Browsing by study line "Cross-disciplinary translational medicine"

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motoneuron disease. ALS is characterized by a progressive loss of upper and lower motoneurons, resulting in muscle atrophy, paralysis and ultimately in death. Approximately 30,000 people die of ALS annually. There is no cure for ALS, and only two drugs - riluzole and edavarone - have been approved for the treatment of the disease. The complex pathology of ALS contributes to the lack of effective treatments. Several cellular pathologies have been suggested to contribute to the pathogenesis, including ER stress, disruption of calcium homeostasis, oxidative stress and excitotoxicity. Here we describe the cytoprotective effects of C-terminal fragments of the novel proteins with neurotrophic factor properties MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) on a toxin model of ALS in vitro. Unlike the classical neurotrophic factors, MANF and CDNF are predominantly localized to the endoplasmic reticulum (ER) and have been shown to alleviate ER stress by keeping the unfolded protein response (UPR) transducers inactive. ER stress is a major component in many neurodegenerative diseases, including ALS, and is a promising therapeutic target for MANF and CDNF. However, the potential of these proteins in ALS treatment remains to be insufficiently described. We used differentiated motoneuron-like NSC-34 cells treated with a range of toxins, modelling different cellular pathologies linked to ALS. After the toxin addition, we treated the cells with MANF and CDNF variants and riluzole and measured the cell viability. The toxin panel consists of tunicamycin, ionomycin and staurosporine. Tunicamycin causes cell death by activating proapoptotic branches of the UPR. Ionomycin is an ionophore and depletes the ER of calcium, thus inducing both UPR-dependent and UPR-independent apoptosis. Less is known about the mechanisms of staurosporine, but it has been shown to induce caspase-3-dependent apoptosis, increase intracellular calcium levels and cause oxidative stress. We hypothesized that both MANF and CDNF variants protect the cells against UPR-dependent apoptosis but not against UPR-independent cell death. We show that MANF and CDNF variants protect the cells against apoptosis induced by tunicamycin, ionomycin and staurosporine. Interestingly, the protein variants mediated the highest protection against ionomycin-induced stress, and they exhibited mild protective effects against staurosporine as well. These findings suggest that MANF and CDNF variants might have a role in maintaining intracellular calcium homeostasis. However, it is possible that staurosporine induces ER stress as well, which would explain the protection conferred by the protein variant. We report that the CDNF variant mediates higher protection at lower concentrations compared to the MANF variant in every toxin assay, whereas the MANF variant mediates higher protection at the highest tested concentration compared to the CDNF variant. We also show that the CDNF variant-mediated protection against staurosporine-induced stress peaked at lower concentrations, and the highest concentration provided distinctively lower, yet significant effect. These data lead us to hypothesize that the protein variants may have a slightly different mode of action, and that they might provide an additive effect when administered simultaneously. We tested a combination of MANF and CDNF variants in cells treated with tunicamycin, ionomycin and staurosporine. However, the combination treatment did not increase the viability more than MANF and CDNF variants independently did. The results answered our questions as well as raised new ones. In the future, the putative calcium-regulating effects of the protein variants should be investigated. The UPR-modifying effects of the drug candidates and toxins need to be assessed by quantifying changes in the UPR marker mRNA and protein expression levels. If it is revealed that the variants have a different mode of action, the possible additive protective effects must be assessed. Finally, a wider toxin panel is needed to fully explore the potential of MANF and CDNF variants in ALS treatment. This study demonstrates the potential of MANF and CDNF variants in protecting motoneurons against several pathological pathways contributing to ALS pathology. However, the mechanisms of action of the variants need further investigation to fully understood their therapeutic potential.

Patients over 65 years are more prone to face difficulties in their care due to not only complex and chronic multimorbid conditions, but also fragmentation of health care services. Current healthcare systems are designed for single-disease conditions that do not align with the care needs of the multimorbid elderly. Multimorbid long-term home care clients use a wide range of home care services but also other health and social services outside of home care, which can lead to fragmentation. The study aimed to map out services used by multimorbid home care clients, present disruptions related to their care, and suggest feasible and scalable solutions for the identified disruptions. Home care professionals (N=10) and clients (N=5) were interviewed focusing on the services and disruptions, and a focus group workshop was conducted for health and elderly care specialists (N=9) for creating the solutions. A total of 38 individual disruptions were discovered, of which 58% (22 cases) mentioned more than one interviewee. The results indicate that multimorbid home care clients faced the most care disruptions when care was prescribed outside of home care and caused fragmentation in the care coordination and sharing of patient information between multiple care providers and actors. Other disruptions were caused by a lack of co-creation of health, inconsistencies within home care protocols, and other factors outside of home care such as rapid workforce turnover. The disruptions discovered were mainly related to healthcare service networks rather than social-related care, due to the healthcare services’ lack of care-related integration at multiple levels and dimensions which was not necessarily needed with social services. Possible solutions suggested by health and elderly care specialists included adapting current healthcare systems to the needs of the home care, improving care coordination through various means, utilizing digital solutions, creating tools to track the status of the client’s care, and increasing co-creation of health with the client. With the current challenges in recruiting and maintaining health care personnel in the home care and constant training of personnel; this study suggests that different types of technological solutions are needed to improve care coordination and integration.

Antibiotic resistance of pathogenic bacteria has increased in recent years. When antibiotics do not work, alternative therapies are developed to prevent major bacterial epidemics. Phage therapy is one of the alternative possibilities to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds or even thousands of phages are required to cover the various bacterial pathogens. For a reliable selection process, high-throughput rapid host range screening of phages is needed to cover the future demands. In addition, collaboration between laboratories is highly important, as the collections of phages of individual laboratories are not broad enough. Thus, the transportation of phages between laboratories is one of the key elements to provide successful phage therapy for patients. The aim of the study was to use gel-based products as protective matrix in phage host-range screening and transportation. The optimal conditions were selected to set a baseline for high-throughput rapid host range screening process, and to set up a ready-to-screen plate assay for phage transportation. In addition, the purpose of the study was to evaluate whether hydrogels could be used as a long-term storage matrix for phages and future product development. Fourteen Escherichia coli phages were used to optimize the liquid culture assay for the E.coli strains. The hydrogel based assays were conducted with two Escherichia and two Staphylococcus phages. For long-term storage, phages were mixed with different consistencies of hydrogels and stored in three different conditions for up to six months at +4oC. The transportation experiments were conducted with phages stored with optimized hydrogel consistencies. The phage viability was measured using liquid culture method. Results show that liquid culture method on microtiter plate is a convenient way to screen bacteriophages in high-throughput assay and that phages can be stored reliably in hydrogel format. When stored in microcentrifuge tubes, phage stability was shown to last for at least six months. When stored as drops on microtiter plate, the phages retained their viability for up to two months. These plates can be used as a robust means for phage transportation.

Mitochondrial disorders form a heterogenous disorder group with symptoms widely varying. They exist because of mutations in mtDNA or nuclear DNA, which encode mitochondrial proteins or regulate their functions. Because mitochondria are present in almost all cells in the human body, symptoms of mitochondrial disorders vary and can manifest in all tissues. The most common manifestation of mitochondrial disorders in adult patients is mitochondrial myopathy, which leads to muscle weakness and fatigue. Pathological findings of mitochondrial myopathy include changed activities in mitochondrial respiratory chain, centrally located nuclei and increased expressions of genes related to integrated mitochondrial stress responses. One of the neurological symptoms of mitochondrial disorders is parkinsonism. Parkinson’s disease is the world’s second most common neurodegenerative disease. Its clinical symptoms include bradykinesia and resting tremor, and its pathological hallmarks include alphasynuclein protein accumulations and loss of dopaminergic neurons in substantia nigra. Despite being described first time over a hundred years ago, the mechanisms behind Parkinson’s disease are still unclear. However, several genes have been associated with Parkinson’s disease. PINK1, encoding PINK1 protein, and PRKN, encoding Parkin protein, are genes, which mutations have been associated to early onset Parkinson’s disease. According to suggested theories, their normal physiological functions include mediating a special form of autophagy, mitophagy, and suppressing mitochondrial antigen presentation. It has been suggested that loss of PINK1 or PRKN could disrupt mitophagy, leading to accumulation of dysfunctional mitochondria in the cell, disrupting its homeostasis and leading to its death. This PINK1/Parkin-mediated mitophagy and its disruption could explain the death of dopaminergic neurons. In addition, loss of PINK1 and Parkin could also activate mitochondrial antigen presentation, which activates autoimmune response and destroys dopaminergic neurons presenting mitochondrial antigens. This suggests that Parkinson’s disease could have autoimmune-like mechanisms behind it. Together, these theories could explain the onset of Parkinson’s disease when PINK1 or PRKN are absent. In this thesis, the main objective was to study, how loss of PINK1 and PRKN would affect on muscle of aged mouse. More precisely, the effects of these genes to mitochondrial homeostasis were studied in terms of mitophagy and mitochondrial respiratory chain activity. In addition, it was studied, if these genes could induce mitochondrial myopathies or affect to existing condition by studying typical signs associated with mitochondrial myopathy, such as changes in activity of mitochondrial respiratory chain subunits, location of nuclei in muscle, changes in lipid and glycogen accumulation and expressions of genes associated to mitochondrial integrated stress responses. Finally, the theory of mitochondrial antigen presentation was studied. The results of this thesis provide more evidence to theory of PINK1/Parkin mediated mitophagy, but also suggest, that these proteins could be compensated. Based on the results, PINK1 seems to be more crucial for healthy muscle, but in already existing mitochondrial myopathy, loss of either of PINK1 or Parkin seems to improve the condition. According to this thesis, both PINK1 and Parkin, and mitophagy seem to be crucial in induction of mitochondrial myopathy. Despite more evidence is now provided to support the roles of PINK1 and Parkin in mitochondrial maintenance, their roles in mitochondrial antigen presentation did not receive support from this thesis.

Bone marrow failure (BMF) is a condition where the bone marrow fails to produce enough functional blood cells leading to peripheral blood cytopenias. Inherited BMF is often a consequence of germline mutations in DNA repair pathway, telomere maintenance, or ribosome biogenesis -related genes and results in up to 20-40% risk of developing a hematological malignancy. Recently, biallelic germline mutations in the gene ERCC6L2 have been identified to cause inherited BMF leading to the accumulation of somatic TP53 mutations and acute myeloid leukemia (AML M6) with dire prognosis. ERCC6L2 is a DNA repair protein that has also been indicated in mitochondrial function. The aim of this thesis was to study the ERCC6L2 protein expression and cellular metabolism in ERCC6L2-derived BMF. The metabolic profile in ERCC6L2-derived BMF was studied in patient-derived fibroblasts using a Seahorse XFe96 Analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at multiple time steps when cells were in standard cell culture (10mM) glucose concentration or low (1mM) glucose concentration. The protein expression was studied in fibroblasts and peripheral blood mononuclear cells (PBMCs) with immunofluorescence assay and Western blotting. The results of this thesis demonstrate a differential metabolic profile in the patient-derived cells. In normal glucose, they thrive exhibiting a higher basal OCR, ATP-related respiration, mitochondrial reserve capacity, and maximal respiratory capacity compared to the control. Contrarily, in low glucose the patient-derived cells struggle and show a lower basal OCR, ATP-related respiration, reserve capacity, and maximal respiratory capacity than the control implying decreased substrate availability in the mitochondrial respiratory chain or mitochondrial dysfunction. Immunofluorescence assay suggests that ERCC6L2 is expressed in the patient-derived cells supporting truncating mutations observed in RNA sequencing. In order to improve the treatment and clinical outcomes in inherited BMF, understanding the role of altered mitochondrial metabolism in ERCC6L2-derived BMF and its progression to AML M6 calls for further studies.

Merkel cell carcinoma (MCC) is a rare, neuroendocrine carcinoma of the skin that is known to have poor prognosis. It is associated with the Merkel cell polyomavirus (MCPyV) and majority of cases harbor this infection. Other risk factors include older age, the male sex, Caucasian skin and increased ultraviolet exposure. Increased lymphocyte invasion into the MCC tumor microenvironment has been reported to infer better survival, but better mechanisms understanding why this occurs this is needed. CCL3 is a chemokine that is implicated in a variety of inflammatory conditions like viral infections and exhibits pro-inflammatory activity mainly through its chemoattractant abilities. In cancer specifically, it functions within the tumor microenvironment by encouraging the trafficking of leukocytes to the tumor site. Transcriptomic data of CCL3 was studied in a cohort of 102 Finnish MCC patients to observe its association with survival, and a variety of clinical-pathological features. The presence of CCL3 in cells was later investigated via immunohistochemistry in 30 formalin-fixed paraffin-embedded Finnish MCC primary tumor tissue samples with varying mRNA expression of CCL3. Macrophages and lymphocytes were found to stain positively for CCL3 and were found exclusively in tumor surroundings. CCL3 was also found to exhibit a MCC-specific survival benefit in patients that harbored higher expression (p=0.031), and was found to be associated with MCPyV positivity (p=0.032). These preliminary findings help establish CCL3’s role in the immune response against MCC and support the need for further studies looking at CCL3 both as a prognostic marker and potential adjuvant therapeutic.

The extraembryonic placenta is composed of trophoblast cells consisting of the proliferative cytotrophoblasts (CTB) and its differentiated subtypes syncytiotrophoblast (SCT) and extravillous trophoblast (EVT). A normal trophoblast development is important as disruptions can lead to pregnancy complications such as pre-eclampsia. Therefore, it is crucial to investigate the underlying causes behind these abnormalities to discover treatments for patients suffering from pregnancy related disorders. Previously placental research was conducted largely on animal models and despite shared conservative pathways with humans, there are differences that exist. Only recently have researchers managed to successfully isolate and culture primary trophoblast stem cells (TSC)s by creating a TSC medium. Due to limited access to placental cells, pluripotent stem cells (PSC)s can be differentiated to TSCs by using the TSC medium. Naïve and primed states are described to be PSCs in different developmental stages, the former representing the pre-implantation state and the latter the post-implantation state. There lacks a consensus on whether both PSC states can be used to generate TSCs that correspond to primary trophoblasts. It has been argued that naïve cells possess more potential to differentiate into TSCs compared to the primed ones. The primed cells have been induced with the bone morphogenic protein (BMP) 4 to generate TSCs. This method is controversial as some suggest the induction resulting in other than TSCs, such as amniotic cells. Therefore, the aim of this thesis was to investigate whether both PSC states could be used to generate TSCs and its subtypes, if at all. Further, the effect of BMP4 was examined in the prime- derived differentiation protocol. The generated cells were then characterized and analyzed using imaging, immunocytochemistry (ICC) and quantitative reverse transcription PCR (RT-qPCR). The thesis found that although TSCs and its subtypes could be successfully generated from both PSC states, differences were observed. In addition to morphological differences, the most significant finding was the expression of the HLA-G gene, an EVT-specific marker, in the prime-derived TSCs (TSC(BMP4)). HLA-G was also significantly more expressed in the prime-derived EVTs (EVT(p)) compared to the naïve-derived EVTs (EVT(n)). Further, MMP2 which is also an EVT specific marker, was significantly more expressed in the EVT(n) compared to the EVT(p). As a result, the research question regarding the validity of the TSCs using both methods and the effect of BMP4 remains open. Further studies are required including single-cell RNA sequencing to obtain a better and broader view of the trophoblast profile and functional assays for subtype differentiation. Additionally, the role of BMP4 should be investigated in more depth.

Recently, several novel post-translational modifications (PTMs) have been identified as important regulators in biology. Succinylation, the reversible addition of a succinyl group from a free succinyl-CoA into a protein lysine, is one such novel PTM. The last decade of research has unveiled succinylation as a powerful regulator of metabolism, prevalent in every organism it has been studied in and with functional effects on target proteins in several key metabolic pathways. A major contribution of this thesis is to catalogue the recent advances in succinylation research into the most comprehensive literary review currently available on succinylation. While the biological role of this PTM is being established, the relevance of succinylation in human disease has remained unclear. Meanwhile, mitochondrial DNA depletion syndrome caused by defective SUCLA2 (SUCLA2 disease) is a progressive hereditary mitochondrial disease with no available treatment. SUCLA2 disease is caused by defective mutations in the ß-subunit SUCLA2 of the TCA cycle enzyme succinyl-CoA synthetase. While the characteristic manifestations, including impairment of respiratory complexes, and the etiological mutations in this disease are well established, the pathogenic model for SUCLA2 disease has remained incomplete. As succinyl-CoA synthetase shares a substrate, succinyl-CoA, with succinylation, this thesis set out to probe SUCLA2 mutants for a potential succinylation phenotype. An extensive hypersuccinylation phenotype was characterized in fibroblasts and tissue samples from SUCLA2 mutant patients by immunochemical methods. The hypersuccinylation target identities in SUCLA2 mutants were revealed with proteomics by mass-spectrometry. Hypersuccinylation in SUCLA2 mutants was shown to be enriched in proteins participating in mitochondrial energy metabolism, including respiratory complex proteins. In addition, several novel metabolic phenotypes were characterized in SUCLA2 mutants with metabolomics by mass-spectrometry, most prominently a significant depletion of aspartate metabolism. While identification of extensive hypersuccinylation in SUCLA2 mutants establishes a novel concept of succinylation relevance in human metabolic disease, the prospect of altered regulation of the respiratory complexes due to hypersuccinylation lays the foundation for a novel pathogenic model for SUCLA2 disease. Meanwhile, the observed novel metabolic phenotypes significantly contribute to the current understanding on SUCLA2 mutant metabolism and inspire a hypothetical model on how the defective succinyl-CoA synthetase could be circumvented in the TCA cycle of SUCLA2 mutants.

Multiple Myeloma (MM) is the second most common hematologic malignancy. Despite the advancements in treatment approaches in the last decade, the prevalence of refractory disease leading to relapsed cases has been a major challenge. A wide range of intricate genetic heterogeneity demonstrated by myeloma patients is a credible explanation for the diverse treatment responses observed in patients sharing the same treatment regimens. Pertaining to this, the study aims to identify predictive gene expression biomarkers that forecast response to BCL2 inhibitor venetoclax and treatment outcome to proteasome inhibitor bortezomib. In this study, samples from MM patients were characterized into sensitive and resistant, (1) based on ex vivo response to venetoclax treatment (Resistant n=21; Sensitive n=21), and (2) based on their bortezomib treatment outcome in clinical profiles (Resistant n=12; Sensitive n=15). Associations between the different gene expressions and drug responses were studied using statistical and bioinformatic tools. As a result, we identified that significant (p-value <0.05) overexpression of 36 genes and downregulation of 38 genes appeared to confer resistance to venetoclax drug response in MM patients. Additionally, the functional association of these genes with pathways was determined using a pathway enrichment tool. Furthermore, the study provided evidence that cytogenetic alterations t(11;14) and t(4;14) are significantly (p-value <0.05) associated with differing venetoclax response in MM patients. These findings demonstrated that gene expression biomarkers and chromosomal translocations play a significant role in regulating venetoclax drug response in MM, which can be further utilized to personalize treatments for patients. The knowledge obtained from this work best applies in personalized medicine; whereby fitting treatments to an individual patient’s genomic landscape will enhance patient outcome.

Liver Kinase B1 (LKB1), also known as STK11, is a well-known tumor suppressor and a metabolic regulator, mutated in Peutz-Jeghers syndrome (PJS) and other sporadic cancers. LKB1 regulates several cellular functions: metabolism, polarity, cytoskeleton organization, differentiation, and proliferation by activating 14 AMPK-related downstream substrates. In a recent study, LKB1 was found to maintain intestinal homeostasis by repressing ATOH1 with the involvement of pyruvate dehydrogenase kinase 4 (PDK4). ATOH1 is a transcription factor and master regulator of secretory lineage in the intestine. It has been reported that ATOH1 is epigenetically regulated in inner hair cells and intestinal epithelium via Polycomb repressive complex 2 (PRC2). However, the repression mechanism of ATOH1 by LKB1 is currently unknown. This study aimed to determine the molecular mechanism of ATOH1 repression by LKB1. In this study, Ls174t, a human colorectal adenocarcinoma cell line, was used to investigate ATOH1 induction by LKB1 signaling from two angles: First, involvement of LKB1 downstream substrates was investigated using shRNA mediated knockdown screen. Interestingly, silencing of either MARK4 or SIK3 alone was found to induce ATOH1 and thus mimic silencing of LKB1 unlike other LKB1 substrates including AMPK kinases. A second angle explored possible epigenetic mechanism in ATOH1 repression pathway, using dichloroacetate (DCA, a PDK4 inhibitor) and GSK126 (a PRC2 inhibitor). DCA treatment resulted in no change on the global levels of histone modifications tested. In addition, GSK126 caused the downregulation of ATOH1 in both control and LKB1 depleted condition. Thus, this study concludes that LKB1 represses ATOH1 through MARK4 and SIK3 and that global changes in the histone modifications investigated are not involved in the mechanism.