Browsing by study line "Cross-disciplinary translational medicine"

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.

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.

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.

Despite recent advances in immunotherapies for lung cancer, their success is still hindered by limited predictability of treatment outcomes in patients, as well as by resistance-conveying tumor mutations such as EGFR. Moreover, due to the vast number of treatment options and their cost, a quick, reliable, and cost-efficient drug screening platform is needed to select the optimal treatments for each individual patient. This thesis focuses on finding the best culture conditions to be used in such a future platform, employing 3D cell cultures and microfluidics to mimic in vivo tumors while saving costs and allowing for high-throughput screening. Image-based analysis showed that culture medium can have significant impacts on both cancer organoid growth and morphology, as well as drug sensitivity to the EGFR-inhibiting drug Osimertinib. Specific medium factors, such as the antioxidant N-acetylcysteine, might be particularly important for the integrity of 3D structures in the platform and help prevent conversion to an adherent morphology. Moreover, flow cytometry analysis of immune cells from pleural effusion samples indicated that medium composition might facilitate creating an inflammatory environment in the platform, and that immune cells should not be cultured longer than one week to maximize their activity. Finally, this thesis compares two microfluidic devices for their suitability to be used in future high-throughput drug-screening applications, by contrasting their ease of handling, applicability in fluorescent imaging-based readouts, and possibility to mimic and study the tumor microenvironment in vitro. The results suggest that the choice of microfluidic device will be dependent on whether microscopy analysis or cell viability assays will be used as the main readout of the drug screening in the future.

Lung cancer remains the leading cause of cancer death worldwide. Cancer immunotherapies have changed the treatment path in some cancers and even led to favorable clinical outcomes in previously incurable cancer types. However, only a fraction of patients benefit from the current immunotherapies. Even though immuno-oncology has great potential, it is facing several challenges including the lack of biomarkers, unknown mechanisms of therapy resistance, complexity of the tumor-immune interactions, and involvement of the complex tumor microenvironment that significantly affects therapeutic efficacy. It remains a great challenge to predict which patients will benefit from immunotherapies, and current immunotherapies are deemed expensive when compared to the more traditional therapeutic modalities. In this work, we aimed to develop platform to study responses to immunotherapy ex vivo in a personalized manner. The platform could enable the study of immune effects and T cell mediated tumor killing in the absence and presence of immunotherapy and other selected drugs. We demonstrate the utility of our ex vivo platform that has potential for personalized drug testing.

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

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.

Lung cancer is one of the most common and deadliest cancers worldwide, but the mechanisms behind different types of lung cancer are still poorly understood. Non-small cell lung cancer makes up 80% of lung cancers, and some epigenetic mechanisms have been proposed for it. Epigenetic modifications are a way of influencing the expression of genes by inhibition or activation. PRC2 is an epigenetic modulator that catalyses the formation of methyl groups on histone 3 lysine 27, which is an epigenetic mark with repressive nature. PRC2 has been proposed to be downstream of AMPK, an energy sensor of the cell, which is phosphorylated by LKB1 under energy stress conditions. Inactivating mutations in LKB1 are known to cause and worsen non-small cell lung cancer, and the overexpression of EZH2, the catalytic subunit of PRC2, has similar effects. Therefore, establishing a novel downstream mechanism linking LKB1, AMPK, and PRC2 together could explain one mechanism for NSCLC tumorigenesis. Changes in metabolism are a feature of cancer cells, and this pathway could also link energy stress and cancer together. Mouse embryonic fibroblast and H358 cell lines overexpressing wild type EZH2, mutant EZH2 and GFP were generated and treated with the glycolysis inhibitor 2-deoxyglucose to study the effects of energy stress. Levels of histone methylation and phosphorylation statuses of AMPK and its downstream target ACC were assessed with Western blotting, and expression levels of potential PRC2 target genes with RT-qPCR. The study setting proved to be functional for the response of AMPK to energy stress conditions, as both AMPK and ACC were phosphorylated in the presence of 2-DG. In mouse embryonic fibroblasts, PIM1 showed different gene expression with wild type and mutant EZH2, suggesting that its activation would be regulated through the phosphorylation of the T311 site of EZH2 during energy stress. The results from histone methylation statuses did not follow the hypothesis, possibly because of the lack of specificity of detecting global H3K27me3. Other target genes besides PIM1 in MEFs did not show significant changes in expression level. Considering that the incorporation of the mutant EZH2 into PRC2 complexes was not validated, additional research would be needed to confirm or deny the explained mechanism between PRC2 and AMPK.

Lymphangiogenesis is crucial for the formation of a fully functional lymphatic vasculature. Dysfunctional lymphatic vasculature is associated with many pathologies including lymphatic malformations, lymphedema, and inflammation. This thesis aimed to elucidate the function of the lymphatic endothelial cell expressed β1-integrin in postnatal lymphangiogenesis in mice. To investigate this, Itgb1 gene was conditionally deleted from the lymphatic vasculature of mouse pups at postnatal day two, using the Cre-Lox recombinase system. Ears of the mice were collected at postnatal day twenty-one, and immunostained for lymphatic vessel, mural cell, and extracellular matrix markers. Relative gene expression analysis was performed from whole ear lysates and isolated endothelial cells. Mice lacking Itgb1 gene presented a disorganized dermal lymphatic vascular network, characterized by dilated capillary and collecting vessels. Collecting vessel specification was abnormal showing persistent LYVE1 expression, lack of V-shaped lymphatic valves, and complete loss of smooth muscle cell coverage. Relative gene expression analysis of isolated endothelial cells revealed a decrease in Pdgfb, a gene associated with smooth muscle cell recruitment. These results indicate a significant role for β1-integrin in postnatal lymphangiogenesis and highlight the complexity of postanal lymph vessel formation. Nevertheless, further studies are needed to specify the mechanisms behind this phenotype. Increasing knowledge in this field is important, because pathologies related to lymphangiogenesis can cause debilitating symptoms and therefore there is a need for therapeutic options.

The APOE4 gene variant of apolipoprotein E (apoE) has been identified as a predisposing factor for late-onset Alzheimer’s disease (AD). ApoE is known to interact with one of the classically characterized hallmarks of AD, the amyloid-beta (Aβ) protein. Aβ activates the classical complement pathway by binding to C1q that may sustain inflammation, hamper Aβ clearance and therefore promote accumulation of Aβ deposits in the brain and affect the integrity of the blood-brain barrier. Depending on context, binding of apoE to C1q can either activate or inhibit complement activation. ApoE can also interact with the complement regulator factor H (FH) which colocalizes with C1q on Aβ plaques. The interactions between these proteins and their role in the AD pathogenesis has not been fully explored. This thesis aimed at unraveling the complex formation between Aβ1-42, apoE, FH, and C1q, and their role in complement activation to provide novel insights on the mechanisms contributing to AD pathophysiology. By conducting enzyme-linked immunosorbent assays and Western Blotting of native PAGE, I was able to show that FH bound to apoE and forms a complex with Aβ1-42/apoE in an isoform- specific manner (apoE2 > apoE3 > apoE4) whereas C1q bound all Aβ1-42/apoE complexes with same affinity. FH did not bind Aβ1-42 alone, thus the results indicated that binding of FH in Aβ1-42/apoE complex took place via apoE2 and apoE3. In the absence of FH and C1q, immobilized Aβ1-42/apoE4 complexes led to higher serum complement activation levels when compared to Aβ1-42/apoE2 and Aβ1-42/apoE3 complexes or Aβ1-42 alone. C1q activated serum complement especially in the presence of Aβ1-42/apoE3 and Aβ1-42/apoE4. Further functional analysis with flow cytometry revealed that in fluid phase the presence of FH could restrict Aβ1-42-induced complement activation, the subsequent formation of C5a and activation of C5a receptor (C5aR) expressed on Human embryonic kidney (HEK) 293 cells. The presence of all apoE isoforms on Aβ1-42/C1q complexes led to reduced C5aR activation. Surprisingly, however, the reduction was the largest with apoE4, and the presence of C1q on Aβ1-42 alone could also decrease C5aR activation. The results of this thesis elucidated the effects of Aβ1-42, apoE, C1q and FH in complement activation and proposed a mechanism by which FH regulates complement activation and cell-mediated inflammation through isoform-specific binding to apoE associated with Aβ1-42. Further work is required to analyze the functional effects of these complexes, whether Aβ1-42, apoE, FH, and C1q can all bind simultaneously to form a large complex and whether this complex affects C5aR activation. This study provides important insights on how APOE4 may predispose to the neurotoxicity and neuroinflammatory changes in AD pathophysiology early in the disease process. Yet, the findings are only preliminary and therefore further studies are required to validate these results and to demonstrate whether promoting FH binding to apoE/Aβ complex could provide a novel target for AD therapy.