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Coming dissertations at MedFak

  • Animal genomics – gene discovery and gene characterization Author: Rakan Naboulsi Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423787 Publication date: 2020-11-25 10:54

    This thesis involves two projects. The aim in the first project was to identify genomic regions associated with spontaneous autoimmune thyroiditis (SAT), which is a hereditary autoimmune disease that affects the obese strain (OS) of chicken, an animal model for human Hashimoto’s thyroiditis (HT). In the second project, we study ZBED6, a highly conserved protein unique to placental mammals. Here we explore the functional significance of ZBED6 in general, and its effect on the regulation of Igf2 and miR483 in specific.

     

    To identify genomic regions predisposing to SAT, a nine-generation intercross between OS and their wild ancestor, the red junglefowl (RJF), was previously generated. In paper I, we developed a cell-based assay to phenotype the F9 chickens by measuring the TSH levels in their serum. We found that 1) SAT is similar to HT in the sense that the serum-TSH levels increase in affected individuals, and 2) that TSH levels in SAT-affected chickens starts to increase after 20 weeks of age. In paper II, a whole genome sequencing experiment was performed to compare a healthy and a severely SAT-affected groups of chicken. This analysis revealed 12 genomic loci to be significantly different between the two groups.

     

    In the second project, we utilized a mouse myoblast cell line, C2C12, to characterize the function of ZBED6. In paper III, we affect ZBED6 function, by either mutating its binding site in Igf2 (Igf2dGGCT), or by completely knocking it out (Zbed6-/-). Functional analysis of the mutant cells revealed that ZBED6 overexpression induces cell cycle arrest and apoptosis, that ZBED6 directly affects mitochondrial activity, and that ZBED6 in myoblast cells mainly exerts its effect through regulating Igf2. In paper IV, we use ZBED6 knock-out and knock-in mice to investigate the effect of ZBED6 on the regulation of miRNA expression. We found that ZBED6 is not a general regulator for miRNA, with the exception of miR483, which exists in an intron of Igf2. Thereafter, we generated miR483-/- cells, using the Igf2dGGCT cell line. In this analysis we found that the main function of miR483 in myoblast cells is to regulate the expression of Igf2, and that ZBED6 partially regulates Igf2 through regulating miR483.

  • Deciphering molecular mechanisms in the evolution of new functions Author: Hind Abdalaal Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423421 Publication date: 2020-11-23 12:17

    The evolution of new genes and functions is considered to be a major contributor to biological diversity in organisms. Through de novo origination, “duplication and divergence”, and horizontal gene transfer, organisms can acquire new genetic material that can evolve to perform novel functions. In this thesis, we investigate how functional trade-offs, “gene duplication and amplification”, and neutral divergence contribute to the emergence of a new function from a preexisting gene.

     In Paper i, we investigated the ability of Salmonella enterica to compensate for the loss of peptide release factor 1 (RFI) and the potential of peptide release factor 2 (RF2) to gain a new function to replace RFI. The amplification of RF2 and accumulated mutations within RF2 were the main evolutionary routes by which the fitness cost was restored. However, further characterization of the evolved RF2 showed a toxic effect to the cell due to the termination on tryptophan codon (UGG). This evolutionary trade-off - which we named “collateral toxicity” - might present a serious barrier for evolving an efficient RF2 to replace RF1.

    In Paper ii, we determined whether we could evolve a generalist enzyme with two functions (HisA + TrpF) from the specialist enzyme HisA, which can only synthesize histidine. In a previous study, we showed that HisA evolved a TrpF activity through strong trade-off trajectories. Here, we developed a selection scheme in which we constantly selected for keeping the original function (HisA), while intermittently selecting for the new function (TrpF). Our results showed that all evolved lineages shared the same “stepping stone” mutations in the hisA gene, which enabled them to grow well in the absence of both histidine and tryptophan. Additional accumulated mutations in the hisA gene gave the strains an increased ability to grow without both amino acids, indicating that the HisA enzyme evolved to be an efficient generalist.  

    In Paper iii, we explored how differences between diverged orthologs influence evolvability. We generated artificial orthologs using a random mutagenesis approach. First, we screened for orthologs with a lower HisA activity and then selected for orthologs with a higher HisA activity; these steps were repeated in alternating rounds. We then tested the ability of each ortholog to evolve  TrpF activity. As expected, the orthologs showed varying abilities to evolve the new function. In particular, orthologs with higher HisA activity levels showed both a higher potential to evolve the new function and a higher TrpF activity when they acquired the new function. 

  • The SHB adaptor protein in human and murine leukemia Author: Maria Jamalpour Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-420489 Publication date: 2020-11-20 13:47

    The SHB adaptor protein operates downstream of tyrosine kinase receptors. It has been found previously that Shb deficiency in hematopoietic stem cells (HSCs) results in less proliferation and failure to maintain the myeloid compartment over time. Based on these findings, I have investigated the effects of Shb deletion on the development of different types of murine as well as human leukemia.     

    The absence of Shb exacerbated p210 BCR-ABL induced myeloid leukemia due to an elevated level of focal adhesion kinase (FAK) activity and high expression of the cytokines Interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF), resulting in an increased number of neutrophils in the blood.

    When the effects of the SHB gene in human leukemia were investigated, it was found that SHB gene expression relates to the survival of patients suffering from acute myeloid leukemia and acute promyelocytic leukemia. Additionally, a group of genes co-expressed with SHB demonstrated immunological phenotypes and vascular and apoptotic characteristics.

    These findings prompted further investigations of the effects of Shb deficiency on neutrophilic, B-cell, and T-cell leukemia. Wild type or Shb knockout bone marrow cells expressing the oncogenes (CSF3R-T618I, p190 BCR-ABL, and Kras-G12D) were transplanted into wild type recipients. As a result, a more aggressive disease with shorter latency and decreased IL-6 and G-CSF expression was observed in the neutrophilic model whereas lower expression of IL-7 and C-X-C motif chemokine 12 (CXCL-12) was observed in the B-cell model in the absence of Shb. In the B-cell and T-cell leukemia models, lack of Shb altered disease characteristics without affecting latency.

    The effect of Shb deficiency in the progression of MLL-AF9-induced mixed-lineage leukemia was also investigated. Bone marrow cells from wild type and Shb knockout mice were transduced with the MLL-AF9 gene. The absence of Shb resulted in a higher cell proliferation rate in in vitro culture, whereas in an in vivo setting, latency was increased compared to the wild type counterparts. Alterations in cytokine expression, especially IL-6 and IL-1b, constituted a likely explanation for this difference.

    In conclusion, SHB plays a pleiotropic role in shifting phenotypic responses in different leukemia models. Therefore, personalized medicine treatment should be planned based on the type of leukemia in relation to SHB gene expression.

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