The studies in this thesis describe the application of genotyping and allele specific expression analysis to genetic studies. The role of the gene NPC1 in Triglyceride metabolism was explored in mouse models and in humans on the population level in study I. NPC1 was found to affect hepatic triglyceride metabolism, and to be relevant for controlling serum triglyceride levels in mice and potentially in humans. In study II the utility of the HapMap CEU samples was investigated for tagSNP selection in six European populations. The HapMap CEU was found to be representative for tagSNP selection in all populations while allele frequencies differed significantly in the sample from Kuusamo, Finland. In study III the power of Allele specific expression as a tool for the mapping of cis-regulatory variation was compared to standard eQTL analysis, ASE was found to be the more powerful type of analysis for a similar sample size. Finally ASE mapping was applied to regions reported to harbour long non-coding RNAs and associated SNPs were compared to published trait-associations. This revealed strong cis-regulatory SNPs of long non-coding RNAs with reported trait or disease associations.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Univ., Diss.--Wien, 2005.

Omkring 200 000–250 000 personer lider av hjärtsvikt i Sverige. Hjärtsvikt är ett tillstånd med nedsatt pumpförmåga hos hjärtat. Tillståndet resulterar i minskad livskvalité, hög morbiditet och mortalitet. Det har gjorts många försök för att hitta lämpliga läkemedelsmål som kan minimera dessa konsekvenser. Neurohormonella kompensatoriska mekanismer, exempelvis renin-angiotensin II-aldosteron-systemet, syftar till att återställa blodtrycket till normala nivåer igen efter för lågt blodtryck men på längre sikt ökar det även belastningen på hjärtat. Via detta system aktiveras hormonet angiotensin II som står för den ökade belastningen. Hormonet har därför varit ett viktigt läkemedelsmål för ACE-hämmare (ACEI) och AT1-antagonister (ARB) för att förhindra blodtryckshöjning. Enzymet neprilysin är ett annat läkemedelsmål, som inhiberas av läkemedelsklassen neprilysin-hämmare. Läkemedlet Entresto är en kombination av ARB och neprilysin-hämmare. Den neprilysin-hämmande komponenten, sakubitril, aktiveras av karboxylesteras 1 (CES1) men mutationer i genen som kodar för enzymet kan leda till utebliven terapeutisk effekt. Dessutom kan patienter med vildtyp CES1 riskera oacceptabla biverkningar som rabdomyolys och Alzheimer’s sjukdom. Syftet med arbetet är att undersöka om Entresto kan ersätta ACE-hämmare vid hjärtsvikt och om den behandlingen är optimal med avseende på farmakogenetiken. Arbetet är en litteraturstudie med vetenskapliga artiklar hämtade från databasen PubMed och via Linnéuniversitetets sökverktyg, OneSearch. I arbetet har fem studier analyserats (I-V). Studierna visar att Entresto är överlägsen enalapril genom att minska hjärtsviktshospitalisering och dödsfall på grund av kardiovaskulära orsaker. Dock är läkemedelseffekten av Entresto beroende av en fungerande CES1-gen eftersom mutationer som G143E orsakar utebliven terapeutisk effekt. Enalapril har visat sig vara oberoende av sådana mutationer. Teoretiskt sett kan en hämning av neprilysin ge upphov till ackumulering av amyloid-β-peptider (Aβ), vilket associeras med Alzheimer’s sjukdom. Studie IV, vars syfte var att undersöka vilken inverkan Entresto har på Aβ-isoformer, visade inga förändrade Aβ-koncentrationer i cerebrospinalvätska. Dock behövs vidare studier med längre durationstid. Däremot visar studie V att en kombination av Entresto och statiner ökar plasmakoncentrationen av statiner, vilket i sin tur ökar risken för att utveckla rabdomyolys. Slutsatsen blir att det inte är optimalt att ersätta enalapril med Entresto vid hjärtsvikt med avseende på farmakogenetik. / Around 200,000–250,000 people suffer from heart failure in Sweden. Heart failure is a condition of impaired heart pumping capacity. The condition results in reduced quality of life, high morbidity and mortality and there have been many attempts to find suitable drug targets to minimize these consequences. Neurohormonal compensatory mechanisms, such as renin–angiotensin–aldosterone system, aim to restore blood pressure to normal levels again but in the long-term it also increases the stress on the heart. The hormone angiotensin II gets activated through this mechanism and is the reason behind the increased stress. Therefore, the hormone has been an important drug target for ACE inhibitors (ACEI) and AT1 blockers (ARB) to prevent antihypertensive effects. The enzyme neprilysin is another drug target whose inhibition is accomplished by using neprilysin inhibitors. Entresto® is a new medication that contains a neprilysin inhibitor and an ARB. The neprilysin inhibitory component, sacubitril, is activated by carboxylesterase 1 (CES1) but mutations in the gene encoding CES1 may cause a non-therapeutic effect. Additionally, patients with wild-type CES1 may risk unacceptable side effects such as rhabdomyolysis and Alzheimer's disease. The objective of this study is to investigate whether replacement of ACE inhibitors with Entresto is optimal in heart failure with regard to pharmacogenetics. This study is organized as a literature study in which five scientific articles (I-V) were analyzed and selected from PubMed database and through Linnaeus University's search engine, OneSearch. The studies show that Entresto is superior to enalapril in reducing the risk for cardiovascular death or hospitalization for heart failure. However, the effects of Entresto is dependent on a functioning CES1 gene because mutations like G143E cause a non-therapeutic effect. Enalapril has shown to be independent of such mutations. Theoretically, inhibition of neprilysin may cause accumulation of amyloid-β peptides (Aβ), which associates with Alzheimer's disease. Study IV, with the purpose to investigate the effect of Entresto on Aβ isoforms, showed no significant change in Aβ concentrations in cerebrospinal fluid. However, further studies with longer duration were suggested. On the other hand, study V shows that a combination of Entresto and statins increases the plasma concentration of statins. That in turn would increase the risk of a development of rhabdomyolysis. The conclusion is that it is not optimal to replace enalapril with Entresto in heart failure with regard to pharmacogenetics.

The unconventional myosin IC has previously been suggested to be a haploinsufficient tumour suppressor. The mechanism for this action has hitherto been unknown, however, and hence we decided to attempt to elucidate the genes involved. The first study involved knock-down of MYO1C using siRNA technology followed by whole transcriptiome microarray analysis performed on samples taken at different time points post transfection. This revealed a cornucopia of differential expressions compared to the negative control, among them we found an early up-regulation of the PI3K/AKT pathway and the pathway for prostate cancer. Among the down regulated pathways we found endometrial-, colorectal cancer and small cell lung cancer as well as the cell cycle pathway which was a little counter intuitive to the hypothesis that MYO1C suppresses cancer. For the next study six different genes (CCND1, CCND2, CDKN2B, CDKN2C, MYC, RBL1) important for the transitions into S-phase of the cell cycle were therefore chosen for validation using qPCR. These six genes and MYO1C were analysed on both the original time series and a new biological replicate as well as a well stratified set of endometrial carcinoma samples. We were able to verify the significant down-regulation of CCND2 in both time series indicating that this is caused by the depletion of MYO1C. In the tumour samples we saw a negative correlation between the expression of MYO1C and FIGO grade corroborating results previously found by our group when looking at protein expression.

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

Bacteria are many, old and varied; different bacterial species have been evolving for millions of years and show many disparate life-styles and types of metabolism. Nevertheless, some of the characteristics regarding how bacteria organize their chromosomes are relatively conserved, suggesting that they might be both ancient and important, and that selective pressures inhibit their modification. This thesis aims to study some of these characteristics experimentally, assessing how changes affect bacterial growth, and how, after changing conserved features, bacteria might evolve. First, we experimentally tested what are the constraints on the horizontal transfer of a gene highly important for bacterial growth. Second, we investigated the significance of the location and orientation of a highly expressed and essential operon; and we experimentally evolved strains with suboptimal locations and orientations to assess how bacteria could adapt to these changes. Thirdly, we sought to understand the accessibility of different regions of the bacterial chromosome to engage in homologous recombination. And lastly, we constructed bacterial strains with chromosomal inversions to assess what effect the inversions had on growth rate, and how bacteria carrying costly inversions could evolve to reduce these costs. The results provide evidence for different selective forces acting to conserve these chromosome organizational traits. Accordingly, we found that evolutionary distance, functional conservation, suboptimal expression and impaired network connectivity of a gene can affect the successful transfer of genes between bacterial species. We determined that relative location of an essential and highly expressed operon is critical for supporting fast growth rate, and that its location seems to be more important than its orientation. We also found that both the location, and relative orientation of separated duplicate sequences can affect recombination rates between these sequences in different regions of the chromosome. Finally, the data suggest that the importance of having the two arms of a circular bacterial chromosome approximately equal in size is a strong selective force acting against certain type of chromosomal inversions.

There is a basic rule to mammalian neocortical expansion: as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.