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

The main development of T cells takes place in the thymus. However, T cells that have just exited the thymus, called Recent Thymic Emigrants, are not yet fully matured and thus have different characteristics and functionalities than mature peripheral T cells. Several biomarkers exist for these Recent Thymic Emigrants in CD4 positive cells, but not in CD8 positive cells. Therefore, human (CD8) RTEs are understudied, despite their critical role in the developing immune system, holding back research in the field of immunological biology, autoimmune disease, cancer, aging and more. Therefore, this thesis aims to identify biomarkers of CD8 positive Recent Thymic Emigrants. To this extent, nearly egress-ready thymocytes, fully egress-ready thymocytes and naïve PBMCs were obtained from pediatric donors, and both bulk and single cell RNA sequencing was performed to provide insight into the gene expression in these stages of T cell development and obtain candidate markers. These candidate markers can then be validated using flow cytometry and T cell receptor excision circle assays (the current golden standard for measuring Recent Thymic Emigrants). Both these methods have been developed and optimized, and some potential candidate markers have been identified. However, some shortcomings like limited sample size and missing single cell data need to be addressed, before the described methods of testing candidate markers can be applied and any markers can be exposed.

Kaposi sarcoma (KS) is a cancer characterized by spindle cells thought to originate from endothelial cells. The most aggressive form is AIDS-related KS, which is one of the most common cancers in Sub-Saharan African regions with high HIV burden. The causative agent of KS, Kaposi Sarcoma Herpesvirus (KSHV), displays a biphasic lifestyle of latent and lytic phases. During the latent program, the viral DNA is tethered to the host chromatin without integration, through the viral protein LANA. This mechanism allows the KSHV genome to exploit the human cellular replication machinery to replicate and maintain viral DNA genomes. Recent, unpublished findings suggest that this mechanism is supported by interactions between LANA and the host transcription factor SOX18. The Ojala lab has shown that this key developmental factor SOX18 is significantly upregulated in KSHV-infected cells. Importantly, SOX18 inhibition by a small molecule inhibitor SM4 decreases the intracellular viral DNA copies and characteristic infected spindle cell phenotype. However, the mechanism behind the upregulation of SOX18 is not understood. Additionally, a previous BioID screen for SOX18 protein interactions discovered that an important structural protein and the ATPase of the host chromatin remodeling cBAF complex, ARID1A and BRG1, respectively, were the highest confidence interaction partners in KSHV-infected cells. Therefore, it was hypothesized that SOX18 and the cBAF complex, and their interactions, co-operate in viral DNA replication and could represent promising therapeutic targets for KS and other SOX18-dependent diseases. This study found that host signaling pathway NF-κB may be responsible for SOX18 upregulation. A BrdU pulldown assay with ectopic SOX18 expression showed enhanced KSHV DNA synthesis, which could be hampered with SM4 inhibition. These results further elucidate the role of SOX18 in viral genome replication. Furthermore, proximity ligation assays and immunofluorescence were performed to investigate interactions between SOX18, KSHV LANA and cBAF in KSHV-infected cells. Cell viability assays, protein expression by Western blotting, and KSHV genome copy quantification upon treatment with cBAF inhibitors were performed. In addition to SM4, inhibition of cBAF led to a regression of the spindle cell phenotype and reduced KSHV genome copies and showed that KSHV-infected cells are sensitized to cBAF inhibition. Infection with a constitutively latent virus supported these results. This thesis sheds light on the mechanism of SOX18 interaction with LANA and host chromatin remodeler in viral latent DNA replication and provides a deeper insight into possible future therapeutic strategies for KS.

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.

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.

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.

Liver transplantation (LT) procedure has evolved tremendously over the last 40 years and is nowadays a standard treatment for both end-stage liver disease and acute liver failure. Improved immunosuppressive medication, surgical methods, imaging and intensive care raise the probability of successful transplantation. Despite good prognosis of survival, acute rejection (AR) is still a significant clinical factor that negatively affects LT outcomes. In the last decade, whole genome-wide incompatibility between recipients and donors has emerged as a potential risk factor in both hematopoietic stem cell and kidney transplantations. The role of non-human leukocyte antigen (HLA) factors in LT outcomes has not been comprehensively established. Thus, the aim of this study is to compare patient and donor genomes pairwise, identify mismatches and further, analyze the effect of these mismatches on AR. After data preparation, 666 LT recipient-donor pairs were included in the analyses. Genotype imputation yielded 8 706 949 variants after quality control. From these variants, 28 225 missense variants were identified and utilized in the genome-wide mismatch analyses. In this study, we calculated genome-wide mismatches of missense variants to identify incompatibilities in all, transmembrane and secreted, transmembrane-only and liver-related proteins between recipients and donors. In addition, we analyzed the effect of mismatches in 40 common deletions and also the sum of deletion-mismatches. We estimated the association of genetic mismatches and time to AR by using Cox proportional hazards model adjusted with recipient and donor age, recipient and donor sex, cold-ischemia time, HLA I eplet mismatch and HLA II eplet mismatch. According to the findings of the study, there is no association between missense variant mismatches coding for either all, transmembrane and secreted, transmembrane-only or liver-related proteins and time to AR. There was also no association between quartiles of missense variant mismatches and time to AR. In the genomic collision model, we found mismatch in deletion-tagging variant rs1523688 to be associated with a decreased risk for AR (adjusted hazard ratio (HR) 0.38, 95% confidence interval (CI) 0.16–0.92, P-value 0.032) and mismatch in rs11985201 to be associated with an increased risk for AR (adjusted HR 1.44, 95% CI 1.04–2.00, P-value 0.030). However, neither of these P-values passed the Bonferroni corrected significance level of 0.00125. No association was observed between deletion mismatch sums and time to AR. In conclusion, we observed no statistically significant association between the genome-wide incompatibility and time to AR in the study cohort of 666 LT recipient-donor pairs. To increase detection power, large-scale studies are required to verify the role of genome-wide mismatches in LTs.

Neuropathic pain is a chronic pain condition, which affects the life quality of almost 10% of the adult population of Europe. Current treatments for neuropathic pain are either not effective enough or have severe adverse effects, which leads to an urgent need for novel and efficient treatment options. Serotonin receptors 5-HT2A have been shown to modulate GABAA receptor activity via KCC2 mediated pathways, which has been suggested to be a possible mechanism behind neuropathic pain. 5-HT2AR agonists LSD and psilocybin activate 5-HT2ARs and acts as a potential novel therapy for neuropathic pain. The aims of this study were to investigate whether 5-HT2AR agonists modulate mechanical allodynia in healthy mice and whether a single administration of 5-HT2AR agonists can reduce mechanical allodynia in mice after SNI. To see whether 5-HT2AR agonists induce mechanical allodynia in healthy mice, baseline response to mechanical stimulus was measured with von Frey filaments with different target forces (0.07 G, 0.16 G, 0.4 G, 0.6 G and 1 G). Mice were treated with LSD, psilocybin, or saline, and after 5 minutes, the von Frey measurements were taken again. The allodynia was induced with SNI, where the common peroneal nerve and tibial nerve were ligated and cut, leaving the sural nerve intact. The development of mechanical allodynia was measured with von Frey filaments before the surgery and on post-operative day 14. On post-operative day 14, after the von Frey measurements, the mice were injected with LSD, psilocybin, pregabalin or saline. After 5 min, post-treatment measurements were performed. The experiments showed that 5-HT2AR agonists do not modulate mechanical allodynia in healthy mice. In SNI mice, psilocybin showed reduced mechanical allodynia between pre- and post-treatment measurements in 0.6 G and in 1 G, while LSD in 0.6 G. When comparing the average effects of 5-HT2AR agonists to allodynia and pain, psilocybin reduced the mechanical allodynia and pain, while LSD only had an effect on pain. The results suggest that 5-HT2AR agonists have analgesic effects after single administration in mice. Overall, this thesis provides insight into the therapeutic potential of 5-HT2AR agonists in the treatment of neuropathic pain and provides interesting viewpoints for the future research in the field.

Hepatoblastoma (HB) is the most common malignant pediatric liver tumor. Although developed treatments have increased the survival rate of HB patients, 20-30 % of the patients show lack of response to the currently used treatment. Due to rapid growth and insufficient blood flow, solid tumors, like HB, develop areas with low oxygen levels. This condition is called tumor hypoxia. Tumor hypoxia correlates with poor prognosis, higher metastasis rate and resistance to cancer treatments. In response to hypoxia, cancer cells start to express carbonic anhydrase 9 (CA9) via HIF1. CA9 contributes to the maintenance of alkaline intracellular pH. That promotes tumor development, while the increasingly acidic extracellular space promotes tumor cell invasiveness. CA9 has historically been related to carcinogenic processes in a variety of cancers, and it has been hypothesized that it may be a possible target for cancer therapy. Substances for the inhibition of CA9 already exist, and one of them, SLC-0111, has given promising results in phase I clinical trials as well as several pre-clinical studies. The aim of this study is to describe the expression of CA9 in HB and study their relationship to pathological features in cellular level, especially the viability and migration of cancer cells. Another purpose of the study is to investigate the effect of SLC-0111 on HB cells and to consider its significance as a potential treatment. CA9 is expressed in two different HB cell lines, HUH6 and HB-303-LEF when exposed to hypoxic conditions. Cells show more aggressive behavior under hypoxic conditions. HB-303-LEF migrates more abruptly in hypoxia compared to normoxic cells. Cells from both cell lines in spheroid modeling, in which CA9 was inhibited by SLC-0111, showed lower viability. HB-303-LEF also showed slower migration in hypoxia where it had received the SLC-0111 inhibitor compared to hypoxic cells. HUH6 results were parallel but not statistically significant. Cells behave more aggressively in hypoxia. The use of SLC-0111 contributes to the reduction of viability and migration. It can be considered an interesting discovery for future treatments against HB.