Neuroendocrine regulation of stress responses

Stres danas predstavlja jednu od glavnih prijetnji homeostazi, a samim time i zdravlju čovjeka. Kada je pod stresom, tijelo pokreće niz obrambenih mehanizama kako bi održalo potrebnu ravnotežu te se oduprlo štetnim utjecajima koji prijete iz okoline. Glavnu ulogu u tome imaju autonomni živčani te endokrini sustav, čija međusobna povezanost pruža organizmu potrebnu zaštitu u stresnim situacijama. Pomoću osi hipofiza – hipotalamus – nadbubrežna žlijezda regulira se lučenje CRH, ACTH te naposljetku glukokortikoida koji sudjeluju u odgovoru organizma na stres. Odgovor, odnosno način na koji će se tijelo nositi sa stresom uvelike ovisi o njegovoj otpornosti na stres. Ona predstavlja sposobnost organizma da se prilagodi traumi te nosi sa kroničnim oblicima nedaća. Osim što je genetički uvjetovana, te ovisi o koncentraciji ključnih neurotransmitera, važnu komponentu otpornosti čine i psihosocijalni faktori te emocionalna mogućnost nošenja sa stresom. Međutim, bitan aspekt u radu mehanizama koji tijelo brane od stresa čini i starost samog organizma. Iako je vrijeme prenatalnog razdoblja prerano za vidjeti trajne posljedice stresa, kronično izlaganje visokoj koncentraciji glukokortikoida u ranom djetinjstvu pokazuje štetne posljedice za organizam u adolescenciji, odrasloj dobi i starosti. Prema tome, jasno je vidljivo da izlaganje kroničnom stresu u bilo kojem razdoblju života kasnije dovodi do nepoželjnih efekata.Nowadays stress represents one of the greatest threats to the homeostasis and by that to human health in general. When under stress, an organism initiates defense mechanisms to maintain the necessary balance and resist the harmful effects from the environment. The main role in that process hold the autonomic nerve system and the endocrine system, coherence of which provides the required protection to the organisms. Stress response is regulated by the CRH, ACTH and glucocorticoids which are all secreted by the hypothalamus-pituitary-adrenal axis. Response or the way that the body handles the stress depends largely on its resilience. It represents the capability of an organism to adjust to trauma and cope with chronic difficulties. Except for being genetically conditioned and depending on the concentration of key neurotransmitters, its important component is also represented by psychosocial factors and emotional capability to cope with stress. However, an important aspect in mechanisms which protect the body from stress is also the oldness of an organism. Although it is too early to see the visible consequences of stress during the prenatal period, the chronic exposure to high concentrations of glucocorticoids in early childhood shows harmful consequences to organism in adolescence, adulthood or old age. It is therefore evident that chronic stress exposure in any life period leads to unwanted effects later in life

Neuroendocrine regulation of stress responses

Stres danas predstavlja jednu od glavnih prijetnji homeostazi, a samim time i zdravlju čovjeka. Kada je pod stresom, tijelo pokreće niz obrambenih mehanizama kako bi održalo potrebnu ravnotežu te se oduprlo štetnim utjecajima koji prijete iz okoline. Glavnu ulogu u tome imaju autonomni živčani te endokrini sustav, čija međusobna povezanost pruža organizmu potrebnu zaštitu u stresnim situacijama. Pomoću osi hipofiza – hipotalamus – nadbubrežna žlijezda regulira se lučenje CRH, ACTH te naposljetku glukokortikoida koji sudjeluju u odgovoru organizma na stres. Odgovor, odnosno način na koji će se tijelo nositi sa stresom uvelike ovisi o njegovoj otpornosti na stres. Ona predstavlja sposobnost organizma da se prilagodi traumi te nosi sa kroničnim oblicima nedaća. Osim što je genetički uvjetovana, te ovisi o koncentraciji ključnih neurotransmitera, važnu komponentu otpornosti čine i psihosocijalni faktori te emocionalna mogućnost nošenja sa stresom. Međutim, bitan aspekt u radu mehanizama koji tijelo brane od stresa čini i starost samog organizma. Iako je vrijeme prenatalnog razdoblja prerano za vidjeti trajne posljedice stresa, kronično izlaganje visokoj koncentraciji glukokortikoida u ranom djetinjstvu pokazuje štetne posljedice za organizam u adolescenciji, odrasloj dobi i starosti. Prema tome, jasno je vidljivo da izlaganje kroničnom stresu u bilo kojem razdoblju života kasnije dovodi do nepoželjnih efekata.Nowadays stress represents one of the greatest threats to the homeostasis and by that to human health in general. When under stress, an organism initiates defense mechanisms to maintain the necessary balance and resist the harmful effects from the environment. The main role in that process hold the autonomic nerve system and the endocrine system, coherence of which provides the required protection to the organisms. Stress response is regulated by the CRH, ACTH and glucocorticoids which are all secreted by the hypothalamus-pituitary-adrenal axis. Response or the way that the body handles the stress depends largely on its resilience. It represents the capability of an organism to adjust to trauma and cope with chronic difficulties. Except for being genetically conditioned and depending on the concentration of key neurotransmitters, its important component is also represented by psychosocial factors and emotional capability to cope with stress. However, an important aspect in mechanisms which protect the body from stress is also the oldness of an organism. Although it is too early to see the visible consequences of stress during the prenatal period, the chronic exposure to high concentrations of glucocorticoids in early childhood shows harmful consequences to organism in adolescence, adulthood or old age. It is therefore evident that chronic stress exposure in any life period leads to unwanted effects later in life

Neuroendocrine regulation of stress responses

Stres danas predstavlja jednu od glavnih prijetnji homeostazi, a samim time i zdravlju čovjeka. Kada je pod stresom, tijelo pokreće niz obrambenih mehanizama kako bi održalo potrebnu ravnotežu te se oduprlo štetnim utjecajima koji prijete iz okoline. Glavnu ulogu u tome imaju autonomni živčani te endokrini sustav, čija međusobna povezanost pruža organizmu potrebnu zaštitu u stresnim situacijama. Pomoću osi hipofiza – hipotalamus – nadbubrežna žlijezda regulira se lučenje CRH, ACTH te naposljetku glukokortikoida koji sudjeluju u odgovoru organizma na stres. Odgovor, odnosno način na koji će se tijelo nositi sa stresom uvelike ovisi o njegovoj otpornosti na stres. Ona predstavlja sposobnost organizma da se prilagodi traumi te nosi sa kroničnim oblicima nedaća. Osim što je genetički uvjetovana, te ovisi o koncentraciji ključnih neurotransmitera, važnu komponentu otpornosti čine i psihosocijalni faktori te emocionalna mogućnost nošenja sa stresom. Međutim, bitan aspekt u radu mehanizama koji tijelo brane od stresa čini i starost samog organizma. Iako je vrijeme prenatalnog razdoblja prerano za vidjeti trajne posljedice stresa, kronično izlaganje visokoj koncentraciji glukokortikoida u ranom djetinjstvu pokazuje štetne posljedice za organizam u adolescenciji, odrasloj dobi i starosti. Prema tome, jasno je vidljivo da izlaganje kroničnom stresu u bilo kojem razdoblju života kasnije dovodi do nepoželjnih efekata.Nowadays stress represents one of the greatest threats to the homeostasis and by that to human health in general. When under stress, an organism initiates defense mechanisms to maintain the necessary balance and resist the harmful effects from the environment. The main role in that process hold the autonomic nerve system and the endocrine system, coherence of which provides the required protection to the organisms. Stress response is regulated by the CRH, ACTH and glucocorticoids which are all secreted by the hypothalamus-pituitary-adrenal axis. Response or the way that the body handles the stress depends largely on its resilience. It represents the capability of an organism to adjust to trauma and cope with chronic difficulties. Except for being genetically conditioned and depending on the concentration of key neurotransmitters, its important component is also represented by psychosocial factors and emotional capability to cope with stress. However, an important aspect in mechanisms which protect the body from stress is also the oldness of an organism. Although it is too early to see the visible consequences of stress during the prenatal period, the chronic exposure to high concentrations of glucocorticoids in early childhood shows harmful consequences to organism in adolescence, adulthood or old age. It is therefore evident that chronic stress exposure in any life period leads to unwanted effects later in life

Nuclear chromosome locations dictate segregation error frequencies

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis [1, 2, 3, 4, 5]. Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer [6, 7] —but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing [8] after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes [9] and aneuploidies in early development [10] occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development. 1. van Jaarsveld, R. H. & Kops, G. J. P. L. Difference makers: chromosomal instability versus aneuploidy in cancer. Trends Cancer 2, 561–571 (2016). 2. Compton, D. A. Mechanisms of aneuploidy. Curr. Opin. Cell Biol. 23, 109–113 (2011). 3. Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015). 4. Ly, P. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat. Genet. 51, 705–715 (2019). 5. Shoshani, O. et al. Chromothripsis drives the evolution of gene amplification in cancer. Nature 591, 137–141 (2021). 6. Davoli, T. et al. Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962 (2013). 7. Knouse, K. A., Davoli, T., Elledge, S. J. & Amon, A. Aneuploidy in cancer: seq-ing answers to old questions. Annu. Rev. Cancer Biol. 1, 335–354 (2017). 8. Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019). 9. Cortés-Ciriano, I. et al. Comprehensive analysis of chromothripsis in 2, 658 human cancers using whole-genome sequencing. Nat. Genet. 52, 331–341 (2020). 10. McCoy, R. C. et al. Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development. PLoS Genet. 348, 235–238 (2015)

Establishment and bioimaging of mouse small intestine organoids culture

Organoidi su novo područje u molekularnoj biologiji koje ima veliki potencijal u temeljnim i primijenjenim istraživanjima. Minijaturni organi uzgajani in vitro značajno nalikuju svojim nativnim dvojnicima u svim fiziološkim, 3D strukturalnim i anatomskim karakteristikama. Već je razvijeno više tipova organoida, uključujući ljudske i mišje organoide tankog crijeva. U ovom radu, ja sam uspješno uzgojila i održala u kulturi organoide mišjeg tankog crijeva. Nakon optimizacije, uzgojeni su organoidi mogli neodređeno dugo rasti i propagirati se u kulturi. Preciznija karakterizacija anatomije i morfologije organoida napravljena je nakon imunofluorescencijskog bojanja i biološkog oslikavanja. Nadalje, razvila sam novi protokol za imunofluorescencijsko bojanje cističnih, šupljih tipova organoida. U odnosu na literaturno dostupne protokole, novi protokol predstavlja poboljšanja u brzini izvedbe i broju koraka koji štede vrijeme i novac. Također, rezultati su pokazali da organoidi mišjeg tankog crijeva mogu dolaziti u različitim oblicima pri čemu veličina stanica unutar organoida ovisi o točnoj poziciji stanice u organoidu te o njihovom proliferacijskom statusu. Na kraju, stopa proliferacije organoida mišjeg tankog crijeva pada s povećanjem broja dana koje su organoidi proveli u kulturi. Zaključno, uspješno je razvijen protokol za izolaciju i uzgoj složenog sustava organoida iz mišjeg tankog crijeva koji ima potencijal brze optimizacije za korištenje na organoidima drugih organskih sustava.A novel field arising in molecular biology, organoids, have a great potential in basic and applied research. Mini organs grown in a dish remarkably resemble their native counterparts in almost every aspect of their physiological, 3D structural and anatomical properties. Many different types of organoids have yet been developed, including mouse and human small intestine organoids. In this thesis, I have successfully established and maintained a culture of mouse small intestine organoids. After some optimization was done, the organoids were able to keep growing and differentiating indefinitely. More precise characterization of their anatomy and morphology was made after immunofluorescent staining and bio-imaging. I have created a novel protocol for immunostaining of cystic, hollow types of the organoids which reduces the time and money consumed. The results have revealed different shapes of small intestine organoids and variation in cell size depending on the position and proliferation status. The proliferation rate, however, decreases as the days in culture increase. To conclude, a novel protocol for isolation and propagation of complex organoid system was successfully developed from mouse small intestine. However, it has potential for fast optimization and adjustment for other types of mouse and human organoids

Establishment and bioimaging of mouse small intestine organoids culture

Organoidi su novo područje u molekularnoj biologiji koje ima veliki potencijal u temeljnim i primijenjenim istraživanjima. Minijaturni organi uzgajani in vitro značajno nalikuju svojim nativnim dvojnicima u svim fiziološkim, 3D strukturalnim i anatomskim karakteristikama. Već je razvijeno više tipova organoida, uključujući ljudske i mišje organoide tankog crijeva. U ovom radu, ja sam uspješno uzgojila i održala u kulturi organoide mišjeg tankog crijeva. Nakon optimizacije, uzgojeni su organoidi mogli neodređeno dugo rasti i propagirati se u kulturi. Preciznija karakterizacija anatomije i morfologije organoida napravljena je nakon imunofluorescencijskog bojanja i biološkog oslikavanja. Nadalje, razvila sam novi protokol za imunofluorescencijsko bojanje cističnih, šupljih tipova organoida. U odnosu na literaturno dostupne protokole, novi protokol predstavlja poboljšanja u brzini izvedbe i broju koraka koji štede vrijeme i novac. Također, rezultati su pokazali da organoidi mišjeg tankog crijeva mogu dolaziti u različitim oblicima pri čemu veličina stanica unutar organoida ovisi o točnoj poziciji stanice u organoidu te o njihovom proliferacijskom statusu. Na kraju, stopa proliferacije organoida mišjeg tankog crijeva pada s povećanjem broja dana koje su organoidi proveli u kulturi. Zaključno, uspješno je razvijen protokol za izolaciju i uzgoj složenog sustava organoida iz mišjeg tankog crijeva koji ima potencijal brze optimizacije za korištenje na organoidima drugih organskih sustava.A novel field arising in molecular biology, organoids, have a great potential in basic and applied research. Mini organs grown in a dish remarkably resemble their native counterparts in almost every aspect of their physiological, 3D structural and anatomical properties. Many different types of organoids have yet been developed, including mouse and human small intestine organoids. In this thesis, I have successfully established and maintained a culture of mouse small intestine organoids. After some optimization was done, the organoids were able to keep growing and differentiating indefinitely. More precise characterization of their anatomy and morphology was made after immunofluorescent staining and bio-imaging. I have created a novel protocol for immunostaining of cystic, hollow types of the organoids which reduces the time and money consumed. The results have revealed different shapes of small intestine organoids and variation in cell size depending on the position and proliferation status. The proliferation rate, however, decreases as the days in culture increase. To conclude, a novel protocol for isolation and propagation of complex organoid system was successfully developed from mouse small intestine. However, it has potential for fast optimization and adjustment for other types of mouse and human organoids

Establishment and bioimaging of mouse small intestine organoids culture

Organoidi su novo područje u molekularnoj biologiji koje ima veliki potencijal u temeljnim i primijenjenim istraživanjima. Minijaturni organi uzgajani in vitro značajno nalikuju svojim nativnim dvojnicima u svim fiziološkim, 3D strukturalnim i anatomskim karakteristikama. Već je razvijeno više tipova organoida, uključujući ljudske i mišje organoide tankog crijeva. U ovom radu, ja sam uspješno uzgojila i održala u kulturi organoide mišjeg tankog crijeva. Nakon optimizacije, uzgojeni su organoidi mogli neodređeno dugo rasti i propagirati se u kulturi. Preciznija karakterizacija anatomije i morfologije organoida napravljena je nakon imunofluorescencijskog bojanja i biološkog oslikavanja. Nadalje, razvila sam novi protokol za imunofluorescencijsko bojanje cističnih, šupljih tipova organoida. U odnosu na literaturno dostupne protokole, novi protokol predstavlja poboljšanja u brzini izvedbe i broju koraka koji štede vrijeme i novac. Također, rezultati su pokazali da organoidi mišjeg tankog crijeva mogu dolaziti u različitim oblicima pri čemu veličina stanica unutar organoida ovisi o točnoj poziciji stanice u organoidu te o njihovom proliferacijskom statusu. Na kraju, stopa proliferacije organoida mišjeg tankog crijeva pada s povećanjem broja dana koje su organoidi proveli u kulturi. Zaključno, uspješno je razvijen protokol za izolaciju i uzgoj složenog sustava organoida iz mišjeg tankog crijeva koji ima potencijal brze optimizacije za korištenje na organoidima drugih organskih sustava.A novel field arising in molecular biology, organoids, have a great potential in basic and applied research. Mini organs grown in a dish remarkably resemble their native counterparts in almost every aspect of their physiological, 3D structural and anatomical properties. Many different types of organoids have yet been developed, including mouse and human small intestine organoids. In this thesis, I have successfully established and maintained a culture of mouse small intestine organoids. After some optimization was done, the organoids were able to keep growing and differentiating indefinitely. More precise characterization of their anatomy and morphology was made after immunofluorescent staining and bio-imaging. I have created a novel protocol for immunostaining of cystic, hollow types of the organoids which reduces the time and money consumed. The results have revealed different shapes of small intestine organoids and variation in cell size depending on the position and proliferation status. The proliferation rate, however, decreases as the days in culture increase. To conclude, a novel protocol for isolation and propagation of complex organoid system was successfully developed from mouse small intestine. However, it has potential for fast optimization and adjustment for other types of mouse and human organoids

A quick, cheap, and reliable protocol for immunofluorescence of pluripotent and differentiating mouse embryonic stem cells in 2D and 3D colonies

Summary: Immunofluorescent labeling is a widely used method to visualize endogenous proteins. It can be expensive and difficult to stain mouse embryonic stem cells (mESCs) because they require expensive growth media, prefer specific substrates, grow in 3D, and have loose cell-substrate adhesion. Here we propose a half-a-day, cheap, easy-to-follow, and reproducible protocol for immunofluorescence of mESCs. This protocol has been streamlined to allow a fast visualization of the investigated proteins, and we provide tips specific to stem cell culture.For complete details on the use and execution of this protocol, please refer to Chaigne et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics