Browsing by master's degree program "Translationaalisen lääketieteen maisteriohjelma (Translational Medicine)"

Lung cancer is one of the most common and deadliest cancers worldwide, but the mechanisms behind different types of lung cancer are still poorly understood. Non-small cell lung cancer makes up 80% of lung cancers, and some epigenetic mechanisms have been proposed for it. Epigenetic modifications are a way of influencing the expression of genes by inhibition or activation. PRC2 is an epigenetic modulator that catalyses the formation of methyl groups on histone 3 lysine 27, which is an epigenetic mark with repressive nature. PRC2 has been proposed to be downstream of AMPK, an energy sensor of the cell, which is phosphorylated by LKB1 under energy stress conditions. Inactivating mutations in LKB1 are known to cause and worsen non-small cell lung cancer, and the overexpression of EZH2, the catalytic subunit of PRC2, has similar effects. Therefore, establishing a novel downstream mechanism linking LKB1, AMPK, and PRC2 together could explain one mechanism for NSCLC tumorigenesis. Changes in metabolism are a feature of cancer cells, and this pathway could also link energy stress and cancer together. Mouse embryonic fibroblast and H358 cell lines overexpressing wild type EZH2, mutant EZH2 and GFP were generated and treated with the glycolysis inhibitor 2-deoxyglucose to study the effects of energy stress. Levels of histone methylation and phosphorylation statuses of AMPK and its downstream target ACC were assessed with Western blotting, and expression levels of potential PRC2 target genes with RT-qPCR. The study setting proved to be functional for the response of AMPK to energy stress conditions, as both AMPK and ACC were phosphorylated in the presence of 2-DG. In mouse embryonic fibroblasts, PIM1 showed different gene expression with wild type and mutant EZH2, suggesting that its activation would be regulated through the phosphorylation of the T311 site of EZH2 during energy stress. The results from histone methylation statuses did not follow the hypothesis, possibly because of the lack of specificity of detecting global H3K27me3. Other target genes besides PIM1 in MEFs did not show significant changes in expression level. Considering that the incorporation of the mutant EZH2 into PRC2 complexes was not validated, additional research would be needed to confirm or deny the explained mechanism between PRC2 and AMPK.

High blood pressure has been shown to increase intestinal permeability, which is associated with several diseases such as inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS). Recently, renin-angiotensin system (RAS) components, the main regulators of blood pressure, have been found to be produced also locally in several tissues e.g. intestine, heart and brain. In the intestine, the local RAS participates in the regulation of inflammation. However, little is known of the functionality of the local intestinal RAS components and their involvement in the regulation of the intestinal barrier function. Conventional angiotensin-converting enzyme (ACE)-angiotensin receptor type 1 (AT1R) axis and the alternative angiotensin-converting enzyme 2 (ACE2)- Mas receptor axis have opposing functions in the body. The disbalance between the two pathways has been associated with different pathophysiological processes. This in vitro study aimed to assess the direct effect of proinflammatory angiotensin II (Ang II) via the activation of AT1R on intestinal permeability of 8-10-week-old Balb/c mice. Jejunum and colon samples were collected and mounted to the Ussing chamber with different Ang II concentrations or a combination of Ang II and AT1R antagonist losartan. Angiotensin (1-7) (Ang (1-7)), a Mas receptor agonist, was also examined for its possible beneficial effect on reducing gut permeability and on alleviating the harmful effects of Ang II. Transepithelial resistance (TER) and short-circuit current (Isc) were analyzed as indicators of the permeability. Given the importance of the tight junction proteins to paracellular permeability, the levels of occludin, claudin-1 and claudin-4 were determined with Western blot from jejunum and colon samples incubated for 75 min under similar conditions used in the Ussing chamber. Ang II increased the paracellular permeability via the activation of AT1R in jejunum. Additionally, Ang (1-7) tended to alleviate the negative effects of Ang II. Changes in tight junction protein levels partly were in accordance with the permeability findings. The fluorescence permeability marker (9Å) used mimics the size of disaccharides. There is evidence that TER measures the changes in the paracellular ion and water transport and as no alterations in TER values were observed we suggest that Ang II increases the flux of macromolecules via the activation of AT1R in jejunum. No significant changes in permeability or in the electrophysiological values were observed in colon after incubation with peptides.

Lymphangiogenesis is crucial for the formation of a fully functional lymphatic vasculature. Dysfunctional lymphatic vasculature is associated with many pathologies including lymphatic malformations, lymphedema, and inflammation. This thesis aimed to elucidate the function of the lymphatic endothelial cell expressed β1-integrin in postnatal lymphangiogenesis in mice. To investigate this, Itgb1 gene was conditionally deleted from the lymphatic vasculature of mouse pups at postnatal day two, using the Cre-Lox recombinase system. Ears of the mice were collected at postnatal day twenty-one, and immunostained for lymphatic vessel, mural cell, and extracellular matrix markers. Relative gene expression analysis was performed from whole ear lysates and isolated endothelial cells. Mice lacking Itgb1 gene presented a disorganized dermal lymphatic vascular network, characterized by dilated capillary and collecting vessels. Collecting vessel specification was abnormal showing persistent LYVE1 expression, lack of V-shaped lymphatic valves, and complete loss of smooth muscle cell coverage. Relative gene expression analysis of isolated endothelial cells revealed a decrease in Pdgfb, a gene associated with smooth muscle cell recruitment. These results indicate a significant role for β1-integrin in postnatal lymphangiogenesis and highlight the complexity of postanal lymph vessel formation. Nevertheless, further studies are needed to specify the mechanisms behind this phenotype. Increasing knowledge in this field is important, because pathologies related to lymphangiogenesis can cause debilitating symptoms and therefore there is a need for therapeutic options.

New treatment methods are urgently needed for glioblastoma (GBM), the most common malignant primary brain tumor in adults, that currently lacks any curative treatment. Targeted therapeutic approaches have shown promising results already, but common drug delivery vehicles come with efficacy issues and are restricted by their safety and toxicity profiles. Exosomes, cell-produced nanosized vesicles, have emerged as a new potential carrier for gene therapies in cancer treatment due to their natural material transport properties, biocompatibility, and specificity in transporting cargo to the target cells. These extracellular vesicles have the additional advantage of being able to cross the blood-brain-barrier (BBB), which makes them especially valuable for brain malignancies, such as glioblastomas. So far, gene therapy approaches in exosomes have focused on RNA in cancer treatment, but research findings are limited with plasmid-based gene therapies using exosomes. The main concern has been whether the increased plasmid size would decrease the transfection efficiency of the plasmid into the exosomes. This study aimed at setting-up exosomes as plasmid-based gene therapy nanocarriers. To achieve this, different plasmid-based gene therapies were tested, including the targeting of common aberrations of GBM cells to impair proliferation and the use of cytotoxins to induce apoptosis in the target cells. The plasmids were transfected into exosomes and subsequently inoculated into patient-derived glioblastoma cells with the aim of decreasing the number of glioblastoma cells. The findings of this study demonstrate a successful set-up of an exosome-based gene therapy in patient-derived glioblastoma cells by using engineered HEK293FT cell derived exosomes consisting of a plasmid-based combination gene therapy encoding the cytotoxins Granzyme B and Diphtheria toxin fragment A.

The APOE4 gene variant of apolipoprotein E (apoE) has been identified as a predisposing factor for late-onset Alzheimer’s disease (AD). ApoE is known to interact with one of the classically characterized hallmarks of AD, the amyloid-beta (Aβ) protein. Aβ activates the classical complement pathway by binding to C1q that may sustain inflammation, hamper Aβ clearance and therefore promote accumulation of Aβ deposits in the brain and affect the integrity of the blood-brain barrier. Depending on context, binding of apoE to C1q can either activate or inhibit complement activation. ApoE can also interact with the complement regulator factor H (FH) which colocalizes with C1q on Aβ plaques. The interactions between these proteins and their role in the AD pathogenesis has not been fully explored. This thesis aimed at unraveling the complex formation between Aβ1-42, apoE, FH, and C1q, and their role in complement activation to provide novel insights on the mechanisms contributing to AD pathophysiology. By conducting enzyme-linked immunosorbent assays and Western Blotting of native PAGE, I was able to show that FH bound to apoE and forms a complex with Aβ1-42/apoE in an isoform- specific manner (apoE2 > apoE3 > apoE4) whereas C1q bound all Aβ1-42/apoE complexes with same affinity. FH did not bind Aβ1-42 alone, thus the results indicated that binding of FH in Aβ1-42/apoE complex took place via apoE2 and apoE3. In the absence of FH and C1q, immobilized Aβ1-42/apoE4 complexes led to higher serum complement activation levels when compared to Aβ1-42/apoE2 and Aβ1-42/apoE3 complexes or Aβ1-42 alone. C1q activated serum complement especially in the presence of Aβ1-42/apoE3 and Aβ1-42/apoE4. Further functional analysis with flow cytometry revealed that in fluid phase the presence of FH could restrict Aβ1-42-induced complement activation, the subsequent formation of C5a and activation of C5a receptor (C5aR) expressed on Human embryonic kidney (HEK) 293 cells. The presence of all apoE isoforms on Aβ1-42/C1q complexes led to reduced C5aR activation. Surprisingly, however, the reduction was the largest with apoE4, and the presence of C1q on Aβ1-42 alone could also decrease C5aR activation. The results of this thesis elucidated the effects of Aβ1-42, apoE, C1q and FH in complement activation and proposed a mechanism by which FH regulates complement activation and cell-mediated inflammation through isoform-specific binding to apoE associated with Aβ1-42. Further work is required to analyze the functional effects of these complexes, whether Aβ1-42, apoE, FH, and C1q can all bind simultaneously to form a large complex and whether this complex affects C5aR activation. This study provides important insights on how APOE4 may predispose to the neurotoxicity and neuroinflammatory changes in AD pathophysiology early in the disease process. Yet, the findings are only preliminary and therefore further studies are required to validate these results and to demonstrate whether promoting FH binding to apoE/Aβ complex could provide a novel target for AD therapy.

IER3IP1 is a protein located in the endoplasmic reticulum (ER) transmembrane, and it is highly expressed in pancreatic beta cells and developing brain cortex. The loss-of-function mutations in IER3IP1 cause monogenic neonatal diabetes together with brain linked diseases such as epilepsy and microcephaly. The aim of this thesis is to study the role of IER3IP1 in the development and function of human beta cells using hESC-derived pancreatic islets. Using CRISPR/Cas9, IER3IP1 knockout (KO) and IER3IP1 loss-of-function mutation knock-in (KI) hESC clones were generated. For KO, the first exon of IER3IP1 was deleted whereas for KI, the 21. valine of IER3IP1 was changed to glycine. The clones together with their unedited controls (H1), were differentiated into pancreatic stem cell (SC)-islets following the optimized 7-stage differentiation protocol. The differentiation was followed during the protocol and the SC-islets were tested at the end of the protocol. In vitro, IER3IP1 KO-islets contained less beta cells and more alpha cells when compared to the H1-islets, as shown by immunostainings for insulin and glucagon. The beta cells of KO-islet accumulated more proinsulin compared to H1-islets and had significantly higher level of ER-stress shown by elevated ER-stress marker BiP. Moreover, the KO-islets showed drastically lower amount of insulin secretion and diminished insulin content. The IER3IP1 KI-islets did not significantly differ from H1-islets. Thus, this master’s thesis shows that IER3IP1 is essential for maintaining normal ER homeostasis and beta cell function in vitro. In future, these results should be confirmed using in vivo model.

Myelin is a lipid-rich substance wrapped around nerve axons that can be adaptively modified in response to neuronal activity and experience. Recent research has revealed myelination of parvalbumin (PV) inhibitory interneurons, critical for brain oscillations and balance. Defects in PV interneuron myelination have been linked to psychiatric disorders, like schizophrenia. Tropomyosin receptor kinase B (TrkB) signaling has been shown to be important for myelination. Moreover, fluoxetine, an antidepressant, binds to TrkB receptors in PV interneurons, enhancing plasticity. While previous studies support the importance of PV interneuron mediated TrkB signaling for anti-depressant induced neural plasticity, its effect on PV interneuron myelination remains unexplored. The objective of this thesis was to investigate whether TrkB signaling, and fluoxetine affect the overall and PV-interneuron specific myelination in the medial prefrontal cortex (mPFC) in mice. Using immunohistochemical analysis, we assessed myelin changes through node of Ranvier morphology and myelin immunostaining intensity in control and in mice with heterozygous conditional TrkB deletion in PV interneurons (hereafter referred to as TrkB KO), with or without fluoxetine. We found that fluoxetine increases node length in TrkB KO mice, while reduced TrkB signaling shortens paranodes in PV neurons compared to controls. Our findings also depict that fluoxetine and PV-mediated TrkB signaling do not alter the overall myelination of the mPFC. The findings of this work provide mechanistic insights into PV interneuron myelination in the mPFC, with potential implications for demyelinating and psychiatric conditions where PV myelination plays a role.

The gastrointestinal (GI) epithelium is composed of a single layer of cells with a turnover time of only a few days. Due to its location at the barrier between GI tract contents and the underlaying mucosa, the epithelium is constantly exposed to stress such as toxic agents and a variety of pathogens and susceptible to injury. Accordingly, the homeostatic growth as well as repair of injury in epithelium must be efficient and strictly regulated. Misregulated repair of the injured epithelium can lead to pathologies such as chronic inflammation or cancer. Underlying stromal cells such as fibroblasts provide growth factors and other signaling molecules regulating the epithelial cell stemness, differentiation and repair, but the stromal regulatory pathways during regeneration are poorly understood. The aim of this study was to establish a consensus view on the heterogeneity of GI fibroblasts, as well as to map potential epithelium derived signals affecting fibroblast function in homeostatic and injury situations using literature review, in silico approaches, and murine primary intestinal fibroblast culture. Seurat and CellChat R packages were used to perform integration and interaction analyses of six previously published mouse and three human single- cell RNA-sequencing datasets of colonic epithelial and mesenchymal cells isolated in homeostatic and/or inflammatory conditions. Murine primary intestinal fibroblasts were treated with identified potential signaling factors ex vivo and 3’RNAseq was performed to identify transcriptional responses. Both mesenchymal and epithelial cell clusters were identified in the scRNAseq data. Interestingly, similar fibroblast populations could be found in the murine and human data. I identified several epithelium-derived signaling molecules potentially targeting GI fibroblasts and focused on Gas6-Axl pathway and lactate. I confirmed high and specific expression of the Gas6 receptor Axl in intestinal fibroblasts, but recombinant Gas6 failed to induce significant changes in cultured primary fibroblasts. Lactate-treated primary intestinal fibroblasts reprogrammed their transcriptome with main alterations in metabolic pathways and induction of neutrophil-attracting chemokines. In this work I suggest a consensus model for GI fibroblast subpopulations and suggest epithelium derived lactate as a powerful means to reprogram fibroblasts.