ER content in the CL during rat pregnancy which further determines the luteotrophic and luteolytic actions of E2. Further, study on expression of E2 responsive genes would help in identifying E2 regulating molecules to get a clear picture on the role of E2 in understanding regulation of the CL function.
The interaction of E2 with growth factor signaling including the IGF pathway has been well established in different species and this interaction is tightly linked to ERα expression, an observation interpreted as physiological coupling of growth factor and stress signaling pathways. Attempts were made towards understanding cross talk between the E2 signaling and the IGF1 signaling in few experiments carried out in Chapter IV. Based on the results, it can be proposed that a causal link exists between reduced ERα transcriptional activity and inhibition of Akt/PKB in the presence of IGFBP5. The present study has shown the activity of IGF on ERα activity mediated partly via PI3K/Akt pathway. Hence, the finding further speculates that inhibitory effect of IGFBP5 on E2 induced ERα function was due to sequestration of IGF1, possibly present in serum or produced within the cells. Another striking observation was the down regulation of glucocorticoid receptor (GR) gene, NR3C1, in the data of earlier studies [Priyanka, 2009, GEO accession number GSE8371 and Kunal, 2014, GEO accession number GSE27961] and the present study has been compared and discussed in this thesis. Glucocorticoids provide key signals for differentiation of fetal and placental tissues. Therefore, regulation of glucocorticoid access to the placenta and fetus is recognized as an important determinant of prognosis outcome and subsequent development of the postnatal phenotype. Differential regulation of these genes in CL post E2 deprivation and replacement further emphasize the regulation of CL via various biological, cellular and molecular functions.
Interestingly, besides transcriptional regulation of IGF axis components, E2 activated ERα also rapidly influence the activity of IGF axis related to signaling proteins in a non-genomic manner, especially by the PI3K/Akt pathway. PI3K/Akt pathway analysis has been carried out in E2 inhibition and replacement experiments. To further confirm the observations of E2 and growth factor interaction, experiments have been set up with exogenous GH for increasing circulating levels of IGF in the system. The findings suggest that the non-genomic signaling pathway activated by the phosphorylation of ERα induced
by E2 gets inhibited in the presence of AI result in increased expression of IGFBP5. The reduction in circulating IGF1 in pregnant rats may be associated with the effect on IGFBP, important for determining biological action of IGF1. The changes observed in the present study emphasize the exclusive effects of the IGFBP5 on the CL function brought about perturbations in luteal E2 content.
The experiments described in the present thesis aim at understanding the mechanism responsible for decreased serum and luteal P4 post PGF2α treatment in buffalo cows, i.e. whether PGF2α acts on biosynthetic or catabolic process of P4. In the present study, experiments were designed to elucidate the role of E2 in regulation of CL function, since down regulation of CYP19A1 gene mRNA was one of the early events observed in buffalo cows post PGF2α treatment. This line of research work was extended to rodents, a species that secretes high levels of E2 during pregnancy. Genome wide transcriptional changes data revealed differential expression of several E2 responsive genes following E2 inhibition and replacement treatments. The results revealed importance of ER-mediated PI3K/Akt signaling essential for regulation of many transcriptional regulatory molecules in the CL and an interesting involvement of IGFBP5 as a link between E2 and IGF signaling. These findings further provide an insight into the role of IGFBP5 in E2-mediated actions in rat CL during pregnancy. In conclusion, the present findings suggest inhibitory effect of IGFBP5 on E2-induced ERα function and hence, its selection as a target molecule for regulation of CL function and for many beneficial processes involved in anti-carcinogenic properties can be thought of.
|
73 |
Uloga insulinskih i IGF1 receptora u regulaciji steroidogeneze i mitohondrijallne biogenze u Leydigovim ćelijama / The role of insulin and IGF1 receptors in regulation of teroidogenesis and mitochondrial biogenesis in Leydig cellsRadović Sava 31 May 2019 (has links)
<p>Leydig-ove ćelije testisa su primarno mesto sinteze muških polnih hormona. Ovi hormoni su neophodani za reproduktivno, ali i za opšte zdravlje budući da su<br />ozbiljni zdravstveni problemi često povezani sa njihovom smanjenom produkcijom. Insulin i insulinu sličan faktor rasta 1, IGF1 <em>(engl.</em> insulin like growth factor 1), i<br />signalizacija koju pokreću preko svojih receptora (INSR i IGF1R), su jedan od ključnih faktora koji regulišu specifični razvoj tkiva, pa i samih gonada. Ipak, uloga i<br />mehanizmi delovanja ovih receptora u steroidogenim tkivima nisu u potpunosti poznati. Stoga je istraživanje uokviru ove doktorske disertacije koncipirano sa ciljem da se, na modelu prepubertalnih (P21) i adultnih (P80) mužjaka miševa sa kondicionalnom delecijom<em> Insr </em>i <em>Igf1</em>r gena u steroidogenim ćelijama (Insr/Igf1r-DKO), definiše uloga INSR i IGF1R u regulisanju diferencijacije i steroidogene funkcije Leydig-ovih ćelija. Pored toga, mužjaci i ženke P21 miševa sa istom delecijom su korišćeni za praćenje ekspresije glavnih markera mitohondrijalne biogeneze i fuzije/arhitekture u Leydigovim ćelijama, ovarijumima i nadbubrežnim žlezdama. Rezultati su potvrdili da delecija Insr i Igf1r u<br />steroidogenim tkivima utiče na diferencijaciju i funkcionalne karakteristike Leydig-ovih ćelija P21 i P80 miševa, upućujući na pojavu tzv. „feminizacije“. Broj<br />Leydig-ovih ćelija izolovanih iz P21 i P80 Insr/Igf1rDKO miševa bio je smanjen, a morfologija i ultrastruktura ovih ćelija izmenjene kod P21 Insr/Igf1rDKO miševa. Steroidogeni kapacitet i aktivnost, kao i ekspresija glavnih elemenata steroidogene mašinerije <em>(Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 i 6, Hsd17b3,</em><br /><em>Sf</em>1) bili su smanjeni u Leydig-ovim ćelijama P21 i P80 <em>Insr/Igf1</em>r-DKO miševa, dok je ekspresija transkripcionih represora steroidogeneze (Arr19 i Dax1) bila povećana specifično u istim ćelijama, ali ne i u ostatku testisa.<br />Transkripcioni profil markera muškog pola (<em>Sry, Sox9, Amh</em>) bio je izmenjen u Leydig-ovim ćelijama P21 i P80 <em>Insr/Igf1r</em>-DKO miševa. Transkripcija markera ženskog pola (<em>Rspo1, Wnt4</em>) u testisima, kao i ekspresija Cyp19a1 i produkcija estradiola (E2) u Leydig-ovim ćelijama, P21 i P80 <em> Insr/Igf1r</em>-DKO miševa bile su povećane. Transkripcija markera mitohondrijalne biogenze (<em>Ppargc1a, Tfam</em>, <em>Mtnd1</em>) bila je smanjena u Leydigovim ćelijama P21 <em>Insr/Igf1r</em>-DKO miševa, dok supromene ekspresije izostale u ovarijumima ženki istog genotipa. Isti markeri su bili povećani u nabdubrežnim žlezdama oba pola. Markeri mitohondrijalne fuzije/arhitekture (<em>Mfn1 i Mfn2)</em> bili su povećani u Leydig-ovim ćelijama P21 <em>Insr/Igf1r</em>-DKO miševa, što je praćeno i narušenom mitohondrijalnom fazom steroidogeneze (produkcija progesterona), kao i brojem i morfologijom ovim organela. Ekspresija istih markera u ovarijumima bila je nepromenjena. Sumirano, rezultati ovog istraživanja su pokazali da su INSR i IGF1R važni za diferencijaciju i steroidogenu funkciju Leydig-ovih ćelija P21 i P80 miševa. Takođe, ovi receptori su važni regulatori markera mitohondrijalne biogeneze i fuzije/arhiteture u steroidogenim ćelijama muških gonada P21 miševa, ali ne i u steroidogenim ćelijama ovarijuma. </p> / <p>Leydig cells of testes are the primary site of the male sex hormones synthesis. These hormones are indispensable for both reproductive and general health since serious health problems are often associated with their reduced production. Insulin and insulin-like growth factor 1, IGF1 (insulin like growth factor 1), and signaling triggered through their receptors (INSR and IGF1R), are one of the key factors that regulate specific development of tissue including gonads. However, the role and mechanisms of these receptors action in steroidogenic tissues are not known enough. This study was designed to observe the role of INSR and IGF1R in regulating the differentiation and steroidogenic function of Leydig cells by using the model of prepubertal (P21) and adult (P80) male mice with the conditional deletion of the Insr and Igf1r genes in steroidogenic cells (<em>Insr/Igf1r-</em>DKO). In addition, male and female P21 mice with the samedeletion were used to monitor the expression of the main markers of mitochondrial biogenesis and fusion/architecture in Leydig cells, ovaries and adrenal glands. The results confirmed that deletion of <em> Insr</em> and<em> Igf1r </em> in steroidogenic tissues influences differentiation and functional characteristics of Leydig cells isolated from P21 and P80 mice, suggesting an appearance of "feminization". The number of Leydig cells isolated from both P21 and P80 <em>Insr/Igf1</em>r-DKO mice was reduced. Morphology and ultrastructure of Leydig cells were disturbed in P21 <em>Insr/Igf1r-</em>DKO mice. Steroidogenic capacity and activity, as well as expression of the main elements of steroidogenic machinery (<em>Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 and 6, Hsd17b3, Sf1) </em>were decreased in Leydig cells from P21 and P80 I<em>nsr/Igf1</em>r-DKO mice, while the expression of transcriptional repressors of steroidogenesis (<em>Arr19</em> and <em>Dax1) </em>was increased in the same cells, but not in the rest of the testes. Transcription profile of the male sex markers (<em>Sry, Sox9</em>, <em>Amh</em>) was altered in Leydig cells from P21 and P80 <em>Insr/Igf1</em>r-DKO mice. Transcription of the female sex markers (<em>Rspo1, Wnt4</em>) in the testes, as well as <em>Cyp19a1 </em>expression and estradiol (E2) production in Leydig cells, from P21 and P80 I<em>nsr/Igf1</em>rDKO mice were increased. Transcription of mitochondrial biogenesis markers (<em>Ppargc1a, Tfam, Mtnd1</em>) was declined in Leydig cells from P21<em> Insr/Igf1r-</em>DKO mice, while changes were absent in the ovaries of the same genotype. Transcription of the same markers was increased in the adrenal glands of both sexes. The mitochondrial fusion/architecture markers (<em>Mfn1</em> and <em>Mfn2</em>) were increased in Leydig cells from<em> Insr/Igf1r</em>-DKO mice and followed by disturbedmitochondrial phase of steroidogenesis (progesterone production), as well as decreased number and disturbed morphology of mitochondria. Expression of the same markers in the ovaries was unchanged. In summary, results of this study showed that INSR and IGF1R are important in differentiation and steroidogenic function of Leydig cells from P21 and P80 mice. Also, these receptors are important regulators of mitochondrial biogenesis and fusion/architecture markers in steroidogenic cells of P21 male mice, but not in steroidogenic cells of ovaries.</p>
|
74 |
Elucidation of the biological roles of Wnt5a signaling in follicle developmentAbedini Najafabadi, Atefeh 08 1900 (has links)
No description available.
|
75 |
Le rôle de Janus Kinase 3 (JAK3) dans le développement folliculaire.Zareifard, Amir 12 1900 (has links)
Janus kinase 3 (JAK3) est un membre de la famille JAK de protéines tyrosine kinase impliquées dans la transduction du signal intracellulaire médiée par les récepteurs de cytokines via la voie de signalisation JAK/STAT. JAK3 s'est avéré exprimé de manière différentielle dans les cellules de la granulosa (GC) des follicules pré-ovulatoires bovins et régulé à la baisse par l'hormone lutéinisante. Ces observations suggèrent que la régulation de JAK3 pourrait moduler la prolifération des GC, l'activité stéroïdienne et l'activation/l'inhibition des cibles en aval. Pour étudier les mécanismes des actions de JAK3 dans GC, nous avons utilisé JANEX-1, un inhibiteur pharmacologique de JAK3, et des traitements FSH et analysé des marqueurs de prolifération, des enzymes stéroïdogènes et la phosphorylation de protéines cibles, y compris STAT3 et les partenaires JAK3 précédemment identifiés CDKN1B/p27Kip1 et MAPK8IP3/JIP3. Les GC en culture ont été traités avec ou sans FSH en présence ou non de JANEX-1. L'ARN total et les protéines ont été extraits et analysés par RT-qPCR, western blot et UHPLC-MS/MS. L'expression de l'enzyme stéroïdogène CYP11A1, mais pas du CYP19A1, était significativement régulée à la hausse dans les GC traités avec la FSH et les deux étaient significativement diminuées lorsque JAK3 était inhibé par rapport au contrôle. Les marqueurs de prolifération CCND2 et PCNA ont été significativement réduits dans les GC traités au JANEX-1 et régulés positivement par la FSH. Les analyses Western blots ont montré que le traitement JANEX-1 réduisait de manière significative les quantités de pSTAT3 tandis que la surexpression de JAK3 augmentait pSTAT3. De même, le traitement à la FSH a augmenté pSTAT3 même dans les GC traités au JANEX-1. Les analyses UHPLC-MS/MS ont montré une phosphorylation et des modifications supplémentaires de résidus d'acides aminés spécifiques dans JAK3 ainsi que ses partenaires de liaison CDKN1B et MAPK8IP3 révélant une activation ou une inhibition possible de JAK3 après des traitements FSH ou JANEX-1, respectivement. L'abondance de la protéine totale JAK3 a augmenté après le traitement par FSH et a diminué de manière significative, avec MAPK8IP3, dans le GC traité par JANEX-1, tandis que l'abondance totale de CDKN1B a été modifiée après FSH et augmentée après JANEX-1. Nous montrons que JAK3 influence l'activité GC par la phosphorylation de protéines cibles en réponse à des stimulations telles que la FSH, ce qui conduit à l'activation de JAK/STAT et module probablement d'autres voies de signalisation impliquant CDKN1B et MAPK8IP3. / Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine preovulatory follicles and downregulated by the luteinizing hormone. These observations suggested JAK3 regulation could modulate GC proliferation, steroidogenic activity and activation/inhibition of downstream targets. To investigate the mechanisms of JAK3 actions in GC, we used JANEX-1, a pharmacological JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3 and previously identified JAK3 partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GCs were treated with or without FSH in the presence or not of JANEX-1. Total RNA and proteins were extracted and analyzed by RT-qPCR, western blotting and UHPLC-MS/MS. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was significantly upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited as compared to control. Proliferation markers CCND2 and PCNA were significantly reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment significantly reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses showed phosphorylation and additional modifications of specific amino acid residues within JAK3 as well as its binding partners CDKN1B and MAPK8IP3 revealing possible activation or inhibition of JAK3 following FSH or JANEX-1 treatments, respectively. Abundance of JAK3 total protein was increased post-FSH treatment and significantly decreased, along with MAPK8IP3, in JANEX-1-treated GC while CDKN1B total abundance was altered post-FSH and increased post-JANEX-1. We show that JAK3 influences GC activity through phosphorylation of target proteins in response to stimulations such as FSH, which leads to the activation of JAK/STAT and likely modulating other signaling pathways involving CDKN1B and MAPK8IP3.
|
76 |
Razvoj bioloških testova za identifikaciju liganada steroidnih receptora i ispitivanje aktivnosti steroidogenog enzima aromataze / Development of biological assays for identification of steroid receptor ligands and determination of activity of steroidogenic enyzme aromataseBekić Sofija 07 August 2020 (has links)
<p>U ovoj doktorskoj disertaciji razvijen je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada ERα, ERβ ili AR i kvantifikaciju njihovog afiniteta vezivanja sa mogućnošću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana je primena optimizovanog biosenzora za procenu estrogenog potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma delovanja odabranih modifikovanih steroida ispitana je njihova antiproliferativna aktivnost prema ćelijskim linijama estrogen receptor pozitivnog kancera dojke (MCF-7) i kancera prostate (PC-3), dok su in silico metodom molekularnog dokinga predviđene energije i geometrije vezivanja ovih jedinjenja za ligand-vezujuće domene ERα i ERβ. Drugi deo ovog rada obuhvata razvoj testa za ispitivanje aktivnosti humanog enzima aromataze, heterologno eksprimiranog u ćelijama kvasca Saccharomyces cerevisiae i/ili bakterija Escherichia coli, u prisustvu ili odsustvu inhibitora. Interakcije modifikovanih steroida, odabranih na osnovu strukture, sa aromatazom ispitane su osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka triptofana iz aktivnog centra usled konformacione promene proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim visoke efikasnosti i bezbednosti po korisnika i okolinu, pokazali specifičnost i ekonomičnost u preliminarnom skriningu liganada steroidnih receptora i inhibitora aromataze. Jedinjenja kod kojih je detektovana značajna biološka 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 this doctoral dissertation, a fluorescent assay in yeast was developed for identification of potential natural or synthetic ligands of ERα, ERβ or AR and<br />quantification of their binding affinity, as well asevaluation of the estrogenic potential of synthetic steroids and selected plant extracts rich in phytoestrogen content. The assay could be used to screen libraries of modified steroids and xenoestrogens. In order to better understand the biomedical potential of selected modified steroids, results were compared to antiproliferative activity against estrogen receptor positive breast cancer (MCF-7) and prostate cancer (PC-3) cell lines. Binding energies and the geometry of binding of these compounds for ERα and ERβ ligand binding domains were predicted in silico by molecular docking methods. The second part of this study includes development of an assay for study of aromatase activity in the presence or absence of inhibitors by heterologous expression of human aromatase in Saccharomyces cerevisiae and/or Escherichia coli cells, as model-organisms. Furthermore, interactions between modified steroids, selected according to their structure, and aromatase were tested using sensitive spectroscopic methods based on ligand-induced changes in the spin state of Fe from the heme group or changes in the fluorescence of a tryptophan residue in the active site. The non-radioactive in vitro assays developed here, besides high efficiency, user and environmental safety, also have greater specificity and are more cost-effective for preliminary screening of steroid receptor ligands and aromatase inhibitors. Additionally, compounds identified to express significant biological activity can serve as a basis for the development of potential therapeutics in the treatment of hormone-dependent diseases and conditions, a global health issue today.</p>
|
Page generated in 0.5004 seconds