The gastrointestinal tract (GIT) regulates nutrient uptake, secretes hormones and has a crucial gut flora and enteric nervous system. Of relevance for these functions are the G protein-coupled receptors (GPCRs) and the solute carriers (SLCs). The Adhesion GPCR subfamily is known to mediate neural development and immune system functioning, whereas SLCs transport e.g. amino acids, fatty acids (FAs) and drugs over membranes. We aimed to comprehensively characterize Adhesion GPCR and SLC gene expression along the rat GIT. Using qPCR we measured expression of 78 SLCs as well as all 30 Adhesion GPCRs in a twelve-segment GIT model. 21 of the Adhesion GPCRs had a widespread (≥5 segments) or ubiquitous (≥11 segments) expression. Restricted expression patterns were characteristic for most group VII members. Of the SLCs, we found the majority (56 %) of these transcripts to be expressed in all GIT segments. SLCs were predominantly found in the absorption-responsible gut regions. Both Adhesion GPCRs and SLCs were widely expressed in the rat GIT, suggesting important roles. The distribution of Adhesion GPCRs defines them as a potential pharmacological target. FAs constitute an important energy source and have been implicated in the worldwide obesity increase. FAs and their ratios – indices for activities of e.g. the desaturase enzymes SCD-1 (SCD-16, 16:1n-7/16:0), D6D (18:3n-6/18:2n-6) and D5D (20:4n-6/20:3n-6) – have been associated with e.g. overall mortality and BMI. We examined whether differences in FAs and their indices in five lipid fractions contributed to obesity susceptibility in rats fed a high fat diet (HFD), and the associations of desaturase indices between lipid fractions in animals on different diets. We found that on a HFD, obesity-prone (OP) rats had a higher SCD-16 index and a lower linoleic acid (LA) proportions in subcutaneous adipose tissue (SAT) than obesity-resistant rats. Desaturase indices were significantly correlated between many of the lipid fractions. The higher SCD-16 may indicate higher SCD-1 activity in SAT in OP rats, and combined with lower LA proportions may provide novel insights into HFD-induced obesity. The associations between desaturase indices show that plasma measurements can serve as proxies for some lipid fractions, but the correlations seem to be affected by diet and weight gain.

Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. / The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.