Browsing by Subject "RNA-seq"
Now showing items 1-4 of 4
-
(2021)MLH1 is a gene that codes for one of the four mismatch repair (MMR) proteins alongside MSH2, MSH6, and PMS2. The main function of the MMR proteins is to recognize base mismatches and insertion-deletion loops formed during DNA replication and aid in their excision. Inherited heterozygous pathogenic variants in any of the four MMR genes lead to Lynch syndrome, an inherited cancer syndrome that predisposes to multiple different cancer types, most notably colorectal cancer. Loss of the expression of an MMR gene causes MMR-deficiency, which leads to microsatellite instability, the accumulation of mutations in microsatellite regions of the DNA. The higher mutational burden caused by MMR-deficiency is thought to be the main driving force of genomic instability and tumorigenesis in MMR-deficient cells. In addition to MMR, MLH1 and the MMR machinery have roles in other anticarcinogenic cellular processes, such as DNA damage signaling and DNA double-strand break repair. Recently, MLH1 has also been shown to have a significant role in regulating mitochondrial metabolism and oxidative stress responses. The identification of MMR-proficient tumors in Lynch syndrome patients begs the question whether the lower amount of functional MLH1 observed in MLH1 mutation carriers could cause problems with these functions and pose alternative routes to tumorigenesis. In line with this, it has been shown that the role of MLH1 in cell cycle regulation in DNA damage signaling is notably more sensitive to decreased amount of the protein compared to its role in MMR. The main goal of the thesis was to study the effects of decreased MLH1 expression on gene expression, cellular functions, and possible alternative tumorigenic pathways. In order to achieve this, the coding transcriptome of human fibroblast cell lines expressing MLH1 at different levels was sequenced and the resulting data analyzed. The study revealed that decreased MLH1 expression affects cellular functions associated with mitochondrial function and oxidative stress responses in cells with functional MMR. Particularly NRF2-controlled cytoprotective defence systems were observed to be downregulated. Decreased MLH1 expression was also observed to affect several cellular functions associated with reorganization of the cytoskeleton and interactions with the extracellular matrix. These results strengthen the recently made notions that MLH1 has a role in controlling the function of mitochondria and in mitigating oxidative stress, and that these two functions are connected. The study also brings to light new information on the possible role of MLH1 in controlling the organization of the cytoskeleton, which has previously received little attention. Dysfunction of mitochondria, increased oxidative stress, and reorganization of the cytoskeleton, as a result of decreased MLH1 expression, could pose events that facilitate malignant transformation of cells prior to the total loss of MMR function.
-
(2023)Animal coloration is as striking as it is diverse; however, the transcriptional basis of coloration is not deeply understood. Cichlid fishes are a tractable system for studying coloration as they exhibit a wide range of phenotypic diversity while remaining genetically similar. This facilitates the study of genotype-phenotype correlations and the identification of causative genes. RNA sequencing is a powerful approach to investigate the genes which characterize chromatophores. However, RNA-seq results can be plagued by the high abundance of rRNA in cells. This thesis aims to investigate differential gene expression between differently pigmented regions as well as explore the effects of tissue treatments and rRNA depletion on gene expression. Gene sets acquired with polyA selection, riboPOOL probes optimized for zebrafish, and zebrafish probes complemented with newly designed riboPOOL cichlid probes were compared to assess the functionality of these different rRNA depletion strategies. The use of zebrafish probes complemented with newly designed cichlid probes captured the greatest diversity of genes, many transcripts of which were missing from the other gene sets. Furthermore, as experiments such as scRNA-seq rely on a dissociation step, the effect of dissociation on gene expression was examined and found to promote the expression of stress response genes. The results of this upstream optimization were applied in the analysis of differential gene expression between the vertical stripes of the cichlid Pseudotropheus demasoni to better understand the molecular basis of vertical striping in fish. The dark stripes exhibited upregulation of melanic marker genes and the light, iridescent stripes showed an increase in iridophore marker gene expression. These findings were corroborated with cell count data from FACS to link transcriptional profiles and cell type quantifications. Overall, the study provides insight into the transcriptional basis of coloration in cichlid fishes and underscores the importance of optimizing methods drawing meaningful conclusions.
-
(2020)MikroRNA:t ovat tärkeitä, transkription jälkeen geenien ilmenemiseen vaikuttavia säätelijöitä. MikroRNA:iden tuotantoa on löydetty konservoituneena useista eri eliöryhmistä ja kudoksista, mikä osaltaan kertoo niiden toiminnan tärkeydestä. Rakenteeltaan miRNA:t ovat lyhyitä, noin 22 nukleotidin mittaisia yksijuosteisia RNA:ita, jotka tuotetaan pidemmistä esi-RNA:sta entsymaattisesti leikkaamalla. Geenien säätelyssä miRNA:iden teho perustuu niiden kykyyn tunnistaa kohdegeenit miRNA:n ja lähetti-RNA:n sekvenssien komplementaarisuuden perusteella. MikroRNA:iden kohteiden tunnistuksen kannalta tärkeätä, 6-8 nukleotidin jaksoa kutsutaan miRNA:n ydinalueeksi (seed-region). Yhdellä miRNA:lla voi potentiaalisesti olla satoja kohdegeenejä, joita se säätelee, minkä vuoksi näitä miRNA-mRNAinteraktioita on pyritty bioinformatiivisesti ennakoimaan useiden ohjelmien ja algoritmien avulla. miRNA:iden toiminnan tutkimista mutkistavat isomiR:it, jotka ovat yleisesti tunnettujen, kanonisten, miRNA:iden vaihtoehtoisia muotoja. IsomiR:ejä tuottavat poikkeamat miRNA:iden esiasteiden prosessoinnissa ja mikroRNA:ihin kohdistuva RNA-editointi. Tuloksena saadaan mikroRNA:ita joiden pituus tai emäskompositio eroavat kanonisista sekvensseistä. Myös miRNA:iden toiminta voi muun muassa alkaa kohdistamaan säätelyä eri geeneihin, kuin mitä kanonisen sekvenssin perusteella voisi olettaa. Tässä pro gradu -projektissa analysoin suurtehosekvensoinnilla tuotettua dataa kahdesta aivoalueesta kuudelta eri laboratoriohiirikannalta. Normalisoin datan ja todettuani sen laadun hyväksi jatkoin miRNA:iden ilmenemisen tarkastelua eri analyyseillä: Selvitin mitkä miRNA:t ilmentyivät tutkituilla aivoalueille jamiten niiden ekspressio erosi eri aivoalueiden ja hiirikantojen välillä. Tämän jälkeen tarkastelin miRNA:sta tuotettujen vaihtoehtoisten sekvenssien, isomiR:ien ilmentymistä. Vertailimme lisäksi aineistossa havaitsemiemme isomiR:ien ekspressiossa eroja aivoalueiden ja hiirikantojen välillä. Lopuksi pyrimme osoittamaan havaittujen isomiR:ejä tuottavien RNAeditointitapahtumien olevan funktionaalisesti merkittäviä. Tätä tarkoitusta varten transfektoimme soluviljelmiä lusiferaasi-reportterivektorilla ja editoiduilla ja editoimattomilla miRNA:ta muistuttavilla sekvensseillä. Analyysien tuloksena havaitsimme lukuisia miRNA:ita, jotka olivat eritavoin ilmentyneitä joko aivoaluiden, hiirikantojen tai molempien välillä. Lisäksi havaitsimme lukuisia isomiR-sekvenssejä: Noin 90%:sta havaittuja miRNA:ita löydettiin vähintään yksi vaihtoehtoinen, ei-kanoninen sekvenssi. IsomiR:ien suuresta määrästä huolimatta,miRNA:iden kokonaisekspressiosta suurin osaoli yleensä vain muutaman isomiR:in aikaansaannosta. Aineistoista löysimme myös runsaasti merkkejä RNA-editoinnista, ja erityisesti ADAR-entsyymin toiminnasta. Funktionaaliset kokeemme antoivat myös vahvoja viitteitä siitä, että miRNA:iden ydinalueisiin kohdistuvat editoinnit ovat funktionaalisesti merkittäviä tapahtumia. Muutosten seurauksena havaitsimme muuttuneiden ydinaluiden tunnistavan eri kohdegeenejä kuin kanoninen muuntelematon miRNA-sekvenssi. Johtopäätöksenä miRNA:iden ja erityisesti niiden isomiR:ien muunteluun on jatkossa syytä kiinnittää nykyistä enemmän huomiota. Parempi ymmärrys isomiR:eistä ja niitä synnyttävistä mekanismeista voivat jatkossa mahdollistaa tehokkaamman koesuunnittelun ja helpottaa saatujen tulosten tulkintaa.
-
(2020)Psychrotrophic lactic acid bacteria (LAB) play a versatile role in research, food, farming and medicinal applications, but also play a role as a source of food spoilage. The effects of temperature changes has yet to be studied in depth. In this study, to analyze in a transcriptome level, cold and heat shock stress to spoilage lactic acid bacteria, a time-dependent RNA-seq for Lactococcus piscium with a temperature of 0 °C and 28 °C was conducted. The data of protein regulation during the experiment shows that Lactococcus piscium has the essential machinery to survive against different types of environmental stress. I observed known heat shock related genes and stress related genes to be present in the regulated response of both temperature extremes. Cold shock upregulation is observed after 35 minutes, which could indicate that the metabolic response at cold temperatures is related to growth rate. With a clear downregulation of pathways of energy consumption and an adaptation in terms of RNA being more prominent that at 28 ºC. Certain surface, cell wall and transport proteins are noticeable more upregulated at 0 ºC in addition to a downregulation of energy related proteins, which in correlation with its growth curve, we can assume it is part of a mechanism of protection against cold environments, in comparison with the regulation of proteins at 28 ºC. This gives an insight of a well controlled preservation mechanism that Lactococcus piscium exhibits that can be linked to its environment. The findings of this research offer new understandings into the survival skills of Lactococcus piscium to a cold and heat shock. Future studies should focus on analyzing the regulation and function of possible new genes for the response to heat stress, as well as the specific function of proteins related to temperature stress in the events where change of temperature plays a constant role in the environment.
Now showing items 1-4 of 4