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

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.

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.

Colorectal cancer (CRC) is the second deadliest cancer in the world. Given the heterogeneity of the disease, a substantial percentage of patients do not benefit from the standard-of-care. The ability to identify patients that most or least likely to respond to the generic therapy prior to its implementation would improve the safety and efficacy of the anti-cancer regimen in CRC.The goal of this study was to assess the suitability of patient-derived organoids (PDOs) as in vitro models to evaluate the efficacy of chemotherapy in CRC. PDOs were generated from surgical tumor specimens of rectal cancer patients. Next, PDOs were treated with standard of care chemotherapeutics for rectal cancer, with or without neoadjuvant radiotherapy, and a commercial cell viability assay was used to assess drug response. PDOs were classified according to the consensus molecular subtype (CMS) system, based on the whole transcriptome sequencing of PDO-derived RNA before treatment. Clinical information was retrieved from the patient records. In vitro drug responses of PDOs revealed heterogeneous drug sensitivity profiles and highlighted patients who would benefit from standard of care. With respect to the consensus molecular subtype (CMS) classification, CMS2 organoids showed highest response to standard of care, while CMS1 PDOs exhibited a less responsive phenotype. For the majority of the PDOs, neoadjuvant radiotherapy prior to drug treatment had no effect on drug response. On the other hand, in certain cases, neoadjuvant therapy sensitized or desensitized PDOs to standard of care chemotherapeutics. This study adds to the literature demonstrating the feasibility of PDOs as platforms for modelling cancer treatment and highlighting their potential to facilitate progress in personalized medicine. More studies involving complex, co-culture PDO models and designed to better reflect the relevant interplay between tumor microenvironment and the anti-cancer regimen are needed to confirm the predictive qualities of the PDOs and inform clinical decisions in CRC.

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.

The infant gut microbiota maturation is central to infant health and well-being and has been suggested to have important health impacts in adulthood. While most of the previous research and description of the dynamics of the gut microbiota acquisition and maturation focused on the prokaryotic community; recent studies have suggested the importance of other microbial members in the community. Viruses, specifically bacteriophages (phages), are found in high abundances in the gut microbiota, and may influence prokaryotic composition and the microbiota’s trajectory during infancy. Phages can shape the bacterial community by killing their cellular host but also by modulating their bacterial host fitness in the ecosystem. Additionally, phages can carry genes which have no role in the viral replication machinery, but instead modulate the cellular host metabolism. These genes, termed auxiliary metabolic genes (AMGs), are largely uncharacterized in the human gut, in particular in the context of the infant gut microbiota maturation. In this thesis, we explored the diversity and persistence of AMGs in infant gut microbiota from 3 months to 2 years old and identified and characterized changes in the AMGs repertoire during the infant gut microbiota maturation. This project utilized a subset of the broader Finnish Health and Early Life Microbiota (HELMi) birth cohort study, a Finnish prospective cohort on early life microbiota and health. In this study, we leveraged whole genome shotgun metagenomes from faecal samples of 475 infants collected at four time points, as well as samples from their mothers (n = 304) and fathers (n = 123). The viral sequences in these metagenomes were then identified, annotated, and characterized, allowing us to build a large catalogue of bacteriophage sequences, called the HELMi Bacteriophage Catalogue (HBaC). Next, we assessed the presence of putative AMGs in this phage collection and determined their prevalence in the viral community and dynamics during the gut microbiota maturation. The HBaC contains 145,818 unique species-like viral OTUs (vOTUs) of which a majority are temperate phages, classified as Caudoviricetes. Notably, we observed an increase in phage diversity and richness during the infant gut microbiota maturation and an increase in relative abundance of virulent phages. Further, the viral community composition strongly associated with the observed prokaryotic faecal community types (FCTs). In our phage catalogue, 8 % of the vOTUs carried at least one putative AMGs and the Demerecviridae, Autographiviridae and Herelleviridae families proportionally carried the most. The most common metabolic pathways encoded by these putative AMGs found in HBaC were organic nitrogen metabolism, transport functions and carbon utilization. Interestingly, we observed a change in putative AMGs abundance and function during the infant gut microbiota maturation. In the future, additional analysis exploring the influence of early life exposures on phage and AMGs prevalence and dynamics could help unravel the complex interaction between phages and their bacterial host in the dynamic infant gut microbiota.

The prevalence of major depressive disorder is increasing despite the increased standard of living. The prevailing hypothesis to explain depression is that there is an unbalance in information processing in relevant brain networks. Antidepressants (SSRIs, SNRIs) have been shown to induce a juvenile-like plasticity state (iPlasticity) in the brain that helps in rewiring the affected neuronal networks when combined with beneficial environmental stimuli (e.g. psychotherapy). However, it takes weeks to see the beneficial effects of conventional antidepressants on mood and they bring relief only to approximately two-thirds of the patients. There is an urgent need for more efficient and rapid-acting antidepressants. Preliminary data suggests that psychedelics may have potential to respond to this need. It is thought that the therapeutic effect of psychedelics rises from the molecular effects leading to structural and functional plasticity and behavioral changes. Molecular effects of psychedelics are believed to arise from the activation of serotonin 2A (5-HT2A) receptors. It is well established that serotonin 2A (5-HT2A) receptor activation lies behind the hallucinogenic effects of psychedelics, but its role in drug-induced plasticity is currently under debate. Signaling of brain-derived neurotrophic factor (BDNF) through its receptor TrkB has been proposed to underlie the plasticity-promoting effects of psychedelics. However, the mechanisms leading to increased BDNF/TrkB signaling after psychedelic administration are poorly understood. This thesis aimed to study the molecular mechanisms associated with psychedelic-induced plasticity in cortical neuronal cultures. The timeline of the effects of LSD was studied by analyzing the phosphorylation of neurotrophic signaling markers downstream of TrkB (mTOR and ERK) in primary neuronal cultures using Western blot. The role of the 5-HT2A receptor was assessed by combining 5-HT2A antagonist M100907 pretreatment with LSD treatment, followed by Western blot analyses of the same signaling markers mTOR and ERK. The degree of molecular effects of psychedelics was compared to the effects of classical antidepressant fluoxetine. Protein-fragment complementation assay (PCA) was used to evaluate the dimerization of the TrkB receptor in the presence of psychedelics and classical antidepressants. In this context, Western blot was also used to assess the phosphorylation of the plasticity-related BDNF signaling markers ERK and two tyrosines of TrkB receptor (Y515 and Y816) that mediate recruitment of neurotrophic signaling pathways. We found that psychedelic treatment promoted phosphorylation of mTOR and ERK significantly. These effects were not affected by pretreatment with M100907, indicating activation of BDNF/TrkB signaling by psychedelics is independent from 5-HT2A activation. Psychedelics were also shown to cause a significant increase in dimerization of TrkB whereas increase caused by fluoxetine was not significant. Lastly, psychedelics were shown to cause increase in phosphorylation of TrkB and ERK that were comparable to those induced by fluoxetine. These results highlight the potential of psychedelics to promote BDNF-mediated neurotrophic signaling associated with juvenile-like plasticity. Interestingly, the results show recruitment of BDNF/TrkB downstream signaling independently from 5-HT2A activation, which suggests that plasticity-promoting effects of psychedelics might be detached from their hallucinogenic effects.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein with pleiotropic therapeutic effects in several disease models, including Parkinson’s disease (PD), diabetes and stroke. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and many GWAS-based genes predisposing for PD are involved in oxidative stress. MANF has been shown to alleviate oxidative stress in PD models, however, the role of MANF in the antioxidant defense and mitochondrial respiration is not fully understood. By performing bulk RNA sequencing on wildtype and MANF knockout (MANF-KO) human embryonic stem cells (hESCs), we uncovered several genes involved in antioxidant defense to be up- or downregulated in MANF-KO hESC. Here we report that MANF-KO hESCs do not express the evolutionary conserved antioxidant enzyme catalase. We show that the loss of catalase makes the MANF-KO hESCs more vulnerable to hydrogen peroxide indued oxidative stress, and that MANF-KO hESCs have a reduced maximal respiration and spare respiratory capacity. Additionally, we examined if the loss of catalase in MANF-KO hESCs inhibits the differentiation of the cells to human dopaminergic neurons in vitro. We show that MANF-KO hESCs differentiate to TH+/MAP2+ cells despite a sustained deficiency of catalase, but the MANF-KO DA cultures tend to have a reduced spare respiratory capacity and higher basal glycolytic activity. To elucidate the structure-to-function relationship of MANF we utilize molecular dynamics simulations in combination with spin relaxation data from nuclear magnetic resonance spectroscopy. By examining the two-domain nature of MANF in different intracellular conditions we provide insight of the biological relevance of MANF interactions. Here we show that MANF conformational ensemble is more compact than previously reported. By simulating MANF in the presence of calcium and ATP, in neutral and low pH, we observed competitive binding of ATP and calcium to MANF. This study provides novel evidence of a regulatory role of MANF in the cellular antioxidant defense and explores the biological relevance of ATP and calcium binding to MANF.

Rapid identification of infectious outbreaks is critical for the timely deployment of containment measures and for better prevention in the future. However, since surveillance mechanisms can be costly and complex to develop, lower-income countries may lack capacity to monitor prevalence data. Outbreaks therefore tend to spread extensively before authorities are notified. To overcome this, patient data can be collected and analysed more thoroughly to yield actionable epidemiological evidence. Using paediatric patient records from western Rwanda, the primary aim of this Thesis was to develop a syndromic surveillance methodology and accompanying visual dashboard to identify localities and times of year with higher prevalence of priority syndromes. The raw dataset of over 100,000 paediatric consultations was collected between December 2021 and July 2023, spanning 31 health facilities in two districts. A secondary aim was to uncover any statistically significant space-time dependencies in a sub-group of these syndromes, allowing for outbreak detection and evidence-based inference regarding seasonal, geographical, or socio-economic risk factors. The surveillance methodology consists of a pipeline of data pre-processing, binary syndromic variable coding and visual dashboard-building for six categories of syndromes: respiratory, febrile, diarrhoeal, nutritional, parasitic, and CNS. The prototype dashboard was built in PowerBI and comprises interactive graphs and maps to present prevalence results in an easily interpretable format for health policymakers. For the secondary aim, two scan statistics models were applied to detect the presence of significant high-prevalence clusters for six top interest syndromes. For each syndrome, spatio-temporal clusters were deemed significant when the p-value < 0.01. The descriptive visualisations generated from our syndromic data revealed several interesting trends. We found that respiratory and febrile syndromes exhibited clearer seasonal fluctuations, particularly increasing at the start and end of the rainy season. Diarrhoeal and malarial syndromes had strong relationships to health facility location, possibly pertaining to factors like elevation and proximity to the lake. On the other hand, nutritional syndromes appeared similarly prevalent throughout the year and across all health facilities. Our statistical dependency analyses also yielded meaningful results, finding at least one significant space-time cluster in four of the six selected syndromes. These results demonstrate the utility of our surveillance pipeline and visual dashboard for uncovering previously unknown epidemiological trends. If data is consistently collected and consulted by policymakers, outbreaks may be caught early and averted ahead of time. They also suggest that the prevalence of certain syndromes is significantly linked to space-time variables like health facility, village of origin and month of occurrence. In the future, further inferential and predictive analyses, like regression modelling, may be applied to evaluate the independent effect of more specific variables like rainfall, temperature, average income and sanitation levels.

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.

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.