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

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  • Jagdeo, Joanna (2020)
    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.
  • Suominen, Peppi Lotta Josefiina (2023)
    Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells in the bone marrow (BM). MM is the second most common hematological malignancy, accounting for about 14% of blood cancers. Despite the improvements in the treatment of MM, the disease remains incurable, and essentially all patients end up relapsing. Deletion of chromosome 16q, the location of tumor suppressor CYLD, occurs in 35% of MM patients. CYLD is a deubiquitinase, most recognized for its function as a negative regulator of the nuclear factor kappa B (NF-κB) pathway. The loss of CYLD is associated with disease progression and worse survival in MM, but its significance in drug response is unknown. As the loss of CYLD is common in MM, determining its effect on drug response is essential. Analysis of data gained from MM patient samples were used to study the effect of CYLD copy number status on drug response. The treatments were selected based on previous research performed by our group. The effect of homozygous deletion was most significant in inducing resistance to BMS-754807, an inhibitor of insulin-like growth factor 1 receptor. To investigate the role of loss of CYLD on drug response in vitro, cell lines with CYLD knockout (KO) were created using the clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR associated protein 9 (Cas9) technology. An established CYLD-KO cell line was treated with carfilzomib, bortezomib, dexamethasone and BMS-754807 to assess the effect of CYLD-KO. The CYLD-KO slightly increased the sensitivity to BMS-754807, but essentially no differences were detected in response to the drugs. The effect of CYLD-KO was additionally explored to NF-κB - and Wnt-pathway activation by Western blot analysis, but due to technical difficulties, the results were inconclusive. The loss of CYLD is a common genetic aberration in MM, giving a survival benefit for the malignant cells. Based on the results from patient data analysis, the loss of CYLD could promote drug resistance to BMS-754807, and the effect should be further studied with more cell lines with CYLD-KO. As the population ages, and as the median age of newly diagnosed patients is 70, the need for efficient MM therapies increases. Studying the mechanisms behind drug resistance and sensitivity is essential in the aim of improving the efficacy of MM therapy and, in the end, the overall survival of the patients.
  • Nowlan, Kirsten Helene Anna-Marie (2020)
    Mucosal associated invariant T (MAIT) cells are a fairly recently described population of innate-like T cells. In humans, MAIT cells represent an exceptionally abundant population in the blood, where they account for 1–10% of all T cells. However, compared to conventional T cells, which can display an almost unlimited T cell receptor (TCR) repertoire, the specificities of MAIT cell TCRs are limited. This evolutionarily conserved subset displays a semi-invariant TCR which recognises riboflavin metabolites, produced by a wide range of bacteria and fungi, and presented on the major histocompatibility complex (MHC)-class I related (MR1) molecule. The function and significance of MAIT cells in health and disease have only started to be unravelled, and it is becoming increasingly clear that MAIT cells are also modulated in non-microbial diseases. Interestingly, MAIT cells have been shown to exhibit a relatively high expression of the transcription factor, Helios, compared to most other T cell subsets. The function of this translational activator and repressor, encoded by the gene IKZF2, in the MAIT cell population remains obscure. This study focused on the functional role Helios may play in regulating the activation of MAIT cells. Thus, by using siRNA to silence Helios expression, and flow cytometry to analyse any potential alterations in MAIT cell activation markers, we aimed to be able to characterise the kinetics and functional role of Helios in peripheral MAIT cells of healthy individuals. Here, we clearly established a striking upregulation of Helios in MAIT cells following 24 hours of stimulation. Moreover, we were able to achieve a >50% knockdown of Helios at the protein level, in this subset of T cells. Nevertheless, no significant difference in any of the activation markers we investigated was present between the MAIT cells with reduced Helios expression and their controls. This could, however, of occurred as a result of the toxicity that the transfection had on the functionally of the T cells. From these results, it is difficult to conclude any clear role for Helios in the activation of MAIT cells, and consequently, further research needs to be performed before any clear conclusions can be drawn.
  • Lapinsuo, Ella (2023)
    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.
  • Arffman, Maare (2021)
    Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.
  • Bouhlal, Jonas Otto Vilhelm (2022)
    Despite of great advancements in the field of cancer therapy in the past decades, the 5-year survival of acute myeloid leukaemia (AML) patients remains low with high mortality especially in elderly patients, in whom the disease is most often observed. Poor prognosis often results from complex heterogenous molecular abnormalities defining the progress of the disease, while making it more difficult to treat due to intensive treatments only being feasible for younger patients. Our increased understanding of cancer immunology and the potential of immunotherapy has, however, led to promising therapeutic innovations, which give hope for discovering long-lasting and effective treatment options. Natural killer (NK) cell-based immunotherapies are amongst the emerging novel therapeutic approaches that aim to target malignant cells with less toxicity and improved applicability. Using high-throughput drug sensitivity and resistance testing combined with single cell RNA (scRNA) sequencing, this study focused on finding drug compounds that could synergise with NK cells to improve their effectiveness in killing leukemic cells. In this study, many drugs showed promising results in being able to potentiate NK cell cytotoxicity, with daporinad and pevonedistat showing the most notable differences when compared to controls. The potentiating effect of Janus kinase (JAK) inhibitors also suggested a method of increasing NK cell activity against leukemic cells through downregulation of major histocompatibility complex (MHC) class I molecules. In conclusion, findings shed light on the synergetic potential of drugs and NK cells, giving hope for clinically relevant findings following further validation and testing.
  • Pällijeff, Pieti (2021)
    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.
  • Siskovs, Klims (2021)
    STK11/LKB1 is a tumor suppressor gene and mutated in 18% of lung adenocarcinomas. Tumor suppressor liver kinase B1 (LKB1) is known to activate adenosine monophosphate-activated protein kinase (AMPK) and 12 AMPK-related kinases (ARKs) by phosphorylating a conserved threonine residue in their T-loop region. A number of studies focused on investigating the influence of LKB1-AMPK signaling on cancer cell proliferation. However, there is no systematic study for identifying the critical LKB1 kinase substrates in suppressing lung cancer cell growth. In this project, the LKB1-deficient lung adenocarcinoma cell line A549 cells were sequentially overexpressed with constitutively active mutants of AMPKα1, AMPKα2, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, SIK1, SIK2, SIK3. The overexpression status was confirmed at both genetic and protein levels by qPCR and Western blotting, correspondingly. In vitro growth assays demonstrated up to 33% reduced growth rate of A549 cells overexpressing AMPKα1, AMPKα2 and NUAK1. Furthermore, siRNA knockdown of the selected substrates in LKB1-overexpressing A549 cells significantly rescued the cell growth defect. These findings suggest, that AMPKα1, AMPKα2 and NUAK1 kinases are critical for LKB1-mediated cell growth defect in lung adenocarcinoma.
  • Tripathi, Shivanshi (2020)
    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.
  • Karttunen, Konsta Eemeli (2023)
    Transposable elements (TEs) are repetitive DNA elements that have an autonomous capability of replicating and inserting themselves into new loci within genomes. TEs have often been thought to be a part of the non-functional “junk DNA”, but advances in next-generation sequencing technology has revealed that TEs have rich biochemical functions: Specific TEs bind a large fraction of transcription factors and harbor marks of active chromatin in human genomes. However, there have been few genome-wide functional enhancer activity studies on the role of TEs in gene regulation, especially in the context of human cancers. In normal cellular homeostasis, TE activity is tightly controlled by epigenetic mechanisms. In contrast, the cell state in cancer genomes is often permissive for TE activation: genome instability and mutations, notably p53 inactivation, and nonmutational epigenetic reprogramming such as DNA hypomethylation are common characteristics of cancers. TE transcription, somatic retrotransposition and activation of cryptic promoter elements occur frequently in tumors. TE activation is highly heterogeneous between cancer types: for example gastrointestinal tract cancers such as colorectal cancer show high somatic insertion activity, whereas retrotransposition is rare in hematolymphoid malignancies. Due to the widespread TE activation in cancers, we posited that this may also be seen in the activity of cryptic TE enhancers, with specific active families of TEs characteristic to different cell types due to lineage-specific TF binding. We asked if TEs contribute to the enhancer landscape of cancers and to which extent, what are the differences between cancers in the activity and transcription factor binding and whether TE enhancers may have a role in tumorigenesis and the regulation of cancer-specific genes. To functionally study TEs, we utilized a high-resolution, unbiased, genome-wide massively parallel reporter assay (MPRA). We utilized colorectal and hepatocellular cancer cell lines to study the differences and similarities between TE activation and combined the MPRA data with orthogonal epigenetic data to study the in vivo signatures of the TEs. We found that both cell lines show common and highly enriched TE subfamilies that were mostly specific for p53, as well as TEs that were highly unique in both cell lines. By using in vitro methylated MPRA libraries, we found that CpG methylation has relatively minor a role in regulating the enhancer activity of some TE subfamilies in the reporter assay. By comparing the epigenetic context of the TEs, we found that especially colorectal cancer has specific highly active TE subfamilies with signatures of canonical active enhancers. We also used an in silico model to predict TE enhancer to gene contacts and found that these subfamilies regulated genes that were frequently overexpressed. Thus, we present the widespread functional activity of TE enhancers in cancers, providing evidence for further functional validation of TEs and their effects on transcriptional programs and especially dysregulation of gene expression in cancer.
  • Mölsä, Riikka (2023)
    Lung cancer is the number one cause of cancer-related deaths in the world every year. Of all non-small cell lung cancers, lung adenocarcinoma is the most prevalent subtype. There is a great need for better treatment options for lung cancer, of which cancer immunotherapy is an attractive option due to the high mutational burden of lung tumors. Patient-derived lung cancer organoids (lung PDTOs) could provide a new testing platform for these studies as the 3D models better represent the original tumor and its microenvironment compared to often used 2D cell lines. One interesting field is immunopeptidomics, which focuses on discovering tumor peptides presented in the HLA-I molecule on the tumor surface that could elicit an immune response. Using lung adenocarcinoma PDTOs, this study aimed to analyze the immunopeptidome of five PDTOs to discover tumor-specific and immunogenic peptides using PeptiCHIP purification and LC-MS analysis. These findings could be used in PeptiCRAd, a novel cancer vaccine comprised of an oncolytic adenovirus coated with tumor peptides. To elucidate the applicability of PDTOs for virotherapy, three oncolytic adenoviruses, D102, Ad5/3Δ24 and Ad5Δ24-RFP, and their ability to infect, kill, and replicate in lung PDTOs was studied. PDTOs were characterized as epithelial, as they presented epithelial cytokeratin and epithelial layer structures, as indicated by cytoskeletal F-actin staining. The three oncolytic adenoviruses were studied by infecting PDTOs and a difference in killing capacity of the three viruses was shown, potentially due to differences in receptor interactions and expressed transgenes. In addition, D102 and Ad5Δ24-RFP were shown to replicate in PDTOs, which is necessary to induce a strong enough immune response against the virus for immunotherapy efficiency. HLA-I expression was high in all tested models, which indicated that antigens could be presented in the tumor cells. Immunopeptidome analysis did not result in a high yield of peptides, likely due to challenges in sample preparation and patient material being scarce. As the HLA-type of each patient was unknown during this study, more data analyses still need to be done to determine the best immunogenic peptides, which could then be further studied in vitro. However, peptides overexpressed in lung cancer and with cancer benefiting properties were found from PDTOs, which already gives promising results. In conclusion, though additional immunopeptidome studies with an increased yield of peptides are needed to select tumor-relevant immunogenic targets for therapeutical use, as well as additional testing on the optimal oncolytic virus for lung cancer targeted PeptiCRAd immunotherapy, this study proved that oncolytic viruses can infect and kill lung PDTOs, and that HLA-I expressed tumor peptides can be identified from them. This is also one step towards finding better and patient-specific research models for testing therapies and discovering and developing personalized cancer treatments.
  • Stadelmann, Christian (2019)
    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by transgenically expressing the four transcription factors OCT4, SOX2, KLF4, and C-MYC. This technology has revolutionised the stem cell field, yet cellular reprogramming is still inefficient and slow. To become fully applicable in regenerative medicine, the robust generation of safe and high-quality iPSCs from patient samples is essential. Various methods and potent reprogramming factors have been described to date. Yet, none have been able to circumvent these limitations markedly. The recently published activator-mediated approach (CRISPRa) is considered to be more physiological compared to the forced transgenic expression as the cell’s own genes are activated. Here, guide RNAs (gRNAs) mediate sequence-specific recruitment of non-cutting Cas9 (dCas9) activator proteins to the promoter region. Unlike other methods, it holds great multiplexing capacity and can also target enhancer and non-coding sequences. CRISPRa reprogramming still needs to be optimised since its efficiency is low. Thus, we aimed at enhancing this aspect and the temporal kinetics by targeting the micro RNA (miRNA) clusters 302/367 and miR-371-373, which both have been described as powerful cell fate regulators. We demonstrate successful reprogramming by targeting the miR-302/367 promoter alongside OCT4, SOX2, KLF4, C-MYC, LIN28A, REX1, NANOG, and EEA-motifs with CRISPRa. Activating the miRNA cluster results in a 2.5 fold efficiency increase in human foreskin fibroblast (HFF) reprogramming compared to the published basal CRISPRa system, quantified by staining for alkaline phosphatase. In HFFs, the CRISPRa efficiency is now comparable to the commonly used transgenic approach. Aiming to clarify the molecular mechanisms of these results, we characterised the expression of direct and downstream targets of miR-302/367 at different time points throughout the reprogramming process. Furthermore, validated with immunocytochemical stainings, the generated bona fide iPSCs express pluripotency markers and spontaneously differentiate into the three germ-layers, both signs of high-quality iPSCs. Beyond that, we report that miR-302/367 activation appears to result in earlier iPSC colony formation resulting in faster proliferating stem cell colonies shown with live-cell imaging. Employing a conditionally stabilised activator construct, we further show that with miR-302/367 targeting, the dCas9 activator expression seems to be required for only a short time period, sufficient to induce pluripotency. At the end of the project, the miR-302/367 cluster targeting was optimised and the best-working gRNAs were selected for further studies, which when combined further increase the CRISPRa-induced expression of the miR-302/367 cluster markedly. All in all, this study demonstrates that non-coding genetic elements like the miR-302/367 cluster can be targeted with CRISPRa, and its targeting significantly improves the reprogramming efficiency. Implications of the study for regenerative medicine and future steps are discussed.
  • Junquera Mencia, Ada (2023)
    High-grade serous ovarian cancer (HGSC) presents a complex clinical challenge as it is often diagnosed at advanced stages. Neoadjuvant Chemotherapy (NACT) is commonly used to treat advanced stage (III and IV) patients who cannot undergo primary debulking surgery. However, 80% of them experience relapse and develop resistance to platinum-based chemo therapies. While NACT alters the tumor immune microenvironment in a treatment response specific manner, its underlying mechanisms remain unclear. Understanding the effect of NACT on tumor microenvironment (TME) is crucial to identify novel biomarkers and develop effective therapeutic strategies. Emerging spatially resolved methodologies, including highly multiplexed-imaging and spatial gene-expression profiling approaches provide novel information regarding spatial interactions and mechanisms operating at single-cell level. However, neither of these methods can offer a comprehensive view of the tumor immune landscape while also revealing the molecular mechanisms behind it. To overcome this limitation, this thesis proposes a novel method integrating tissue cyclic immunofluorescence (tCycIF) and GeoMx DSP spatial transcriptomics. tCycIF is a high-throughput multiplexed imaging method, while GeoMx DSP offers sequencing information at near to single-cell resolution in well-defined regions of interest (ROIs), enabling the dissection of the underlying molecular mechanisms. Here I set out to explore the effect of NACT on tumor stroma interface (TSI) and the surrounding tumor and immune cells, particularly IBA1+ macrophages, and CD8+ T cells. HGSC patient-derived pre- and post-NACT FFPE blocks were acquired from Helsinki University Biobank. To preserve morphological and spatial features, adjacent tissue sections underwent tCycIF and GeoMx experiments. Potential regions for spatial transcriptomics (Pre ROIs) were successfully delineated based on tCycIF staining patterns, enabling the identification of IBA1+ macrophage and CD8+ T cell rich areas within the TSI. By overlaying a crop of the tCycIF image onto the GeoMx scan, areas with distinct similarity degrees between tCycIF and GeoMx were generated. The GeoMx-ROIs were ultimately selected based on the Pre-ROIs and their similarity degree with tCycIF scanned images. Tumor and stroma segmentation was performed through a custom segmentation method guided by tumor specific marker pan-cytokeratin staining in GeoMx images, defining distinct spatial compartments for sequencing. Finally, the image-derived data from both techniques was integrated in a single file, enabling a combined subsequent analysis. The novel pipeline developed in this study opens promising research possibilities, as tCycIF-guided ROI selection allows for precise targeting of cell neighborhoods and structures after performing a thorough microenvironment exploration. This approach can potentially be adapted for other uses in a wide range of biomedical fields, beyond the focus of HGSC.
  • Naddafi, Seyedehshima (2022)
    Autophagy is an essential pathway that evolved to sustain cellular integrity by removing damaged and aged organelles. During this process, our cells sense, encapsulate and deliver defective cellular components to the lysosome for destruction. Over the past decade, many laboratories have demonstrated that damaged mitochondria can be selectively eliminated, during a process known as "mitophagy". Mitophagy senses, targets, and engulfs defective mitochondria for elimination via lysosomal hydrolysis. The identification of factors that promote or prevent mitophagy has high therapeutic relevance, particularly those that alter PINK1/Parkin-independent mitophagy. Recent research in the McWilliams lab uncovered a novel role for lipid metabolism in the regulation of PINK1/Parkin-independent mitophagy. Briefly, the team discovered that DGAT1-dependent lipid droplet (LD) biosynthesis occurred several hours upstream of mitochondrial clearance, with LDs accumulation upon iron chelation. LDs accumulate in a DGAT1-dependent fashion as mitochondria are eliminated. Pharmacological or genetic inhibition of DGAT1, restricts mitophagy levels in vitro and in vivo. However, the mechanism that linked defective lipid metabolism to reduced mitophagy remained mysterious. We hypothesized that defective lipid signalling may compromise lysosomal activity leading to reduced levels of mitophagy. Accordingly, my project examined the functional contribution of DGAT-dependent LD biogenesis to lysosomal homeostasis in the context of PINK1/Parkin-independent mitophagy. After first verifying the DGAT1-dependent nature of LD accumulation in human cells, I established assays to investigate lysosomal homeostasis in the context of iron chelation-induced mitophagy. Using a variety of labelling approaches, live cell imaging experiments revealed a significant displacement of endolysosomes upon DGAT1/2 inhibition, in addition to possible alterations in lysosomal dynamics. My data suggest that loss of DGAT1 activity impairs lysosomal homeostasis when iron levels are low. This likely explains the mitophagy impairments and might account for additional phenotypes of impaired cell viability upon DGAT1 inhibition. Changes in lysosomal acidity were inconclusive, indicating further timepoints may need to be analysed to detect transient impairments in hydrolysis. My results emphasize the importance of organelle crosstalk in mitophagy and the emerging role of LDs in cellular integrity. These data further highlight that targeting lipid metabolism may provide a means to sustain efficient mitochondrial turnover.
  • Oostveen, Mayke (2023)
    Head and neck cancers form the seventh most common type of cancer globally, and 90% of these cancers are classified as head and neck squamous cell carcinomas (HNSCC). The 5-year survival rate for head and neck cancers remains around 50%, indicating a need for new treatment strategies. This thesis aimed to examine the potential of BH3 mimetics A 1155463, A-1331852, and navitoclax combined with irradiation, as a treatment for HNSCC. BH3 mimetics are compounds able to bind to anti-apoptotic proteins, thereby blocking their ability to exhibit anti-apoptotic effects. A high-throughput screening method was used to characterize the synergistic effects of the BH3 mimetics with different irradiation doses on the viability of twelve HNSCC, normal (NOF) and cancer-associated fibroblastic (CAF), and HPV-16 immortalized oral dysplastic keratinocyte (ODA) cell lines. Additionally, the effects of A-1155463 and A-1331852 on apoptosis and proliferation of two HNSCC cell lines (UT SCC 40 and UT-SCC-42A), in combination with 8 Gy irradiation, were measured. Effects on invasion of UT-SCC-42A cells with the irradiation-mimetic combination were tested in a 3D spheroid model. Lastly, the effect of A 1331852 and navitoclax in combination with immunotherapy drug nivolumab on donor-derived immune cell migration and apoptosis of UT-SCC-40 was assessed with a microfluidic chip assay. All three BH3 mimetics in combination with irradiation synergistically reduced viability in six to ten HNSCC cell lines and ODA cells, but not in NOF or CAF. A-1155463 and A-1331852 induced apoptosis and reduced proliferation in both tested cell lines. The addition of irradiation to the compounds significantly increased the apoptotic ratio in both cell lines and compounds. In the spheroids, A-1331852 significantly reduced cancer cell area and length of invasion both with and without irradiation. A-1155463, unlike navitoclax, had a trend in reducing HNSCC invasion. The combination of A-1331852 with nivolumab increased the immune cell migration of one out of the three donors. These results show the synergistic and apoptosis-inducing effects of the BH3 mimetics combined with irradiation, proving them plausible candidates for HNSCC treatment. The reduction of invasion by A 1331852 suggests a role for Bcl-xL in HNSCC invasion. It is proposed to further investigate the properties of A-1331852 in in vitro cocultures and in vivo studies, with and without irradiation.
  • 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.
  • Tohmola, Tiialotta (2022)
    The current 5-year survival rate of OSCC patients is around 50%. There are no diagnostic tests or markers and the diagnosis is based on histopathologic samples only, which postpones the time of detection and worsens the prognosis of the patient greatly. New diagnostic methods and tools are required to improve the survival rate. This study aimed to find possible biomarkers and develop a new diagnostic method for OSCC by comparing the serum proteomic expression of tongue cancer patients and healthy controls with label-free liquid-chromatography mass spectrometry. The results showed small, but statistically significant difference in protein expression between the patients and healthy controls, as well as a clear separation between the groups based on the peptide data. In addition, no specific networks or cellular pathways were highlighted for OSCC compared to other types of cancers. These results didn’t introduce considerable advances into the diagnostics of OSCC but showed a possibility for finding further distinctive differences between the OSCC patients and healthy controls.
  • 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.
  • Peletier, Pim (2023)
    Cardiovascular diseases are reported to be the main cause of death. Inducing the growth of blood vessels, called angiogenesis, holds promising potential for improved vessel reperfusion after myocardial infarction. The vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are important regulators of blood vessel development, growth, and maintenance. VEGF-A is the protagonist of the family, but as a therapeutical measure, severe side effects impede its use. On the contrary, VEGF-B, which is mainly expressed in the heart and skeletal muscle, lacks a general pro-angiogenic effect. However, overexpression seems to carry angiogenic promise by increasing VEGF-A availability for VEGFR-2 through competition for VEGFR-1 binding. VEGF-B transcripts undergo alternative splicing, resulting in two isoforms, namely VEGF-B167 and VEGF-B186. The different isoform properties affect the VEGF-B bioavailability; thus, they should hold different translational potentials. In vivo, adeno-associated viral vector-mediated transduction of the VEGF-B isoforms resulted in cardiac hypertrophy and increased proliferation of endothelial cells. Both were more potently induced by VEGF-B186 than VEGF-B167, and the proliferation was mostly detected in the sub-endocardial region of the heart. Although the transcript levels were comparable between the isoform groups, the protein level of VEGF-B186 was much greater than VEGF-B167, implying a difference in VEGF-B isoform degradation and receptor binding dynamics. In vitro, endothelial cell regulation of the VEGF-B isoforms suggested a faster degradation of the VEGF-B167 protein. Blocking of neuropilin-1, a VEGFR-1 co-receptor, decreased the amount of VEGF-B167 protein, bound to cultured endothelial cells, whereas blocking of VEGFR-1 increased it, indicating internalization and subsequent degradation through VEGFR-1. Intracellularly, the VEGF-B167 protein increased upon blocking of ubiquitin-mediated degradation using MG132, suggesting that the protein is targeted by the ubiquitin-proteasome system. Thus, overexpression of VEGF-B stimulated a pro-angiogenic response, but of the two isoforms, VEGF-B186 had a more potent effect in the heart, presumably because VEGF-B167 was degraded more rapidly by the endothelial cells. Besides further validation of the in vitro degradation dynamics, live imaging of VEGF-B and its binding targets fused with fluorescent proteins could visualize the binding dynamics. Understanding the different properties and degradation patterns of the VEGF-B isoforms should aid in the clinical translation of their angiogenic potential, but further work is needed to elucidate the function, binding targets, and turnover of VEGF-B.