Department of Biological Sciences (BIO)

Molecular Biology Master theses submitted in 2018

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Jonelle Dickow Villar

Jonelle Dickow Villar, Anne Kristin Stavrum, Stephanie Le Hellard og Kari Ersland

Identification of Epigenetic ModificationsFollowing Treatment with Olanzapine

No chronic disease burdens the world more than psychiatric disorders (Collins et al., 2011), with an estimated 40% of the population in 30 European countries affected in any given year (Insel et al., 2012). Current medical treatment for schizophrenia (SCZ), bipolar disorder (BPD) and major depressive disorder (MDD) is based upon well-established antipsychotic drugs and mood stabilizers. Treatment efficacy (30-40%) (P. Lowe et al., 2017) and potentially serious side effects (Leucht et al., 2013) often challenge medication compliance, adding an additional challenge in the path towards wellness. New drugs are required to address the burden of psychotic disorders, and yet a new science is required to address the interplay between the heterogeneous nature of psychotic disorders and drug mechanisms.Epigenetic mechanisms, particularly alterations of methylation patterns at CpG sites have been shown to alter gene expression in humans, animal models and in vitro cell cultures. It is believed that epigenetic modifications induced by antipsychotic drugs plays a role in therapeutic response. Identification of pathways implicated by epigenetically modified genes, including the dopaminergic pathway, for example, has enhanced our understanding of the therapeutic mechanism of the antipsychotic drug olanzapine (Melka et al., 2013).In the current study, we aimed to identify differentially methylated regions induced by olanzapine. 82 European patients adhering to monotherapy were selected through the TOP Cohort (Thematically Organised Psychosis). Methylation data derived from blood samples was assessed genome-wide using the Illumina 850K EPIC array. The statistical model was corrected for gender and smoking. Following identification of differentially methylated positions (DMPs) in patient blood, we exposed a cultured cell line (HepG2) to verify the modifying effect of olanzapine on DNA methylation levels.The results of our study provide evidence of differentially methylated positions and regions in the blood of patients adhering to olanzapine monotherapy. A comparison of models adjusting for cell type composition provided evidence of improved p-values when cell type adjustment was included in the model. This finding was in concordance with state-of-the-art epigenome-wide-association (EWA) studies. Our results showed concordance between blood and brain for two identified differentially methylated regions, including the Trio and F-actin binding protein (TRIOBP) shown to be relevant in schizophrenia (Nicholas J. Bradshaw et al., 2014). The pathways implicated by the differentially methylated genes showed evidence of alterations in immune pathways and the possible mediating effect of olanzapine.

Anni Sofie Geithus

Anni Sofie Geithus, Thomas Arnesen, Rasmus Ree

Characterization of NAA10 mutations in patients exhibiting phenotypes associated with N-terminal acetylation deficiency

Approximately 80% of the proteins found in human cells are acetylated on their N-terminus, either partially or completely, by a group of enzymes called N-terminal acetyltransferases (NATs)1. To date, seven NATs, NatA-NatF2–7 and NatH8, have been identified in humans; NatA-NatE associate with ribosomes and acetylate their substrates co-translationally, NatF associate with the Golgi apparatus membrane and acetylate membrane proteins post-translationally, and NatH is found in the cytosol where it acetylates actins, also post-translationally8,9.The NatA complex, consisting of catalytic subunit NAA10 and auxiliary subunit NAA15 facilitating the association of the complex with the ribosome, has the most protein N-termini targets among the NATs, and is also the most studied complex2,9. The function of NatA and uncomplexed NAA10 in the cell is gradually being characterized by an increasing number of studies on patient identified NAA10 mutations10–14. The patients share a wide spectrum of phenotypes, most commonly developmental delay, growth deficiency, post-natal growth failure, and cardiac and skeletal anomalies13.This thesis’ main focus is to functionally characterize two novel NAA10 missense mutations, C21G and R83H, identified in three male patients presenting with typical NAA10 deficiency. Characterization involves in vitro acetylation assay of uncomplexed recombinant MBP-NAA10, cellular stability assay of NAA10 and NatA, and bioinformatic assessments of structural functions as well as conservation and in silico prediction of disease. The work presented here, demonstrates that both mutations are found in highly conserved regions in NAA10 involved with Ac-CoA and substrate binding as well as NatA complex formation. The acetylation assay show that while the R83H mutant has profoundly reduced catalytic activity, the C21G mutant show an increased activity. From the cellular stability assay, none of the mutations show any apparent destabilizing effects. However, to draw conclusions about the functional impact of these mutations on the NAA10 and NatA and their link to human health, more research is needed than is presented here in this thesis.

Morsal Saba

Morsal Saba, Marios Chatzigeorgiou, Riccardo Esposito

Ca2+-signalling in C. intestinalis notochord cellsduring embryonic development

C. intestinalis notochord is an essential structure during embryonic development, conveying position and fate information, in addition to serving as a structural support and enabling locomotion during larval stage. Researchers have previously presented Ca2+-signalling as a key element in development of many organisms, but few have performed detailed studies on C. intestinalis. This thesis aimed to elucidate the mechanism underlying Ca2+-signalling in developing C. intestinalis notochord, through four sub-aims: 1) Determine whether the notochord cells exhibit Ca2+-signalling activity during development, 2) Determine the contribution of various subcellular compartments, known to store/release Ca2+, to the observed Ca2+-signalling activity, 3) Study the mechanisms underlying Ca2+-signalling in the notochord cells, and 4) Study the effects of calcium pathway on the actomyosin network, which mediates cell motility and cell shape changes.The results present evidence of Ca2+-signalling in C. intestinalis notochord cells at all studied developmental stages; as continues blips, waves, and short/long lasting blinks in individual cells, and/or blinks travelling in-between cells. Waves seem to dominate stages 22-24, whereas blinks appear to dominate stages 24-26. For the sake of simplicity, the Δ used in this context stands for “change in”. There is a significant difference in terms of number of peaks, (Δ) peak intensity and (Δ) duration, between stage 22 and 23, and from stage 24 and onwards, but not between stage 23 and 24. The number of peaks appears to depend on cation channels and gap junctions, whereas the (Δ) signal intensity seems to rely on cation channels, gap junctions, IP3R and SOCE. As for (Δ) signal duration, it might depend on IP3R and SOCE as well. Furthermore, calmodulin is indicated to be important during stage 23 but not during stage 22, insignificant for number of peaks except for stage 23, and its significance various from stage 24-26. With regards to SERCA, stage 22, and 24-25 suggest independence, whereas stage 23 and 26 seem to depend to some degree. Calmodulin and SERCA data also indicate cell non-autonomous behaviour, as it is hinted in calmodulin RNAi with regards to differences in both AP and M compared to wild type. Ca2+-signalling inhibition also affects the notochord actomyosin network. Specifically, Gado3+ and 2APB affect the cell structures and protein expression already from 3hpi and onwards. Carbenoxolone inhibition, on the other hand, leads to visually different protein expression and cell shape from 6hpi and onwards. Lastly, this study further presents Ca2+-signalling in epidermal and muscle cell, implying importance in tail relaxation, twitching, and movement during embryonic development.

Camila Cimadamore-Werthein

Camila Cimadamore-Werthein, Mathias Ziegler, Marc Niere

The generation and maintenance of the mitochondrial NAD pool is independent of mitochondrial NAD biosynthetic enzymes

Nicotinamide adenine dinucleotide (NAD) is a vital regulator of cellular homeostasis due to its involvement in many key metabolic and signaling pathways. In the compartmentalized eukaryotic cell, many NAD-dependent processes occur in different organelles. In redox reactions, NAD+ is reversibly converted to NADH, whereas NAD-dependent signaling events lead to a net-consumption of this small molecule. Thus, there is constant need for replenishment of the NAD pools, of which the mitochondrial is the predominant one. As the chemical properties of NAD prevent its diffusion through biological membranes, the generation and maintenance of individual NAD pools must occur either via autonomous biosynthetic routes or by NAD transporters.In this study, the possible contribution of two NAD biosynthetic enzymes to mitochondrial NAD biosynthesis was investigated. To this end, a hitherto uncharacterized isoform of nicotinamide riboside kinase 1 (NRK1), which may be encoded by a newly identified alternatively spliced transcript, was analyzed for localization to mitochondria. A mitochondrial NRK1 would provide the substrate for the mitochondrial nicotinamide mononucleotide adenylyltransferase isoform 3 (NMNAT3). Therefore, the role of NMNAT3 in mitochondrial NAD biosynthesis was studied in CRISPR-Cas9-engineered human NMNAT3 knock-out cells.RT-PCR analysis demonstrated the presence of the new NRK1 transcript variant in various cell lines. Therefore, subcellular localization studies using the protein expressed from recombinant DNA were conducted. Unexpectedly, transfection of a plasmid encoding the C-terminally FLAG-tagged protein did not result in any detectable FLAG immunoreactivity in cells or cell lysates. Control experiments established that the coding sequence was properly transcribed, irrespective of the presence of endogenous regulatory elements in the 5’-untranslated region in the eukaryotic expression vectors.C-terminal fusion of EGFP to the full-length NRK1 protein variants indicated that the cellular distribution of the protein encoded by the new transcript variant does not differ from the established nuclear-cytosolic localization observed for the protein translated from the canonical transcript. Additional EGFP-fusion constructs also revealed that the N-terminal amino acids of the protein encoded by the new NRK1 transcript variant do not serve as a mitochondrial targeting sequence.


Henrikke Nilsen Hovland

Henrikke Nilsen Hovland, Anders Molven, Kelly Velasco

Functional characterization of rare variants of SCHAD, a protein involved in unregulated insulin secretion

Congenital Hyperinsulinism of Infancy (CHI) is a group of rare inherited disorders characterized by persistent hypoglycaemia due to inappropriate elevated secretion of insulin from the pancreatic beta cells. CHI can be caused by abnormalities in at least 10 genes. One of them, HADH, encodes the mitochondrial enzyme SCHAD, which has two functions: it catalyses the third step in the oxidation of fatty acids, and it has been reported to negatively regulate the enzyme glutamate dehydrogenase (GDH). Inactivating mutations in HADH lead to a loss of the inhibitory protein interaction of SCHAD, which then cause increased insulin secretion due to overactive GDH.We here aimed to understand the functional impact of rare SCHAD variants present in human populations. We have explored the effect of four pathogenic SCHAD variants with regard to the level of expression, subcellular localization and enzymatic activity. Two of the variants (G34R and I184F) showed protein instability and had clearly reduced level of protein expression in HEK293 cells. The mRNA levels appeared normal, which indicated that the cause of instability lies downstream of the transcription. The two other variants (K136E and M188V) had normal protein expression levels in HEK293 cells, but a severely reduced enzymatic activity compared to the wild type protein. The four SCHAD mutations did not affect the mitochondrial localization of the protein. In addition to the four pathogenic variants, protein stability of 11 other rare SCHAD variants was tested in HEK293 cells by the use of the protein synthesis inhibitor cycloheximide. Four additional variants were found to be unstable (I33M, H170R, P258 and G303S). The effect of the different SCHAD variants on GDH interaction was evaluated by co-immunoprecipitation. Protein:protein interaction could be detected for the SCHAD wild type protein, whereas several of the other variants seemed to have reduced or lost binding to GDH. The data gathered throughout the study were in combination with other relevant information used to reclassify the pathogenicity of a total of 16 rare SCHAD variants present in human populations. Taken together, our results illustrate the extensive work necessary for evaluating the functional impact of rare variants that are identified in high-throughput sequencing of human genomes.


Victoria Smith Arnesen

Victoria Smith Arnesen, Aurelia Lewis, Fatemeh Mazloumi Gavgani

Role of the class I PI3K p110β and PtdIns(3,4,5)P3 in rRNA transcription in the nucleolus

The phosphoinositide 3-kinase (PI3K) signalling pathway is one of the most altered pathways in human cancer. It is a complex pathway, when considering that ubiquitously expressed isoforms regulate different routes with their own cellular outcomes. The majority of research has been focused on the PI3K p110α isoform, due to frequent findings of mutations in different human cancers. The PI3K p110β isoform has received less attention, however it has been found to be tumorigenic when overexpressed in its wild-type form, which has been linked to its lipid kinase activity. Our group has focused on p110β and shown that the mRNA for the p110β-coding gene PIK3CB are elevated in endometrial cancer cell lines. Also, our group has demonstrated that the nuclear levels of PtdIns(3,4,5)P3 (PIP3), the lipid product of p110β, is high in the endometrial cancer cell line RL95-2. Both p110β and PIP3 have been found to localise in the nucleus and nucleolus of some cell lines, though their role in the nucleolus is still unknown.During this study, p110β and its lipid product PIP3 was confirmed to localise within the nucleoli of RL95-2 cells. To determine what the purpose of p110β and PIP3 is within nucleoli and ribosomal biogenesis, p110β was inhibited by Kin193, a specific p110β inhibitor. The results show a decrease in 47S rRNA transcription, the initial ribosomal transcript. Labelling nascent rRNA with and without Kin193 showed that inhibiting p110β indeed led to decreased rRNA fluorescent signal in human cells. These findings further validate Kin193 as a potential anti-cancer drug in patients with endometrial cancer. The experiments were also performed on two mouse cell lines, one p110β wild type (WT) and a p110β catalytic mutant (KI), though there were no significant differences between them.For PIP3 to function as a signalling lipid, it must bind and recruit proteins. During a PIP3 lipid pull-down from the nuclei of HeLa cells, our group found a cohort of potential PIP3 effector proteins. One of these was poly(ADP-ribose) polymerase I (PARP1), which is involved in single stranded DNA break repair, amongst other roles. PARP1 has also been found to localise in the nucleolus, along with PIP3, and PTEN-deficient endometrial cancer cells have been shown to be sensitised to PARP1 inhibition. PARP1 contains multiple KR-motifs, which are known to bind PIP3. The results show that fragments 1, 2 and 3 bind a variety of lipids, including PIP3. Fragment 3, which contains a KR-motif, was also analysed in NMR spectroscopy, where the KR-motif was confirmed to be part of PIP3 interaction.


Ranveig Seim Brekke

Ranveig Seim Brekke, Anders Molven, Bente B Johansson, Karianne Fjeld

Characterization of insertion variants of the carboxyl-ester lipase (CEL) gene - A role in pancreatic disease?

Carboxyl ester lipase (CEL) is a digestive enzyme that is mainly expressed in the acinar cells of the pancreas. The CEL gene is highly polymorphic due to a variable number of tandem repeat (VNTR) region in the last exon. Diseases that are associated with CEL are related to alterations in the VNTR region. Single base insertions in the VNTR cause a premature stop codon and a truncated protein. It is not clear whether these insertions are linked to pancreatic disease.In this study, we wanted to investigate the location of the insertions and the frequency by sequencing human DNA samples. We further aimed to test tail-specific antibodies for the normal CEL protein (CEL-WT) and the CEL insertion variants (CEL-INS9 and CEL-INS11) by western blot, immunocytochemistry and immunohistochemistry. Moreover, we wanted to study the impact of CEL insertion variants in a cellular model system, thereby evaluating if insertions in the CEL VNTR potentially could be pathogenic.We sequenced 50 human DNA samples from the Pancreas Biobank at Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen. We noticed five cases with INS9, one case with INS10 and one case with INS12, which results in a carrier frequency of 14 %. However, we observed that the five cases with insertion in repeat 9 all had a VNTR length of 13 on one of the alleles. We further investigated this by sequencing all remaining DNA samples in the Pancreas Biobank with a 13 repeat VNTR length (N=25) and observed that in total, 23 of 31 samples had insertion in repeat 9 and a VNTR length of 13 on one of the alleles.The CEL-WT protein has a C-terminal that ends with the six amino acids, PAVIRF. However, if an insertion occurs in the CEL VNTR it results in a truncated protein and a C-terminal that ends with the sequence PRAAHG. Tail-specific antibodies have been produced towards these C-terminal ends. We tested the tail-specific antibodies by western blotting, immunocytochemistry and immunohistochemistry. The tail-specific antibodies were highly specific in the western blots, but did not display the same specificity by immunocytochemistry and immunohistochemistry.Finally, we studied the effect of insertions in the CEL VNTR on protein secretion and aggregation in HEK293 cells. We observed that the insertion variants were similarly detected4in the lysate and the medium. However, we detected more of the CEL-INS9 variant, than CEL-INS11 and CEL-WT in the pellet fraction.In summary, we have shown that there is an association between insertion in repeat 9 of the CEL VNTR and a VNTR length of 13. We also found that the tail-specific antibodies had the highest specificity in western blot analysis. Moreover, our data indicated that insertions may affect the distribution of the CEL protein in different cellular fractions, thereby implicating that the VNTR region is important for the cellular properties of CEL.