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

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  • Lahtinen, Emilia (2022)
    The early life gut microbiota plays a major role in establishing neonatal immunity and child’s long-term health. However, relatively little is still known about the role of individual bacteria as most studies so far have focused on characterizing the diversity and the individual and temporal variations of the infant gut microbiome. The genus Bacteroides is of particular interest since its abundance is remarkably decreased in infants born via C-section, and relatively little is known about the genomic and phenotypic characteristics of early Bacteroides colonizers despite their anticipated role in the increased morbidity following C-section birth. This thesis aims to contribute to the isolation and characterization of Bacteroides strains from infant and mother stool samples from the Health and Early Life Microbiota (HELMi) cohort study using culture-based and metagenomic approaches. Gram-negative bacteria were isolated from stool samples of 9-week-old infants and identified by Sanger sequencing. In total, seven isolates identified as unique species of Bacteroides, isolated from infant samples or previously from mother samples in late pregnancy, were then characterized for their potential to activate innate immunity in vitro by using HEK-Blue™ hTLR2-hTLR6 reporter cells either as live cells or filtered culture media. Whole genome shotgun sequenced stool metagenomes obtained from 88 infants during the first year of life were leveraged as well. A computational pipeline able to scale to the large size of the dataset was developed to obtain metagenome assembled genomes (MAGs) from the metagenomes. MAGs obtained from Bacteroides species were further taxonomically and functionally annotated. Among the seven Bacteroides spp. isolated from HELMi mother and infant samples, the majority were able to activate the TLR2/6 receptor in vitro. The isolates varied in their potential to activate the receptor via their cell surface molecules and substances they excreted to the culture media. In addition, over 2500 MAGs could be retrieved from the infant metagenomes, of which 18 belonged to Bacteroides spp. Based on predicted open reading frames, majority of the identified proteins of these MAGs were involved in housekeeping functions. Most of predicted proteins involved in cellular metabolism were, however, related to carbohydrate metabolism, amino acid metabolism, and glycan metabolism, stressing the role of Bacteroides spp. in the gut as important and versatile carbohydrate consumers. The results indicate that the Bacteroides spp. colonizing infant gut have an immunologically and metabolically active role. Further work is needed to characterize the molecules responsible for the TLR2/6 activation as well as the nature of the downstream immune responses elicited by the isolated Bacteroides spp.
  • Sirc, Neja (2022)
    Large granular lymphocytic (LGL) leukemia is a rare form of chronic lymphocytic leukemia, that is characterized by clonal expansion of mature cytotoxic T- or natural killer (NK)- cells. As the white cell count in patients is predominantly not distinguishably altered, it often goes underdiagnosed or is diagnosed accidentally. T lymphocytic LGL leukemia (T-LGLL), that makes up 85% of all LGL leukemia (LGLL) cases is characterized by a prolonged expansion of peripheral blood T-lymphocytes, mostly CD8+ lymphocytes. 40 % of T-LGLL patients harbor mutations in the Signal Transducer and Activator of Transcription 3 (STAT3) gene. Y640F mutation of STAT3 (STAT3 Y640F) is the most commonly occurring alteration, present in approximately 17% of all T-LGLL patients, and 42% of patients that bear a mutation in STAT3. Furthermore, a higher prevalence of rheumatoid arthritis (RA) can be observed in patients with mutated STAT3 (26% vs 6%, p=0.02). As T-LGLL patients with the Y640F mutation have a higher incidence of co-occurring RA, we aimed to understand the possible role CD8+ T-cell clones carrying somatic mutation of STAT3 may play in the autoimmune process. We applied lentiviral vectors to express STAT3 wild type (wt) and STAT3 Y640F in murine and human CD8+ T cells. We were able to show their successful integration into the host genome using droplet digital PCR (ddPCR). ddPCR showed high selectivity in its ability to differentiate between the hosts’ gDNA and virally inserted cDNA. The custom-designed probes showed high specificity for either STAT3 wt or STAT3 Y640F, proving the functionality of the assay. Sensitivity studies provided us with accurate quantification even with the presence of STAT3 wt or STAT3 Y640F cDNA under 1%, displaying successful detection of rare variants in low concentration samples. In our expression studies, using Flow cytometry and Western Blotting (WB), we detected a modest rise in STAT3 expression in the virally transduced CD8+ cells. We hypothesized that the CD8+ cells were successfully transduced, but unable to accommodate sufficient STAT3 expression.To determine the role of Y640F mutation in the migration of CD8+ lymphocytes in different tissues in vivo, we injected lentivirally transduced cells, mixed in a 1:1 ratio (wt:mut), into the mice. Unfortunately, our ddPCR method was not sensitive enough to reliably quantitate the transduced cells in the diverse tissue samples. Consequently, we decided that any further mouse experiments cannot be justified. In conclusion, we present successful integration of lentivirally expressed wt and mutant STAT3 in both human and mouse primary CD8+ T lymphocytes and human peripheral blood mononuclear cells. The successfully constructed and optimized ddPCR assay was not, however sensitive enough for in vivo quantification of the transduced cells. As the lentivirally mediated expression of STAT3 variants was low, new approaches and tools are needed to study the role of STAT3 mutated T cells in the pathogenesis of RA.
  • Koppinen, Tapani Kalle (2019)
    Multiple sclerosis (MS) is a demyelinating autoimmune disease in which peripheral immune cells infiltrate the CNS and damage the insulating myelin sheaths surrounding neurons, creating demyelinated lesions in the spinal cord and the brain. MS is an incurable, life-long disease which causes a range of symptoms resulting from CNS degeneration. Current treatments mostly focus on preventing autoimmune attacks and the formation of lesions, but do not reduce the damage caused by the attacks, or impact the gradual degeneration of the axons of MS patients. This study aimed to establish the potential of MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) as treatments for MS. MANF and CDNF are endoplasmic reticulum (ER) located proteins with unique structure and mode of action. UPR is a cellular stress response that, when triggered by inflammation in MS, can cause the apoptosis of myelinating oligodendrocytes and neurodegeneration. MANF and CDNF are also capable of modulating immune responses and improving regenerative processes in damaged tissues. The capability of these two molecules to protect CNS tissue was tested on mice induced with experimental autoimmune encephalomyelitis (EAE), a disease model for MS. Intravenous injections of MANF or CDNF in two doses were performed every 2nd day for 28 days after disease induction. Behavioral testing (rotarod and open field tests) indicated that both proteins improved motor function before the onset of paralysis. Daily clinical scoring showed a brief therapeutic window after the onset of paralysis, during which MANF and CDNF were able to halt disease progression. Flow cytometry analysis of mice spleens and brains showed no effect on immune cell populations at the end of the 28-day testing period. Immunohistological staining at the end of the experiment showed no differences in levels of neuroinflammation between treatment groups and control mice but showed that treatment with MANF and CDNF clearly reduced the formation of demyelinated lesions over the duration of the disease. These findings suggest the improved motor performances and protection from paralysis provided by treatment by MANF and CDNF may be due to their ability to protect CNS tissue from UPR caused by autoimmune demyelinating attacks. Further research is required to elucidate the mechanics behind this neuroprotective ability, and lead to more effective use of MANF and CDNF.
  • Järvinen, Elli Katariina (2021)
    Ischemic stroke is a complex disease involving multiple pathophysiological mechanisms. To date, many therapeutic intervention strategies such as anti-inflammatory treatments have been tested, but none of them has been successful. Previous studies have shown that mesencephalic astrocyte-derived neurotrophic factor (MANF) improves stroke recovery and increases the expression of phagocytosis related genes. In this study, the phagocytic and inflammatory effect of monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), complement component 3 (C3), adhesion G protein-coupled receptor E1 (ADGRE1), MER receptor tyrosine kinase (MerTK) and mesencephalic astrocyte-derived neurotrophic factor (MANF) on microglia were studied simultaneously for the first time. The phagocytosis related genes were transiently transfected into a microglial cell line and studied in vitro utilizing phagocytosis assay, fluorescence-activated cell sorting, Western blot and enzyme-linked immunosorbent assay. MCP-1, M-CSF and C3a were shown to enhance microglial phagocytosis without inducing a pro-inflammatory response. In addition, MerTK induces phagocytosis and the synthesis of pro-inflammatory cytokines. In conclusion, the real therapeutic potential of MCP-1, M-CSF, C3a and MerTK in stroke treatment should be further characterized and tested in vivo.
  • Paech, Jennifer Bianca (2020)
    Cardiovascular diseases are the leading cause of death globally. Especially pathological cardiac hypertrophy can be a trigger for severe pathological conditions, such as congestive heart failure. Previously, overexpression of vascular endothelial growth factor B (VEGF-B) in cardiomyocytes has been shown to lead to cardiac hypertrophy, but in a reversible, physiological way. Furthermore, VEGF-B overexpression leads to significant expansion of the coronary vascular tree. This study compares transcriptomics of postnatal and adult murine cardiac endothelial cells (ECs) and examines the transcriptional changes in response to VEGF-B transgene, plus the effect of the VEGF-B transgene on recovery of the murine cardiac ECs from myocardial infarction (MI). I analyzed isolated ECs from VEGF-B transgenic and AAV-VEGF-B transduced mice with single-cell RNA sequencing. The markers used for identification of the cell types applies to all experimental groups, although the proportions of cells differ among the conditions. The myocardial VEGF-B transgene promotes EC proliferation during development and boosts endothelial proliferation also in adult mice both in physiological conditions and after MI. Trajectory analysis indicates that ECs from the VEGF-B treated mice follow a distinct trajectory to enter the cell cycle after MI. These results suggest VEGF-B gene therapy as a new tool for coronary vessel remodeling, which could open new perspectives in the prevention and treatment of myocardial infarction.
  • Bobik, Nina (2022)
    Despite recent advances in immunotherapies for lung cancer, their success is still hindered by limited predictability of treatment outcomes in patients, as well as by resistance-conveying tumor mutations such as EGFR. Moreover, due to the vast number of treatment options and their cost, a quick, reliable, and cost-efficient drug screening platform is needed to select the optimal treatments for each individual patient. This thesis focuses on finding the best culture conditions to be used in such a future platform, employing 3D cell cultures and microfluidics to mimic in vivo tumors while saving costs and allowing for high-throughput screening. Image-based analysis showed that culture medium can have significant impacts on both cancer organoid growth and morphology, as well as drug sensitivity to the EGFR-inhibiting drug Osimertinib. Specific medium factors, such as the antioxidant N-acetylcysteine, might be particularly important for the integrity of 3D structures in the platform and help prevent conversion to an adherent morphology. Moreover, flow cytometry analysis of immune cells from pleural effusion samples indicated that medium composition might facilitate creating an inflammatory environment in the platform, and that immune cells should not be cultured longer than one week to maximize their activity. Finally, this thesis compares two microfluidic devices for their suitability to be used in future high-throughput drug-screening applications, by contrasting their ease of handling, applicability in fluorescent imaging-based readouts, and possibility to mimic and study the tumor microenvironment in vitro. The results suggest that the choice of microfluidic device will be dependent on whether microscopy analysis or cell viability assays will be used as the main readout of the drug screening in the future.
  • Ranta, Amanda Katrianna (2020)
    Ex vivo drug sensitivity testing is used widely in studies aiming at personalizing medicine for acute myeloid leukemia (AML) patients. However, different conditions, such as cytokines used in media and cryopreservation of cells, as well as varying readout methods can affect primary cell viability, cell composition and sensitivity results. Such affects have been previously studied in some AML treatments, however, not with flow cytometry or with venetoclax. In this thesis, we studied the responses of AML patients to venetoclax using ex vivo drug sensitivity testing with various settings. We first tested three media and two sensitivity readout methods on 29 fresh primary AML samples to determine the optimal media and method for determining ex vivo drug sensitivity. We then tested these same variables on 16 cryopreserved samples and compared these results to their fresh counterparts. Finally, we applied our platform to clinical use and tested its capability to predict in vivo responses to venetoclax in ten AML patients. Our platform was able to predict venetoclax responses in nine out of ten patients using condition media coupled with a flow cytometry-based method, determined as optimal in the first phase. Sensitivity results as well as cell composition obtained after cryopreservation differed from their fresh counterparts and, therefore, we conclude that cryopreserved samples should not be used in guiding treatment ex vivo. Our results give valuable information about sources of error associated with ex vivo drug sensitivity testing. Consideration of these results when designing preclinical studies will enhance their reliability and relevance. Ex vivo testing could be in the future implemented into clinical practice in guiding treatment, saving society and patients from costs and unnecessary adverse effects.
  • Azam, Shadi (2022)
    Background: Oral contraceptive (OC) use may increase the risk of specific cancers and mortality. The aims of this study were to investigate the association between OC use and its duration with the risk of breast cancer, to examine the overall mortality associated with OC use and its duration, and finally to identify sociodemographic characteristics of OC use. Methods: Data are derived from the Older Finnish Twin cohort consisted of monozygotic and same-sexed dizygotic twin pairs born before 1958. We included N = 9,607 Finnish twin women aged 18 – 49 years old with information on OC use and other covariates. The information on OC use, reproductive, and lifestyle factors was collected using a mailed questionnaire. The information on breast cancer incidence was obtained from the Finnish Cancer Registry and the data on mortality was collected from the national Population Information System and Statistics Finland. We used Cox proportional hazards regression to estimate the association between OC use and its duration with risk of breast cancer and overall mortality while controlling for potential confounders. Also, we used logistic regression to identify sociodemographic characteristics of OC use. All tests of statistical significance were two-sided. Results: A total of 758 women developed breast cancer during median follow-up of 42.6 years. Women who ever used OC had 20% greater risk of developing breast cancer than women who never used (HR =1.20, 95% CI = 1.02 to 1.40, P = 0.02). Women who used OC for more than 5 years had greater risk of developing the disease than those who used OC for less than 2 years (HR = 1.11, 95% CI = 0.85 to 1.46), however, the results did not reach the statistical significance. Mortality did not significantly different between women who had ever used OC with those who had not used OC while controlling for potential confounders. Current smokers and women who consumed alcohol more than 10 gram/day had the highest odds of ever using OC. Conclusion: Our results suggest that OC use slightly increases the risk of breast cancer, however, no evidence from this study indicates that OC use adversely affect long-term risk for mortality.
  • Tonttila, Kialiina (2021)
    Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.
  • Alsaed, Bassel (2022)
    Lung cancer remains the leading cause of cancer death worldwide. Cancer immunotherapies have changed the treatment path in some cancers and even led to favorable clinical outcomes in previously incurable cancer types. However, only a fraction of patients benefit from the current immunotherapies. Even though immuno-oncology has great potential, it is facing several challenges including the lack of biomarkers, unknown mechanisms of therapy resistance, complexity of the tumor-immune interactions, and involvement of the complex tumor microenvironment that significantly affects therapeutic efficacy. It remains a great challenge to predict which patients will benefit from immunotherapies, and current immunotherapies are deemed expensive when compared to the more traditional therapeutic modalities. In this work, we aimed to develop platform to study responses to immunotherapy ex vivo in a personalized manner. The platform could enable the study of immune effects and T cell mediated tumor killing in the absence and presence of immunotherapy and other selected drugs. We demonstrate the utility of our ex vivo platform that has potential for personalized drug testing.
  • Sorri, Selma (2022)
    Diffuse large B-cell lymphoma (DLBCL) represents the most common diagnostic entity of lymphoid malignancies. As only 60% of the patients are cured with the current standard of care R-CHOP immunochemotherapy, the quest for better biomarkers and targeted therapies continues. Non-synonymous mutations in the WWE1 domain of an uncharacterized E3 ubiquitin ligase Deltex-1 have been associated with poor outcomes in DLBCL patients. Thus, to elucidate molecular features underlying this observation, this Master’s thesis set out to characterize the expression and subcellular localization of Deltex-1 in a panel of DLBCL cell lines, and to investigate the interaction partners of Deltex-1 in the activated B-cell like (ABC) DLBCL cell line context. The study aimed to gain further knowledge to understand the role that Deltex-1 plays in the pathogenesis of DLBCL, which could be used for inspecting its future possibilities as a prognostic marker or a drug target. Western blot analysis of the cell lysates revealed variable levels of Deltex-1 expression, especially between the ABC-DLBCL cell lines in comparison to germinal centre B-cell like (GCB) DLBCL cell lines. Western blots of separate cytoplasmic and nuclear fractions of the cells showed that Deltex-1 was expressed both in the cytoplasmic and the nuclear fractions of the cells, and the expression levels were reflecting the levels of the whole cell lysates of the same cell lines. The more exact localization of Deltex-1 was observed with immunofluorescence staining and microscopy of fixed cells from a few chosen cell lines. A distinct plasma membrane localization was detected in an ABC-DLBCL cell line U2932. The protein-protein interaction partners of Deltex-1 in the U2932 cell line were screened using proximity-dependent biotin labelling and affinity purification mass spectrometry. The experiments revealed novel associations between Deltex-1 and B-cell receptor signalling regulators, such as B- lymphocyte antigen CD20 and tyrosine protein kinase Lck. Though additional research is needed to define the functional mechanisms of these interactions, these findings might lead to the discovery of the connection between Deltex-1 and lymphomagenesis. In conclusion, this study provides novel information on Deltex-1 expression in the DLBCL context and describes previously unidentified associations of Deltex-1 with B-cell receptor signalling. Yet, more functional experiments are required to clarify the nature of these interactions.
  • Hotakainen, Ronja (2019)
    Diabetes is a group of chronic metabolic disorders caused by the inability of the body to produce or utilize insulin efficiently. Globally, diabetes affects over 422 million people (WHO 2014) and one third of the patients suffer from diabetes-related complications, which cause a considerable economic burden on the healthcare. Diabetic kidney disease (DKD) is one of the most severe complications, since one in five patients develop end-stage renal disease, which requires dialysis or kidney transplantation for survival. In addition, diabetes is a risk factor for cardiovascular disease (CVD), the most common cause of mortality among individuals with diabetes. Conventional clinical risk factors for both DKD and CVD have been established and include an altered lipoprotein profile, an abnormal glucose balance and hypertension. While the clinical risk factors are fairly well recognized, the genetic background of both DKD and CVD is rather unknown. The aim of this thesis was to study the effects of rare genetic variants altering lipids and other cardiometabolic risk factors and to determine their impact on diabetic complications. This study focused on loss of function and missense variants from whole exome- (N=500) and whole genome sequencing data (N=600) in type 1 diabetics from the Finnish Diabetic Nephropathy Study cohort. Single variant and gene-based association analysis were used to detect lipid-associated genetic variants and suggestive genes involved in lipid metabolism. Meta-analysis of whole exome- and whole genome single variants was performed to increase the sample size and detect additional lipid-associated variants. Three lipid-associated variants were genotyped in a cohort of 3000 patients to confirm the detected associations. Single variant association analysis detected a novel, previously unpublished, 21bp deletion located in the RBM47 gene, which was associated with lower apoC-III serum concentrations. To fully understand the impact of the 21bp deletion in RBM47 on apoC-III, further studies investigating the role of RBM47 in lipid metabolism are requested. Furthermore, single variant meta-analysis detected several lipid-associated variants. We showed that the rs451195 in PPIC was significantly associated with DKD. This study sheds light on the genetic background of diabetic dyslipidemia.
  • Colanglo, Kia Kristiina (2021)
    High-Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological cancer in developed countries. Due to lack of early detection methods or targeted treatment options the mortality has not reduced significantly in decades. Standard treatment includes surgery and platinum-taxane chemotherapy, the treatment is very seldom curative. More studies are needed to understand the biology and mechanisms defining chemoresistance, and to develop more personalized treatment schemes. Cancer stem cells are known to resists chemotherapy and therefore this study focuses on the expression of putative cancer stem cell biomarkers in HGSOC. Using Immunohistochemistry (IHC) and Immunofluorescence (IF), a Tissue-Micro Array (TMA) containing 95 patients’ samples was generated and tested for four different potential biomarkers: SOX2, BMI1, C-MYC and ALDH1A1. Scanned slides were evaluated, and results were analyzed using Rstudio as well as Excel analytics. We report that chromogenic IHC staining of individual markers revealed no major differences between expression and Platinum-Free Interval (PFI). Instead, some of the co-expressions and especially triple expressions analyzed with IF resulted in major difference in PFI. Beyond that, ALDH1A1 and SOX2 were found together extremely rarely, and it indicates that it is possible that these two biomarkers are normally not expressed in the same tumor cells. Further study options as well as possible implications are discussed along with the clinical value of the findings.
  • Dürnsteiner, Pia Karoliina (2022)
    Multiple sclerosis (MS) is one of the most common reasons for neurological disability in young adults, yet the aetiology of the disease remains to be discovered. MS involves an autoimmune reaction in the central nervous system, which results in demyelination, axonal degradation, and inflammation. These result in various symptoms, such as motor and sensory disturbances, cognitive symptoms, fatigue, and problems with balance. MS is chronic and progressive, and medications are used to slow the neuronal damage and reduce relapses. The most evident risk factor for MS is Epstein-Barr virus (EBV) infection, as nearly 100% of MS patients are seropositive for the virus. However, the mechanism how EBV contributes to the disease is not known. A highly sensitive quantitative multiplex PCR method was used to examine reactivation of EBV and eight other human herpesviruses in the saliva of MS patients (n=9) and healthy controls (n=7). Single-cell RNA sequencing methods were used to study the cell composition and expression patterns of cerebrospinal fluid (CSF) in treatment-naïve MS patients at the diagnostic phase (n=4) and in controls (n=4). EBV was found to be shedding in eight out of nine MS patients and in only one control, and the viral load was significantly higher in MS patients. Single-cell sequencing of the CSF revealed that MS induces expansion of antibody producing and cytotoxic cell types. Differential expression analysis found that MS CSF B cells significantly express EBNA1BP2, which plays a crucial role in the replication and partitioning of EBV episomes in infected cells. These results support the involvement of EBV in MS. Better knowledge of the viral role in the onset of MS will be useful in the development potential antiviral drugs and EBV vaccination that could even prevent the disease.
  • Harkki, Juliana Sade Maria (2020)
    Background: Alcohol dependence is a chronic severe substance use disorder that has devastating personal and public health consequences. The efficacy of the current pharmacotherapy options for the treatment of alcohol dependence are modest at best, therefore better alternatives are greatly needed. Lysergic acid diethylamide (LSD) has shown promise in treatment of alcohol dependence in several clinical trials. A sigle high dose of LSD has been suggested to have a treatment effect that last for at least six months, indicating a remarkably better efficacy than the currently available methods. LSD itself has been reported to have a low addiction potential. In mouse models, acute LSD has been demonstrated to reduce ethanol consumption. Yet, the mechanism of action behind these effects has remained largely unknown. LSD is an agonist of serotonin’s 5-HT2A and 5-HT2C receptors which have been shown to modulate the dopaminergic activity of the reward circuitry, a crucial brain area in the initiation of addiction. Intracranial self-stimulation (ICSS) is a procedure for a quantitative assessment of reward behavior in animal models. In ICSS, laboratory rodents self-administer electric stimulation to the dopaminergic pathways of the reward circuitry inducing a reinforcing effect similar to drug reward. Aim: The aim of the current body of work was to use ICSS to assess the acute effects of LSD on reward behavior in C57BL/6JRj mice. This was done to improve the understanding of the mechanism of action of LSD and to evaluate whether the ethanol-consumption-reducing effect of LSD in mice is mediated through the reward mechanism. Methods: Bipolar electrodes targeting the medial forebrain bundle were implanted in the brains of C57BL/6JRj mice in a stereotaxic surgery. The animals were trained to acquire the self-stimulation in the discrete-trial current-intensity procedure. First, the possible dose-dependent acute effects were tested with three different doses of LSD. Next, the acute effect of LSD on amphetamine-induced changes in ISCC were tested. Lastly, a small preliminary test on the effects of LSD on lipopolysaccharide (LPS) -induced changes on ICSS were conducted. Results and conclusions: Acute LSD did not affect reward behavior in ICSS on any of the tested doses. Accordingly, LSD did not affect the facilitation of ICSS induced by acute amphetamine. The results of the LPS experiment were likely to be skewed by the development of tolerance to LPS, therefore the evaluation of the possible effect of LSD was not possible. These findings suggest that the previously reported LSD-induced reduction in ethanol consumption in mice, is not mediated through alteration of the reward mechanism. At the same time, these findings provide further evidence supporting the suggestion that LSD itself does not induce facilitation of the reward circuitry needed for the development of addiction.
  • Saikkala, Minea (2021)
    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.
  • Laukkanen, Liina (2021)
    This study investigated the in vitro and in vivo effects of direct angiotensin II (ANG) receptor type 2 (AGTR2) agonist Compound 21 (C21). The blockade of ANG receptor type 1 (AGTR1) by AGTR1 antagonists has long been associated with antidepressant and anxiolytic effects. Furthermore, it has been suggested that the therapeutic effects of the AGTR1 antagonists are partially dependent on enhancing the signaling through neuroprotective AGTR2. This suggests that as a specific AGTR2 agonist C21 could be used as a potential therapeutic tool to treat mood disorders that would greatly benefit from new effective treatments. Brain-derived neurotrophic factor (BDNF) is a neurotrophic that binds to tropomyosin receptor kinase B (TRKB). This study aimed to test how C21 affects BDNF:TRKB signaling that has been shown to regulate the therapeutic effects of different antidepressants that act on mood disorders. In vitro effects of C21 on BDNF:TRKB signaling were investigated with ELISA in the cortical cell cultures. Acute AGTR2 stimulation significantly elevated the amount of surface TRKB whereas a prolonged treatment of C21 for three consecutive days induced activation of TRKB. Similarly, combined treatment of C21 and a non-therapeutic treatment of BDNF induced TRKB activation, further linking the AGTR2 stimulation by this compound to the BDNF:TRKB signaling. In vivo effects of C21 on conditioned and unconditioned fear were investigated in mice by using contextual fear conditioning and elevated plus-maze (EPM) respectively. The therapeutic effect of C21 protected mice from conditioned fear but failed to provide similar results for unconditioned fear in the EPM. Interestingly, these stress-protective effects of AGTR2 stimulation were lost in the BDNF-deficient animals. To conclude, AGTR2 stimulation by C21 elevates the amount of surface TRKB that enhances the BDNF:TRKB signaling similar to antidepressants, which further leads to the therapeutic, stress-protective effects. Furthermore, these AGTR2-induced effects were absent without exposure to stress or when BDNF was diminished, indicating that both fear conditioning and BDNF are crucially involved. This study suggests that the AGTR2 is indeed a potential therapeutic target for treating mood disorders, and that in the future C21 could be translated for this use. To achieve this result, the cell types that regulate this effect need to be identified.
  • Launonen, Hanna (2020)
    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.
  • Sokka, Laura (2021)
    Lactase is a digestive enzyme, and its principal function is to break down lactose, a disaccharide found in milk. The main site for lactase expression is the intestines, however, it is also expressed in other tissues, including the brain. Because the primary substrate, lactose, is not present in the central nervous system, it can be assumed that lactase serves another function besides lactose breakdown outside the digestive system. In C57BL/6NCrl mice, lactase expression is higher in the ventral hippocampus after chronic social defeat stress in comparison to controls. This suggests that lactase expression is to some extent affected by stress. Although lactose metabolism is only necessary for mammals, some other animals – including the zebrafish (Danio rerio) – possess a gene that codes for lactase. Research on the zebrafish lactase gene is scarce, and the expression pattern of its two transcripts, the primary lct-201 and the secondary lct-202, is not known. This study focused on measuring lactase expression in adult wild type zebrafish – both on the gene and on the protein level as enzymatic activity. The effect of stress on lactase expression was also examined by applying two different stress models: netting handling stress as a form of physiological stress, and chronic social defeat as a model for psychosocial stress. Real-time polymerase chain reaction (q-RT-PCR) showed lct-201 expression in all five tissues investigated in this study – the forebrain, the mid-hindbrain, higher intestines, lower intestines, and skeletal muscle, whereas lct-202 was only expressed in the higher and lower intestines. The expression level of lct-201 in the muscle was only fifth of that in the lower intestines. Lactase activity assay on the whole brain and whole intestines displayed enzymatic activity in both tissues, with the activity in the intestines being more than seven-fold compared to the brain. q-RT-PCR on both stressed and control fish whole brain and intestines revealed higher lactase expression in the stressed fish intestines, however, the effect was only seen with a primer pair targeting both transcripts simultaneously, and not for either of them separately. Lactase expression was on average approximately 40 % higher in physiologically and 55 % higher in psychosocially stressed fish in comparison to their respective controls. Neither physiological nor psychosocial stress affected lactase expression in the brain. These findings suggest that the two zebrafish lactase transcripts have distinct expression patterns, which might imply different functional roles for lct-201 and lct-202. Furthermore, these results indicate that lactase is expressed in the zebrafish brain, suggesting that it has a specific function in the central nervous system. Based on the findings in this study, lactase gene expression might be connected to experienced stress – both physiological and psychosocial.
  • Bütün, Felicia (2021)
    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.