Characterization and pathways of synhronization of peripheral biological clock and steroidogenesis in rat Leydig cells

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), gena uključenih u metabolizam holesterola (Lipe, Soat2, Scarb1) i steroidogenih gena, (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4)...Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4). Abolishment of melatonin, a main cue that spread information of light regime via cAMP signaling, stimulated expression of clock (Bmal1/BMAL1, Per1/2) and steroidogenic (Star/StAR, Hsd3b/HSD3B) elements..

Estimation of the Genetic Diversity in Tetraploid Alfalfa Populations Based on RAPD Markers for Breeding Purposes

Alfalfa is an autotetraploid, allogamous and heterozygous forage legume, whose varieties are synthetic populations. Due to the complex nature of the species, information about genetic diversity of germplasm used in any alfalfa breeding program is most beneficial. The genetic diversity of five alfalfa varieties, involved in progeny tests at Institute of Field and Vegetable Crops, was characterized based on RAPD markers. A total of 60 primers were screened, out of which 17 were selected for the analysis of genetic diversity. A total of 156 polymorphic bands were generated, with 10.6 bands per primer. Number and percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon's information index were used to estimate genetic variation. Variety Zuzana had the highest values for all tested parameters, exhibiting the highest level of variation, whereas variety RSI 20 exhibited the lowest. Analysis of molecular variance (AMOVA) showed that 88.39% of the total genetic variation was attributed to intra-varietal variance. The cluster analysis for individual samples and varieties revealed differences in their population structures: variety Zuzana showed a very high level of genetic variation, Banat and Ghareh were divided in subpopulations, while Pecy and RSI 20 were relatively uniform. Ways of exploiting the investigated germplasm in the breeding programs are suggested in this paper, depending on their population structure and diversity. The RAPD analysis shows potential to be applied in analysis of parental populations in semi-hybrid alfalfa breeding program in both, development of new homogenous germplasm, and identification of promising, complementary germplasm

Karakteristike mišljenja dece uzrasta od šest do devet godina

Мишљење је психичка функција која се надограђује на чулна сазнања и представља извор човековог сазнања света, омогућавајући му да схвати узрочност у свету, као и интеракцију и међусобну условљеност свих предмета и појава. Као основни инструмент мишљења, издваја се појам, док расуђивање представља логичко повезивање идеја. Главни циљ овог истраживања је утврђивање карактеристика мишљења и нивоа развоја мишљења деце узраста од шест до девет година. Узорак чини укупно 74 деце типичног развоја, оба пола, од чега 20 испитаника похађа прeдшколски програм, 27 похађа први, a 27 похађа други разред редовне основне школе. За процену мишљења код испитаника коришћен је Протокол за процену когнитивних способности. Резултати нашег истраживања показују да су испитаници имали више успеха на задацима који се решавају уз визуелну подршку (компарација слика, стрип, елиминација, довршавање низа) у односу на „чисто“ вербалне задатке (компарација речи, вербална аналогија, прича са поуком). Утврђена је статистички значајна разлика у средњим вредностима резултата на тесту између групе предшколаца и ученика другог разреда (р=0,00) и резултата ученика првог и другог разреда (р=0,022). Такође, ученици другог разреда су чешће бирали таксативни одговор у односу на ученике првог разреда (р=0,019) и таксативни (р=0,032) и тематски (р=0,041) у односу на предшколце. С друге стране, ученици првог разреда су чешће бирали функционални одговор од ученика другог разреда (р=0,017) али и перцептивни одговор од предшколаца (р=0,025). Предшколци су статистички значајније чешће бирали функционални одговор у односу на ученике другог разреда (р=0,002). Вредност аритметичке средине укупног резултата већа је у групи старијих ученика у односу на групу млађих (АС=53,43 према АС=49,09), и та разлика је статистичких значајна (р=0,031). Резултати су показали да је вредност аритметичке средине укупног резултата већа код девојчица у односу на дечаке (АС=53,31 према АС=49,34), и та разлика је статистичких значајна (р=0,049). Најбоље резултате на тесту постигли су ученици који потичу из породица са надпросечним материјалним примањима и утврђена је статистички значајна ралика у постигнућима ове групе и групе ученика који потичу из породица са просечним примањима. Није потрврђена статистички значајна разлика у постигнућима у односу на ниво образовања родитеља, нити у односу на школски успех. Закључак нашег истраживања је да су старији ученици, другог разреда, имали боље резултате од млађих ученика који се тек припремају или су започели процес школовања, што иде у прилог схватању да је мишљење у функцији сазревања и процеса образовања.Thinking is a mental function which is upgraded to sensory perception and represents a source of human knowledge of the world enabling humans to apprehend causality in the world as well as the interaction and mutual dependency of objects and phenomena. The main tool of thinking is a concept, whereas reasoning is a logical linking of ideas. The aim of this research is determining the characteristics of thinking and levels of thinking development within the children between the age of six and nine. The sample consists of 74 typical children, of both sexes, of which 20 participants attend preschool program, 27 are in year one and 27 are in year two of elementary school. The Cognitive Abilities Evaluation Protocol was used for evaluation of participants’ thinking. The results of the research show that the participants were more successful while solving the tasks using visual support (image comparison, comics, elimination, series completion) compared to “purely” verbal tasks (comparison of words, verbal analogy, fables with morals). Statistically significant difference in average values of the test results of preschoolers and the second grade pupils (p=0,00) and the first and second grade pupils (p=0,022) is confirmed. Moreover, the second grade pupils chose a taxative answer more often than the first grade pupils (p=0,019) and taxative (p=0,032) and thematic (p=0,041) more often than preschoolers. On the other hand, the first grade pupils chose a functional answer more often than the second grade pupils (p=0,017), and a perceptive answer more often than preschoolers (p=0,025). Preschoolers chose a functional answer more often than the second grade pupils (p=0,002), which is statistically significant. The arithmetic mean of the total result is higher in the group of older pupils than in the group of younger pupils (M=53,43 to M=49,09) and the difference is statistically significant (p=0,031). The results demonstrate that the arithmetic mean of the total result is higher with the girls than the boys (M=53,31 to M=49,34), which is statistically significant difference (p=0,049). The pupils coming from the families with above average income achieve the best results on the test and statistically significant difference in the achievement between this group and the group of pupils coming from the families with average income is determined. Statistically significant difference is not confirmed within the achievement regarding the level of parental education as well as school achievement. The conclusion of the research is that older pupils, the second grade pupils, have better results than younger pupils, who are just preparing or have started schooling, which contributes to the fact that thinking stimulates development and educational process

Characterization and pathways of synhronization of peripheral biological clock and steroidogenesis in rat Leydig cells

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), gena uključenih u metabolizam holesterola (Lipe, Soat2, Scarb1) i steroidogenih gena, (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4)...Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4). Abolishment of melatonin, a main cue that spread information of light regime via cAMP signaling, stimulated expression of clock (Bmal1/BMAL1, Per1/2) and steroidogenic (Star/StAR, Hsd3b/HSD3B) elements..