Browsing by study line "Molecular and Analytical Health Biosciences"
Now showing items 1-20 of 32
-
(2022)While there is no current consensus on what characterizes a heathy gut microbiome, there are evidence supporting the association of high microbial diversity with health stability in the host species. A growing literature suggests that microbial communities can withstand short-term anthropogenic changes with resilience, however enduring long-term changes might have a negative impact on the natural composition of host microbiome. Parasitism and anthropogenic disturbances resulting in habitat degradation might represent two of such long-term challenges to the invisible diversity of microbial communities, with consequences for the fitness of their host species. In this study, I tested whether parasitism by a specialist parasitoid and ecological changes due to anthropogenic habitat degradation affected the microbial community associated with the diapausing larvae of the Glanville Fritillary butterfly (Melitaea cinxia). The larval samples were collected in September 2012 from seven different communes across the Åland islands, from two extreme types of habitat patches (i.e., highly human-impacted habitats versus natural meadows). The parasitoid wasp Hyposoter horticola naturally parasitize approximately a third of the M. cinxia larvae in Åland, giving an opportunity to further investigate how parasitism might affect the larvae-associated bacterial community in this system. The DNA from the gut of each larva was extracted, and the bacterial V5-V6 region of the 16S gene was amplified and sequenced at the Finnish Institute for Molecular Medicine. The sequencing data was prepared for analysis by processing it through Mothur and QIIME2 data cleaning protocols. According to alpha- and beta-diversity metrics analyses, anthropogenic degradation of the habitat did not cause significant changes in the bacterial composition of the microbiota associated with diapausing larvae of M. cinxia, however, parasitism with the parasitoid wasp has a significant negative effect on the beta diversity of the microbial communities associated with the diapausing larvae.
-
(2021)Adeno-Associated Viruses (AAVs) are quickly becoming one of the most applied vectors for gene therapy applications. In the recent years three new AAV-based gene therapies have been approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The regulatory bodies require accurate and reliable characterisation of the clinical grade viral vectors during and after production. Analytic methods measuring the purity, potency and safety of the product support the up-stream and down-stream processes during the production and are used for final-drug substance characterisation. Median Tissue Culture Infectious Dose (TCID50) is a well-established method for measuring the infectious titer of a virus. Here, an assay for determining the infectious titer of AAVs, which has previously been used to characterise the existing AAV2 Reference Standard Material (AAV2RSM) was set up and optimised for research use at Kuopio Center for Gene and Cell Therapy (KCT). The assay utilizes the HeRC32-cell line, a HeLa clone, that stably expresses AAV Rep and Cap -proteins and in presence of adenovirus, enables the replication of recombinant AAV-vectors. The cells were grown in 96-well plates and infected with 10-fold dilution series of AAV vectors (AAV2 and AAV6) using human adenovirus type 5 as the co-infector. 72 hours post infection the vector genome replication of AAV was detected with quantitative PCR (qPCR). Thresholds for qPCR determined copy number and cycle threshold (Ct) were set and used for the determination of infection-positive wells. The 50-percent endpoint was observed and used to calculate the infectious titer according to the Spearman-Kärber method. The assay was set up and optimised with the AAV2 Reference standard material (AAV2RSM) using two different primer-probe sets (targeted sequences were; SV40 polyadenylation signal and AAV inverted terminal repeats (ITRs)). Plates infected with AAV2RSM were analysed separately with both primers resulting in mean infectious titers of 8.07 ± 3.13 x 108 TCID50 Infectious Units (IU) / mL (n = 9) and 1.27 ± 0.464 x 109 TCID50 IU/mL (n = 9) for SV40 and ITR, respectively. After the assay was set up with the AAV2RSM, an in-house AAV6 product was analysed with the ITR primers yielding 6.09 ± 3.94 x 109 TCID50 (IU) / mL (n = 5). The assay protocol was successfully set up for research use at the KCT laboratory. Improvements were added to the original protocol to increase assay robustness, accuracy and precision as well as to minimize the possibility of over-estimation of the infectious titer. The assay can be further optimised for a particular therapeutic AAV product in the research laboratory or technology transferred to a production facility for optimisation and validation for the analytics needs of a production pipeline.
-
(2019)Amelogenesis Imperfecta (AI) is a group of hereditary diseases where tooth enamel is abnormally formed. This disease has been found in Lancashire Heeler dogs. However, none of the mutations known to cause AI in canine, explain the disease in the Lancashire Heeler population. A research study was initiated to investigate the genetic background of the disease in the breed. Based on the pedigree data the disease was suggested to be inherited as an autosomal recessive disease. Whole-exome sequencing of two affected dogs was performed which revealed a plausible candidate variant in a gene regulating intracellular calcium concentration. The aim of this study was to examine whether the mutation in the candidate gene causes AI. The DNA were extracted from blood samples of 249 Lancashire Heelers. A genome-wide SNP genotyping was conducted on four cases and a homozygosity mapping was performed. The candidate variant was genotyped in a large cohort of Lancashire Heelers (n=249) and dogs from related breeds (n=91) to confirm the association with the disease. The expression of the candidate gene in lymphocytes was studied with RT-PCR. Clinical studies were performed to evaluate the clinical features and serum calcium levels were measured. The segregation of genotypes and phenotypes of the dogs was complete. The identified variant is predicted to cause a premature STOP codon, which, if translated, leads to a truncated protein lacking protein transmembrane domains. According to the results acquired from RT-PCR, it is possible that the protein will complete the translation despite the mutation. Clinical studies did not reveal other clinical features than enamel defects and serum calcium levels were normal. According to the results, we suggest that the identified variant is causal for AI in Lancashire Heelers. The results have significant impact because no other genetic connection between the gene and AI has been found before. Consequently, the development of a gene test is possible, creating multiple opportunities for researchers and veterinary medicine. The results are applicable also to human medicine. However, we need more research to achieve a complete understanding of the mutation and its effects.
-
(2024)Immunopeptidomics research, focused on HLA-presented peptides, indicates that identifying neoantigens – unique peptides from cancer cell mutations – holds potential for personalized cancer treatment. PeptiCHIP, a compact microfluidic device from ValoTx, facilitates neoantigen identification in small tumor samples. This study evaluates PeptiCHIP against traditional immunopurification methods, assessing its advantages and commercial potential through stakeholder interviews guided by the double diamond model of design thinking. The aim is to establish PeptiCHIP’s unique advantages in immunopeptidomics research and precision oncology. Data for this research were gathered through semi-structured interviews and comparative laboratory analyses. The interviews revealed a positive reception among 19 stakeholders towards PeptiCHIP, highlighting a strong interest in its potential to accelerate workflows and enhance the personalization of cancer treatment. Laboratory comparisons confirmed that PeptiCHIP offers greater sensitivity than competitor products in detecting antigens, pinpointing the most immunogenic peptides from individual tumor samples. This suggests that the data could be effectively utilized as a targeted treatment approach in precision oncology therapies. The results indicate an eagerness to adopt PeptiCHIP, driven by its enhanced detection speed and accuracy. This research not only underscores the technological and commercial potential of PeptiCHIP in impacting precision oncology but also highlights the importance of aligning technological innovations with user needs and market demands. For ValoTx, a strategic commercial approach is needed to set a benchmark in precision oncology innovation.
-
(2024)The significance of adhesion to the basement membrane (BM) for the growth and survival of healthy mammary epithelial cells is well-established. In ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer, luminal cells detach from the BM and proliferate within the ductal lumen of the mammary gland. This suggests that malignant cells are capable of evading detachment induced cell death, called anoikis during DCIS. The alterations that contribute to anoikis resistance may be essential for anchorage-independent survival, a phenomenon that remains largely unexplored in the context of BM. Hence, in this study, I aimed to investigate the association between anchorage-independent survival of breast cancer cells and the expression of BM components. I utilized a low-adhesion 3D suspension culture devoid of any matrix to evaluate the efficiency of mammosphere formation in luminal A and triple-negative breast cancer cell lines. To understand the ability of cancer cells to survive in an anchorage-independent environment, I investigated the expression profiles of BM components (including laminins and type IV collagen) across various breast cancer cell lines. Gene expression analysis of BM components was conducted in conventional 2D monolayer cultures, while immunofluorescence analysis was performed on mammospheres or single cell clusters derived from low-adhesion 3D cultures. Furthermore, to explore their functional significance, I examined the effect of laminin α5 (LAMA5) downregulation on the spheroid formation ability of luminal A cell line ZR75-1. The primary results of the study highlighted the ability of breast cancer cells to survive in low anchorage conditions by forming mammospheres or as single-cell clusters. Significant variations in the expression of BM components were observed across breast cancer cell lines and within the subtypes. Moreover, I observed the atypical expression and localization of BM components produced by the various breast cancer cell lines in the 3D culture. The potential functional relevance of BM was demonstrated by the decreased mammosphere-forming ability of the ZR75-1 with LAMA5 downregulation. Although further investigations are needed, these observations suggest a crucial role of BM produced by tumour cells in facilitating anoikis resistance and anchorage-independent survival of cancer cells.
-
(2022)Formation of template switching mutation has previously been proposed as a mechanism of RNA evolution. TSM mechanism may contribute to the creation, maintenance, and modification of the RNA Hairpin. The finding of de novo TSM in RNA sequences will provide evidence for this hypothesis. Ribosomal RNAs (rRNAs) appear in multicopy clusters on different chromosomes and evolve through concerted evolution. To study the properties of de novo TSM and the dynamics of the concerted evolution of rRNA, we developed a computational tool to analyze pairwise differences and the phylogenetic relationship of rRNA genes on different chromosomes. The genome assemblies that are based on traditional short-read sequencing methods have limitations on studying long tandem repeat rDNA, because the reading length is shorter than on the rRNA gene. To overcome this limitation. PacBio Hifi long-read sequencing data for human rRNA 18S and 28S genes were studied. By analyzing the diversity of rRNA genes between individuals and families, single nucleotide mutations, multiple nucleotide insertions, and deletions were identified. As expected, genetic variations in ribosomal genes were detected both within and between individuals. A larger sample size may be required for TSM identification. The finding of this research that related to the dynamics and concerted evolution of human rRNA may contribute to a better understanding of rRNA mutation-related genetic disorders.
-
(2024)Primary human complement deficiencies are rare conditions, which affect the functions of the immune defense. Central complement component C3 deficiencies manifest in early childhood mostly as recurring bacterial infections. The severity of the infections showcases the importance of the human complement system in antimicrobial defense and overall immunity. Overall, there are only about 30 reported cases of total C3 deficiency worldwide. This study introduces two Finnish siblings with no detectable C3 in the plasma of either the proband or their sibling. Primary C3 deficiency was suspected in the proband (elder sibling) due to the relapses of severe pneumococcal infections (meningitis, sepsis, pneumonia) under the age of two. We set out to study the genetic basis of the deficiency. The proband's C3 gene was sequenced and results validated with Whole Genome Sequencing. C3 protein deficiency was confirmed via immunoblotting of serum and peripheral blood mononuclear cell lysate samples. Comprehensive clinical data was obtained from patient physicians. In genetic studies, we observed 1) a maternal truncation at exon 9 of the human C3 gene and 2) a paternal indel disrupting a splice site motif at exon 29. Together, they create compound heterozygosity resulting in total C3 deficiency. Total C3 deficiency was confirmed by the lack of C3 protein by immunoblotting of plasma and of blood mononuclear cell lysates. Compound heterozygosity in the patients lead to a complete deficiency of mature, functional C3. The lack of C3 in serum was first described in Pekkarinen et al. (2013) and now confirmed through immunoblotting. Also, no intracellular C3 was detected in similar experiments. The complete lack of C3 in the patient does not corroborate extracellular or intracellular C3 being obligatory for life.
-
(2024)Exercise-induced hyperinsulinism (EIHI) is a pathological condition characterized by aberrant insulin secretion triggered by physical exercise or pyruvate exposure. The monocarboxylate transporter protein (MCT1), encoded by SLC16A1, is ubiquitously expressed in almost all cell types except pancreatic islet cells. In patients with EIHI, mutations in the regulatory regions of the SLC16A1 gene are thought to lead to the unwanted expression of MCT1 on the beta cell membrane, allowing the influx of elevated lactate and pyruvate blood levels during exercise. These substrates feed into the Krebs cycle, increasing insulin release. This excessive insulin secretion can lead to hypoglycemia during exercise, causing weakness, syncope, and confusion. Since EIHI has never been studied using a human stem cell-derived islets, this thesis aims to establish a robust model in which to investigate the disease mechanism in detail. To achieve this, we reprogrammed EIHI patients’ fibroblasts into a stable pluripotent state and further differentiated them into functional pancreatic stem cell-derived islets (SC-islets) in vitro. These SC-islets were then matured further in vivo (in immunocompromised mice) and compared to healthy SC-islet controls. Rigorous quality control measures were implemented throughout the differentiation process to ensure its efficacy and the expression regulation of SLC16A1 was studied during SC-islet development. Extensive phenotypic characterization was conducted using immunohistochemistry, quantitative gene expression level analysis, and insulin secretion assays with glucose and pyruvate. Contrary to expectations, the results of this study demonstrated that despite the SLC16A1 promoter mutation, the expression of SLC16A1 was downregulated similarly to the control cell line during development in vitro, resulting in similar pyruvate-stimulated insulin secretion to the control cells. Interestingly, immunohistochemical analysis of in vivo implanted SC-islets showed a clear phenotype with an increased number of MCT1-positive cells only in the mutant grafts, some of which were endocrine cells. In conclusion, the phenotypic manifestations of EIHI were not visible in the setting of in vitro modeling, which was attributed to the similar expression levels of MCT1 in both the mutant and control cell lines. However, following in vivo implantation, there was a noticeable increase in MCT1 expression exclusively in the mutant cells. This finding suggests a distinct regulatory mechanism of MCT1 expression, which might be impacted by the in vivo surroundings and the maturation state of human islets.
-
(2019)Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
-
(2023)Lynch syndrome (LS) is the most common cancer predisposition disease caused by dominantly inherited pathogenic variant (PV) of a mismatch repair (MMR) gene leading to a defective gene allele. The four major MMR genes encode MMR proteins – MSH2, MSH6, MLH1 ja PMS2 – that participate in the proofreading and repairing of the daughter strand for mismatches after every replication. The inherited PVs predispose to cancer development as only one somatic allele loss is required for biallelic loss according to the Knudson’s “two-hit” hypothesis. The biallelic loss of an MMR-gene leads to disrupted protein function altering the MMR process. When mismatches are left unrepaired, genomic instability is caused, which can eventually lead to tumorigenesis. Especially, the risk of colorectal cancer (CRC) and endometrial cancer (EC) is increased in LS. The predisposition syndrome, LS, is important to detect as early as possible to decrease the risk of cancer by prevention and surveillance. The MMR genes and their defects vary in their consequences to the repair process considerably, and thus, it is crucial to know the different characteristics and functional effects of them when estimating the level of cancer risk. Variants of uncertain significance (VUS) are especially prevalent among LS variants. More information about their impact to the disease can be acquired by in vitro and in silico methods, for instance. The main goal of the efforts for early detection and prevention is to reduce cancer morbidity and mortality. In this thesis, the pathogenicities of MSH2 and MSH6 variants were studied with DiagMMR assay, which has been developed for studying the protein function of these genes. In addition to the traditional agarose gel electrophoresis (AGE), the samples were also analyzed by a fragment analyzer, Labchip, that bases its function on capillary electrophoresis. This way the MMR detection efficiency of the methods could be compared. Samples were collected as skin biopsies from controls and LS patients with known MMR gene variants by Helsinki University Central Hospital (HUCH). InSiGHT database, that collects the different MMR-gene variants and their pathogenicity classification, was used to ensure that different kinds of variations, both pathogenic (class 5) and currently internationally unlisted variants, were analysed. The skin samples were cultured to acquire primary fibroblasts for nuclear protein extraction. The level of pathogenicity was revealed by MMR-protein activity when substrate DNA with a mismatch was added to the extract. Then, restriction enzymes were used for producing fragments of different lengths, depending on the repair action, and the MMR efficiency was visualized by both electrophoretic methods. Additionally, MAPP-MMR tool was used for studying the MSH2 mismatch variants in silico. By comparing the results from these two methods, we show that the more quantitative Labchip brings diagnostic value to DiagMMR suggesting 100% specificity (n=10) and 90,9% (n=11) sensitivity in reference to the variant information. For example, MSH6 c.3103C>T, which is listed as pathogenic in InSiGHT, was more consistent in giving a MMR deficient (dMMR) result with Labchip. Difference in the functional detection could be seen particularly with the MSH6 variants, but the differences were less notable when Labchip results were compared to the previous interpretations of the samples made based on the validated DiagMMR protocol. With the unlisted MSH6 variants, c.3139dupT was detected as dMMR by Labchip which was in unison with the previous interpretation. Another one, MSH6 c.551delA, was seen as MMR proficient (pMMR) in all the results by both the methods, and with the previous interpretation being unclear, which highlights the importance of further testing of this variant. There was also one unlisted variant (c.1805T>C) among MSH2 for which we got uniform dMMR results in two patients. The high MAPP-MMR score (25.150) for the MSH2 p.Leu602Pro amino acid change also supported the evidence gained of the pathogenic nature of this variant. As a conclusion, DiagMMR can be used reliably for MMR efficiency analysis, especially when performed together with a more quantitative analysis method.
-
(2021)Skeletal dysplasias are a group of rare monogenic bone disorders affecting joints and the skeleton. An increasing number of gene defects have been associated with skeletal dysplasias, but many cases remain without a known cause or a clear diagnosis. Exome sequencing data of the family with two siblings affected with an undiagnosed type of bone dysplasia was examined in this study with the aim of determining the genetic cause behind the phenotype. The causal variant was assumed to be in a novel disease-causing gene, since a previously performed gene panel of skeletal disease-causing genes had not revealed any positive results. The search for potential rare pathogenic variants in genes linked to the skeleton was done with VarAFT filtering software. The search revealed a short list of candidate variants confirmed first with Broad Institute’s Integrative Genomics Viewer (IGV) and then with targeted Sanger sequencing. Conservation analysis on the affected amino acids, in silico functional analysis on the variants and a comprehensive literature review on all candidate genes were performed to evaluate the likelihood of them being the variant behind the phenotype. A shortlist of three genes were obtained with the analyses, with one of them seeming to be the most likely candidate. However, to assuredly identify the disease-causing variant, further testing should be performed. Functional analyses should be done to test the functions of the proteins encoded by the candidate genes and the consequences of the pathogenic variants.
-
(2021)Mature T-cell leukemias/lymphomas (MaTCLs) represent a rare and clinically heterogenous group of diseases that vary from indolent stages to aggressive malignancies with very limited therapy options due to the resistance development against conventional chemotherapies. T-cell prolymphocytic leukemia (T-PLL) is the most common subtype of MaTCLs and was therefore selected as a model disease to study the genetic mechanisms leading to the resistance development with eight clinically relevant cancer drugs. In this Master’s thesis genome-wide CRISPR/Cas9 knock-out screen with Brunello lentiviral library was used to study genetic aberrations that could induce resistance or sensitivity towards the selected cancer drugs. Two main pathways were enriched in multiple drug conditions including p53 regulating genes as well as epigenetic regulation, suggesting these pathways could be related to resistance development mechanisms. p53 pathway is dysregulated in most cancer types and therefore it can also be seen as a positive control of the screen. Epigenetic modulations play an important role regulating cell proliferation and genes related to this pathway are frequently altered in MaTCL patients. The screening results show enrichment of epigenetic regulation and suggest that it has a role in drug resistance development. This thesis work included results only from one genome-wide screening experiment and in the future is important to compare the findings with replicate screen results as well as with other cell lines to confirm the findings. In addition, validation of the most biologically interesting genes should be performed, most optimally in primary cells.
-
(2021)Tumors contain variable number of different immune cells that infiltrate the tumor microenvironment, such as tumor infiltrating lymphocytes (TILs). More research is needed to understand the functional and clinical importance of various TIL subgroups in cancer. Understanding the differences between individual cancer patients will help development of new treatment methods and discovering why only some patients respond to immunological treatments. Renal cell carcinoma (RCC) is the most common kidney cancer type with good overall survival prognosis when the tumor is surgically removed before it has metastasized. However, the prognosis of RCC is significantly decreased when the cancer has spread. The aim of this master’s thesis project was to characterize the tumor infiltrating lymphocyte populations in patient derived RCC samples. Characterization was done with flow cytometry and a custom antibody panel designed to detect various lymphocyte subpopulations. We also wanted to further study the TILs by expanding the lymphocytes from the tumor samples and test their function in an impendence-based assay against matched autologous tumor cells. Based on the flow cytometry results, the different RCC subtypes in the cohort showed some variation in TILs. Still, more research is needed to investigate these differences. We were able to culture the TILs from the RCC tumor samples, and most of them were CD4+ T cells expressing memory markers CD45RO and CCR7. Some expanded TILs expressed markers related to T cell activity and terminal differentiation. In conclusion, this thesis provided material and insights for future RCC TIL experiments as well as considerations for optimization needed in further studies.
-
(2024)Hypercholesterolemia is characterized by elevated levels of low-density lipoprotein (LDL) cholesterol in the bloodstream and contributes substantially to the global burden of atherosclerotic cardiovascular disease (ASCVD). Low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) is an endocytic adaptor protein which assists LDL internalization by promoting LDLR localization in clathrin-coated pits and mutations in LDLRAP1 are associated with autosomal recessive hypercholesterolemia. However, the clinical relevance of many LDLRAP1 variants is yet to be fully determined. The aim of this master’s thesis is to adopt an analysis pipeline for bulk generation and functional characterization of such LDLRAP1 variants. To accomplish this goal, a cell model with impaired LDLR internalization is validated and the activity of LDLRAP1 and LDLRAP1 variants are evaluated within this system. This involves implementing a semi-automated variant generation pipeline to produce LDLRAP1 variants in large-scale, and LDLRAP1 variants are introduced into this cell system. The main finding of this thesis is that impaired LDLRAP1 function results in LDL-LDLR complexes to be retained on plasma membrane. The expression of LDLRAP1-GFP in internalization deficient cells reduced LDL and LDLR accumulation on plasma membrane. These features were evaluated for variant characterization, revealing three variants that may have impaired LDLRAP1 functionality. In conclusion, the findings of this thesis underscore the imperative for large-scale variant generation efforts and highlight the pivotal role of LDLRAP1 functionality in LDL internalization.
-
(2022)Abstract Faculty: Faculty of Biological and Environmental Sciences Degree programme: Genetics and Molecular Biosciences Study track: Molecular and Analytical Health Biosciences Author: Annie Stendahl Title: Measurement repeatability of flow cytometry and nanoparticle tracking analysis for optimization of extracellular vesicle measurements Level: Master’s thesis Month and year: 11/2022 Number of pages: 92 Keywords: extracellular vesicles, repeatability measurements, metrology, traceability, flow cytometry, nanoparticle tracking analysis, reference material, METVES Supervisor or supervisors: Virpi Korpelainen, Katariina Maaninka and Pia Siljander Collaborative partner: VTT Technical Research Centre of Finland Ltd. Where deposited: E-thesis Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles secreted by all cells, containing variable cargo from nucleic acids and proteins to carbohydrates, metabolites, and lipids. EVs are considered to be involved in many physiological and pathological cell functions. Due to their presence in biofluids hence enabling semi-invasive liquid biopsies, EVs have indicated great promise for utilization as biomarkers in clinical settings. The innate properties of EVs and their cargo could also be harnessed into therapeutic use. However, the current methods and reference materials for determining EV concentration and size have not yet achieved the metrological level of repeatability and traceability, which is needed for EV measurements to be utilized in clinical settings. The aim of this thesis project was to evaluate repeatability of the methods typically used for EV quantification and size determination, flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The repeatability was analyzed with reference material made of hollow organosilica beads and biological EV test samples, both developed in an ongoing EU metrology-project METVES II for EVs. A similar biological EV test sample was also prepared as part of the thesis project. Finally, the repeatability measurements were conducted with calibration beads recommended by the instrument manufacturers. The calibration beads gave repeatable results with FCM and one of the two NTA instruments tested, but neither the reference beads nor the biological EV test samples produced repeatable results to enable determination of repeatability. However, valuable understanding was gained on what can be optimized during the measurements and operation of the instruments to generate more repeatable results with FCM and NTA in EV analysis. Prior knowledge of both the sample type and method used for measuring would enable optimization of the measurement and instrument operation. Whether the aim is EV quantification or size determination, instrument errors and bias could then be minimized by adjusting the settings according to sample type. Furthermore, EV quantification and size determination would benefit from combining different methods to ensure more reliable and repeatable results. It is clear that more research needs to be done, for i.e., the tested reference beads need to be further developed to be established as EV reference material and enabling standardization of EV measurements. Standardizing EV quantification and size determination is required to achieve metrological repeatability and ultimately, traceability, and thus for EVs to be utilized in clinical settings as biomarkers or therapeutic use.
-
(2022)Chemoresistance is a significant contributor to the lethality of high-grade serous ovarian cancer (HGSOC). Treatment response to traditional platinum-based chemotherapy is poor, and the need for improvement is urgent, as more than 50% of the patients pass within 5-years from diagnosis. Mitochondrial metabolism has emerged as a potential target in HGSOC, and enhanced capacity in mitochondrial oxidative phosphorylation (OXPHOS) has been shown to correlate with a better chemoresponse. The vital metabolic cofactor for mitochondrial enzymatic reactions, during e.g. OXPHOS, is nicotinamide adenine dinucleotide (NAD+). It is now well-established that NAD+ precursor supplementation can boost intracellular NAD+ content and, consequently, mitochondrial function. In cancer, NAD+ boosting shows mitochondrial activation mediated anticancer and chemosensitizing effects and presents an intriguing route to modulate cancer metabolism and treatment response. In HGSOC, NAD+ metabolism and its association with tumours’ metabolic profile is poorly understood. Also, the impact of mitochondrial activation on HGSOC chemoresponse remains unexplored. This thesis aimed to evaluate patient-derived HGSOC tumour NAD metabolite content and its association with OXPHOS. Also, the aim was to explore whether in vitro NAD+ boosting promotes mitochondrial function and subsequently enhances chemosensitivity to platinum-based treatment. Thus, I measured the NAD metabolite concentrations in HGSOC tumours and two HGSOC cell lines, OVCAR-5 and COV318. The impact of NAD+ boosting on HGSOC cells OXPHOS and chemoresponse was assessed with respirometry and cell viability assays. I found that the HGSOC tumours presented alterations in NAD metabolite content, with an increase in the reduced forms and a decrease in the metabolite redox ratios. Also, the change in the NAD metabolite seemed to be impacted by the tumours’ anatomical location and OXPHOS capacity. In vitro HGSOC cells differed in their OXPHOS capacity, with the OXPHOS-high cell line exhibiting enhanced sensitivity to chemotherapy. The NAD+ boosting increased intracellular NAD+ content and mitochondrial OXPHOS without impacting the cells’ chemoresponse or growth. In conclusion, the altered NAD+ metabolism in HGSOC tumours presents potential target pathways for the disease with poor treatment response. The NAD+ boosting mediated metabolic modulation increased the OXPHOS capacity independently of the cell lines’ OXPHOS-status. In OXPHOS-low cells’ mitochondrial activation enhanced OXPHOS to the level of chemosensitive OXPHOS-high cells but did not alter the cell lines’ chemoresponse within a short-term treatment period. These observations have increased the understanding of NAD+ metabolism. Also, as a proof-of-principle, NAD+ boosting was presented as a tool for mitochondrial activation and metabolic modulation in HGSOC cells, opening an intriguing approach to explore HGSOC mitochondrial function and chemoresponse.
-
(2021)Growth Differentiation Factor-15 (GDF15) is a neurotrophic factor associated with anorexia and weight loss. It is elevated in obesity and various diseases. It signals by forming a tripartite complex with the coreceptor Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-like (GFRAL) and the receptor Rearranged during transfection (RET). Targeting this pathway has therapeutic potential for the treatment of obesity and anorexia-cachexia syndrome, but many aspects are still unclear. What is the affinity of binding between these proteins? Does GDF15 induce dose- and time-dependent RET phosphorylation and activate intracellular signaling pathways, and are there differences between GDF15 and GDNF signaling, as the different bend angles of their complexes suggest? Can soluble GDF15-GFRAL mediate the effects of GDF15 outside of the brainstem, and what is the function of the short cytoplasmic domain of GFRAL? Furthermore, how well is the pathway evolutionally conserved between species? Binding affinities were assessed with microscale thermophoresis, whereas RET phosphorylation and intracellular signaling assays were performed utilizing immunoprecipitation and western blotting. GFRAL-RET binding is low-affinity (350 nM ± 223) similarly to GFRα1-RET binding (GDNF family receptor alpha-1), whereas GDF15-RET binding without GFRAL does not occur. GDF15 appears to compete for binding to GFRAL or RET, differing from GDNF mechanisms, but noise in the data may have affected the results. The data provide ideas about the ligand-receptor complex formation. Furthermore, RET phosphorylation by GDF15 is dose- and time-dependent. Firstly, the strongest RET and ERK activation occur at GDF15 concentrations typical of disease states. Secondly, RET activation by GDF15 is rapid and sustained like by GDNF activation, whereas ERK activation by GDF15 is rapid and much more transient than by GDNF. Thirdly, AKT activation by GDF15 is much weaker than by GDNF. The differences may be caused by different conformations of binding surfaces for adaptor proteins being available on RET because the bend angles of the complexes are different. Moreover, soluble GDF15-GFRAL does not activate RET, although soluble GDNF-GFRα1 does. Also, the short cytoplasmic domain of GFRAL is not necessary for activating AKT and ERK pathways, but may be needed to activate RET. Furthermore, GDF15 from cynomolgus monkey, but not rat or mouse, activates RET with human GFRAL, indicating sequence similarity in the active site of GDF15. In conclusion, novel aspects of GDF15 signaling and differences between GDF15 and GDNF signaling were discovered.
-
(2024)Coronary artery disease is a leading cause of death and disability globally. The complement system has an important role in vascular health by defending against pathogens and regulating inflammation. Dysregulation of complement activation can lead to inflammation and the weakening of the vascular wall. The study investigated polymorphisms in the complement factor H (CFH) - complement factor H related (CFHR) genes region, which can predispose to inflammation and thus lead to coronary artery diseases. This study specifically focused on the CFHR 1 gene region because it competes with complement factor H, which plays a protective role. The protein structure of CFH and CFHR1 is similar. CFH protects the complement attacks, and on the other hand, CFHR1 does not prevent complement activation. 225 samples were selected for this study to evaluate the CFH-CFHR gene region in different patient groups from the COROGENE Cohort. Patients were assigned for coronary angiograms at the Helsinki University Central Hospital, and samples were collected every 5 years within 12 months to study trends in heart disease and coronary risk factors. Samples were analysed by SALSA MLPA (Multiplex Ligation-dependent Probe Amplification) to detect the presence of copy number variations (CNVs) in the CFH-CFHR gene region. The results of analysis indicate that the mortality rate was higher in this follow-up among males with CFHR 1 deletion compared to males with a normal genotype (p= 0.013). Furthermore, males with myocardial infarction (MI) and CFHR 1 deletion have a higher mortality rate than males with a normal genotype (p = 0.032). The results of logistic regression analysis indicate that CFHR 1 deletion, combined with other risk factors, is more likely to lead to non-ST elevated myocardial infarction (NSTEMI), than ST elevated myocardial infarction (STEMI). NSTEMI is more difficult to detect on an ECG compared to STEMI. A limitation of this study was the small sample size in each group. This study adds to the understanding that STEMI and NSTEMI have different risk factors. These differences should perhaps be considered in diagnostics and treatment once we understand them.
-
(2020)Human induced pluripotent stem cells (hiPSC) can be propagated in a long-term culture and further differentiated into many cell types, including cardiomyocytes (CM) and endothelial cells (EC). Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are promising tools in cardiac research, since they retain the original genotype of the individual donor and thus enable the use of patient- and disease specific cells. Crucial for the optimal use of hiPSC-CMs in experiments are methods for assessing cardiomyocyte phenotype. Contraction is a prominent feature for CMs, and it is essential that contraction can be quantified accurately. Reliable quantification is relevant when hiPSC-CMs are used for studying disease phenotypes, cardiac safety pharmacology, genotype-phenotype correlations, cardiac disease mechanisms and cardiac function over time. In this thesis project, contractile behavior of hiPSC-CMs was analyzed using video microscopy and online tool MUSCLEMOTION. Contraction parameters were obtained from hiPSC-CMs derived from patients with hypoplastic left heart syndrome (HLHS) and healthy controls on multiple timepoints during differentiation. In addition, contraction was analyzed in iPSC-CMs cocultured with induced pluripotent stem cell derived endothelial cells (iPSC-ECs), since it has been suggested that ECs can promote morphological and functional maturation of CMs in culture. Contraction duration (CD), time to peak (TTP), relaxation time (RT) and contraction amplitude (CA) was compared between different timepoints as well as between CMs cocultured with ECs and CMs cultured alone. Compared to control cell lines, HLHS patient hiPSC-CMs exhibited longer CD, TTP and RT as well as higher CA values. This difference was present in most of the timepoints, suggesting slower contractile kinetics in HLHS patient iPSC-CMs compared to control iPSC-CMs. Significant changes were also observed in contraction parameters when comparing hiPSC-CMs in coculture and monoculture. Contraction parameters of coculture iPSC-CMs changed in a relatively consistent manner over time, increasing or decreasing throughout the monitoring period whereas in hiPSC-CM monoculture there was more variation between timepoints. This project and results support the use of modern methods in detailed functional characterization of hiPSC-derived cells. In addition, it highlights the potential of coculture in disease modeling and the fact that hiPSC-CMs express variation in phenotypes. However, experiments should be repeated, and additional methods should be used in order to further validate the results and conclusions.
-
(2022)Suuresta tarpeesta huolimatta, ei työvälineitä geeniekspression endogeeniseen säätelyyn ole nykypäivänä tarjolla lääketieteellisiin eikä tutkimuksellisiinkaan tarkoituksiin. Vuonna 2017 karakterisoitiin uuden CRISPR-Cas tyypin VI kompleksit. Nämä kompleksit hyödyntävät endonukleaasiaktiivisuudessaan uudenlaista Cas13 -efektoria, jonka kohteena toimii yksijuosteiset RNA-molekyylit. Cas13 tunnistaa kohteensa opas-RNA:n (gRNA) avulla. Tätä komplementaarisuutta hyödyntämällä on onnistuttu kohdistamaan Cas13:n aktiivisuus spesifisiin lähetti-RNA molekyyleihin nisäkässolukokeissa ilman kohde-RNA:n ulkopuolisia hajoamistuotteita. Tämän lisäksi nukleaasiinaktivoituja Cas13 efektoreita (dCas13) on käytetty esimerkiksi kohdennettuun nukleotidien deaminaatioon ja vaihtoehtoisen silmikoinnin indusointiin. Tämä todistaa dCas13 efektorien kyvyn toimia ohjelmoitavina apuproteiineina, joiden avulla voidaan ohjata kompleksiin liitetyn proteiinin toiminta spesifisille kohde-RNA:n alueille. Tässä tutkimuksessa, kaksi eukaryoottien translaatioon osallistuvaa proteiinia, ELAVL1 ja EIF4E, yhdistettiin dCas13b efektorin kanssa C- ja N-terminaalisesti, tarkoituksena lisätä kohteena olevan lähetti-RNA:n ekspressiota parantamalla tämän translatiivisuutta tai stabiliteettia. Koeasetelmassa vertailtiin näiden neljän fuusioproteiinin sekä aktiivisen Cas13b:n vaikutusta lusiferaasin ekspressioon käyttäen kuutta eri gRNA:ta aktivaation ohjaamisessa. Kokeet suoritettiin in vitro ympäristössä HEK293T-soluissa. Natiivi Cas13b vähensi merkittävästi lusiferaasisignaalia, kun taas samassa asetelmassa C-terminaalinen dCas13b-ELAVL1 fuusio lisäsi tätä tilastollisesti merkittävästi eli aikaansai kasvaneen lusiferaasiekspression. Vastaavaa vaikutusta ei ollut havaittavissa muissa fuusioproteiineissa. Tulokset todistavat onnistuneen Cas13b-välitteisen degradaation, sekä dCas13bELAVL1-välitteisen lisääntyneen lusiferaasitranskriptin translaation. Jatkotutkimusta vaaditaan fuusioproteiinin toiminnallisuuden ja varsinaisen kohteen ulkopuolisen aktiivisuuden määrityksessä. Siitä huolimatta tutkimuksessa esitelty fuusoproteiinikonstrukti voisi hyvin olla toimiva työkalu geeniekspression spesifin lisäyksen mahdollistamisessa endogeenisessa kontekstissa
Now showing items 1-20 of 32