Potilaasta johdettujen indusoitujen pluripotenttien kantasolujen käyttö liikunnasta aiheutuvan hyperinsulinismin (EIHI) tautimallinnuksessa

Liikunnasta aiheutuva hyperinsulinismi (EIHI) on sairaus, joka ilmenee epänormaalina insuliinin erityksenä rasituksen aikana. Monokarboksylaattien kuljettajaproteiini (MCT1), jota koodaa SLC16A1-geeni, on laajalti ilmentynyt lähes kaikissa kudoksissa paitsi haiman saarekesoluissa. Henkilöillä, jotka sairastavat EIHI:ä, SLC16A1 säätelyalueen deleetion uskotaan johtavan MCT1:n virheelliseen ilmenemiseen beta soluissa, mikä mahdollistaa kohonneiden laktaatti- ja pyruvaattipitoisuuksien virtauksen solukalvon läpi rasituksen aikana. Nämä substraatit käynnistävät Krebsin kierron, mikä lisää insuliinin eritystä. Ylimääräinen insuliinin eritys aiheuttaa alhaisen verensokeritason rasituksen aikana, mikä johtaa heikkouteen, pyörtymiseen ja sekavuuteen. Koska EIHI sairauden mekanismia ei ole aikaisemmin mallinnettu, tämän Pro-gradu tutkielman tavoitteena oli mallintaa taudin toimintamekanismeja sairaasta henkilöstä johdettujen indusoitujen pluripotenttien (iPS) kantasolujen avulla. Työssä uudelleenohjelmoitiin EIHI:ä sairastavan henkilön fibroblasteja iPS-soluiksi käynnistäen pluritpotenssia säätelevät geeniverkostot uudelleen. Tämän jälkeen sairaan henkilön sekä terveen henkilön (kontrolli) iPS solut erilaistettiin toiminnallisiksi haiman saarekesoluiksi in vitro ympäristössä rinnakkain. Seuraavassa vaiheessa soluja siirrettiin immuunipuutteisiin hiiriin, jolloin solut kypsyivät edelleen in vivo ympäristössä. Erilaisia laadunvalvontamittauksia (virtaussytometria) tehtiin erilaistamisprosessin aikana. Lisäksi tutkittiin SLC16A1-geenin ilmentymistä erilaistamisen eri vaiheissa. Lopuksi haiman betasoluiksi erilaistettujen solujen ominaisuuksia tutkittiin muun muassa mittaamalla insuliinin eritystä, sekä glukoosi-että pyruvaattistimulaation jälkeen, sekä MCT1-proteiinin immunohistokemiallisella analyysillä. Vastoin oletuksia, tutkielman tulokset osoittivat, että SCL16A1 promoottorialueen deleetiosta huolimatta sairaan henkilön beta solut erittivät insuliinia samalla tavalla kuin terveen henkilön kontrollisolut. SLC16A1-geenin ilmentyminen myös aleni vastoin odotuksia samalla tavalla kuin kontrollisoluilla. MCT1-proteiinin immunohistokemialliset värjäykset hiiren munuaiskapselin alle siirretyistä kantasolusaarekkeista kuitenkin osoitti selkeän eron; deleetion sisältävät solut ilmensivät MCT1:stä selkeästi enemmän kuin kontrollisolut. Yhteenvetona voidaan todeta, että sairauden oletetut patologiset piirteet eivät olleet selkeästi havaittavissa in vitro -mallissa. Kuitenkin munuaiskapselin alle tehtävän siirron jälkeen, in vivo -mallissa, mutanttisoluissa havaittiin MCT1:n ilmentymisen lisääntyneen huomattavasti. Tämä löydös viittaa siihen, että MCT1-ilmentymistä säätelee erillinen mekanismi, johon vaikuttaa ympäristöolosuhteet. Tulokset luovat hyvän pohjan tulevaisuudessa näitä mekanismeja tutkiville tutkimuksille.Exercise-induced hyperinsulinism (EIHI) is a pathological condition characterized by aberrant insulin secretion triggered by physical exercise or pyruvate exposure. The monocarboxylate transporter protein (MCT1), encoded by SLC16A1, is ubiquitously expressed in almost all cell types except pancreatic islet cells. In patients with EIHI, mutations in the regulatory regions of the SLC16A1 gene are thought to lead to the unwanted expression of MCT1 on the beta cell membrane, allowing the influx of elevated lactate and pyruvate blood levels during exercise. These substrates feed into the Krebs cycle, increasing insulin release. This excessive insulin secretion can lead to hypoglycemia during exercise, causing weakness, syncope, and confusion. Since EIHI has never been studied using a human stem cell-derived islets, this thesis aims to establish a robust model in which to investigate the disease mechanism in detail. To achieve this, we reprogrammed EIHI patients’ fibroblasts into a stable pluripotent state and further differentiated them into functional pancreatic stem cell-derived islets (SC-islets) in vitro. These SC-islets were then matured further in vivo (in immunocompromised mice) and compared to healthy SC-islet controls. Rigorous quality control measures were implemented throughout the differentiation process to ensure its efficacy and the expression regulation of SLC16A1 was studied during SC-islet development. Extensive phenotypic characterization was conducted using immunohistochemistry, quantitative gene expression level analysis, and insulin secretion assays with glucose and pyruvate. Contrary to expectations, the results of this study demonstrated that despite the SLC16A1 promoter mutation, the expression of SLC16A1 was downregulated similarly to the control cell line during development in vitro, resulting in similar pyruvate-stimulated insulin secretion to the control cells. Interestingly, immunohistochemical analysis of in vivo implanted SC-islets showed a clear phenotype with an increased number of MCT1-positive cells only in the mutant grafts, some of which were endocrine cells. In conclusion, the phenotypic manifestations of EIHI were not visible in the setting of in vitro modeling, which was attributed to the similar expression levels of MCT1 in both the mutant and control cell lines. However, following in vivo implantation, there was a noticeable increase in MCT1 expression exclusively in the mutant cells. This finding suggests a distinct regulatory mechanism of MCT1 expression, which might be impacted by the in vivo surroundings and the maturation state of human islets

Gendered support to older parents: do welfare states matter?

The aim of this study is to examine the association of welfare state policies and the gendered organisation of intergenerational support (instrumental help and personal care) to older parents. The study distinguishes between support to older parents provided at least weekly, i.e. time-intensive and often burdening support, and supplemental sporadic support. Three policy instruments were expected to be associated with daughters’ and sons’ support or gender inequality in intergenerational support respectively: (1) professional social services, (2) cash-for-care payments and (3) legal obligations to provide or co-finance care for parents. The analyses based on the Survey of Health, Ageing and Retirement in Europe showed that daughters provided somewhat more sporadic and much more intensive support than sons throughout Europe. While about half of all children who sporadically supported a parent were men, this applied to only one out of four children who provided intensive support. Logistic multilevel models revealed that legal obligations were positively associated with daughters’ likelihood of giving intensive support to parents but did not affect the likelihood of sons doing so. Legal obligations thus stimulate support in a gender-specific way. Both legal obligations and cash-for-care schemes were also accompanied by a more unequal distribution of involvement in intensive support at the expense of women. Social services, in contrast, were linked to a lower involvement of daughters in intensive support. In sum, the results suggest that welfare states can both preserve or reduce gender inequality in intergenerational support depending on specific arrangements

The impact of low-frequency and rare variants on lipid levels

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing

The impact of low-frequency and rare variants on lipid levels

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing

The impact of low-frequency and rare variants on lipid levels.

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing

Cardiovascular Efficacy and Safety of Bococizumab in High-Risk Patients

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