Browsing by master's degree program "Genetiikan ja molekulaaristen biotieteiden maisteriohjelma"

Human induced pluripotent stem cells (hiPSCs) are derived from adult differentiated somatic cells and reprogrammed to an embryonic-like state. Pluripotent stem cells can be differentiated into almost any somatic cell type by using directed differentiation methods, but the differentiation efficiency often varies depending on the cell type. hiPSCs and cells differentiated from them can be used as a disease model carrying the patient’s phenotype and genotype. Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease where both upper and lower motor neurons degenerate, leading to paralysis. There is no curative treatment for ALS, and it leads to the death of the patient in 3 to 5 years on average from the first symptoms. The most common genetic cause of familial ALS is a hexanucleotide repeat expansion in C9orf72-gene. ALS pathology is strongly linked to endoplasmic reticulum (ER) stress, which affects cell homeostasis and proteostasis, and leads to apoptosis when prolonged. The primary aim of this research is to characterize the differentiation of four hiPSCs lines towards lower motor neurons and to study the neuroprotective effects of cerebral dopamine neurotrophic factor (CDNF) and CDNF-derived peptide on ER stress and cell viability. This experiment used two control cell lines from two healthy donors and two patient cell lines from two different ALS patients carrying the C9orf72-mutation. To evaluate the efficiency of the differentiation towards motor neurons, molecular markers for pluripotent and neural progenitor cells as well as for maturated motor neurons were analyzed. Relative gene expression levels were measured from weekly time points with qPCR. Immunocytochemical (ICC) antibody staining was performed during differentiation. Endogenic CDNF levels were analyzed from differentiating cells at weekly time points and the effect of CDNF on Thapsigargin (TG) -induced ER stress in motor neurons was analyzed. In addition, cell viability was analyzed in TG-CDNF treatment. All pluripotent and progenitor markers were downregulated in differentiated cells, and the expression of the mature motor neuron markers was upregulated. Mature motor neuron markers were also expressed at the protein level. The endogenous CDNF levels were highest at the progenitor cell stage. The ER stress response was upregulated in TG-treated cells, and there were no differences between treatments against ER stress. Furthermore, TG and growth factor treatments differentially affected the viability of the control and patient cell lines. Treatment decreased viability in control cell lines and increased viability in patient cell lines. Pluripotent stem cells were successfully differentiated toward motor neurons. The differentiation was performed twice, and the results were similar on both individual biological repeats. Analysis of endogenous CDNF expression levels was performed for the first time on hiPSCs lines. In this study, CDNF or its derivate didn’t reduce ER stress but it influenced cell viability, especially in patient cell lines with growth factor treatment. In the future, TG-treatment could be optimized regarding timing and growth factor treatment, or the toxin could be changed to another ER-stress inducing toxin. In addition, the C9orf72 pathology should be identified in order to use differentiated motor neurons as a pre-clinical disease model.

Chimeric antigen receptor (CAR) T cells are genetically modified usually autologous T cells expressing de novo designed CAR that binds a specific antigen on the surface of the cancer cells, inducing T cell receptor-independent activation and cytotoxic response against the targeted cancer cells. While CAR T cells have been shown to offer effective treatment in acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma, several resistance mechanisms can lead to CAR T cell exhaustion characterized by impaired functions and the expression of inhibitory receptors. The Finnish Red Cross Blood Service has developed novel CARs, differing in structure from the ones currently published. Since the evasion of CAR T cell exhaustion is considered one of the key objectives in the development of CAR T cell therapy, this Master’s thesis project aimed to create a working method to determine the exhaustion of CAR T cells in vitro after long-term repeated stimulation. In order to induce and measure exhaustion, CAR T cells were produced and activated ex vivo in the presence of IL-2 or IL-7/IL-15 cytokines, cultured long-term and repeatedly stimulated by exposure to target cells. CAR T cell cytotoxicity and expansion were determined and the expression of various inhibitory receptors was analyzed. The method enabled the comparison of the designed CAR T cell candidates and the positive control CD19-CD28ζ CAR T cells in long-term cytotoxic potency. In addition, it helped to reveal the surprising difference between IL-2 and IL-7/IL-15 cytokines and their impact on CAR T cell exhaustion. Although CAR T cells produced with IL-2 had poorer expansion during CAR T cell production than CAR T cells produced with IL-7/IL-15, they showed lower expression of exhaustion-related markers supported by better survival, proliferation and cytotoxic activity during long-term repeated stimulation assay.

Background– The BCL-2 protein family members are major regulators of apoptosis, and the anti-apoptotic (pro-survival) members of the family is commonly targeted with BH3 mimetic drugs in haematological cancers. However, these treatments have not been very impactful when administered as single agents and they have long been investigated for combination therapy with other agents. Acute myeloid leukaemia (AML) is one of the difficult-to-cure haematological malignancies. A recently approved therapy for AML consists of the combinatorial administration of venetoclax (a selective BCL-2 inhibitor) and a DNA methyltransferase (DNMT) inhibitor such as azacitidine or decitabine. Although this novel therapy has shown promising clinical results, the majority of the patients still relapse under this treatment. These relapsed patients typically become highly resistant to treatment and have poor prognosis, emphasising the need for new effective drug combinations. Apart from BCL-2, other family members like BCL-xL and MCL1 are also common targets of BH3-mimetic drugs. This project thus aims to understand and characterise the resistance against BH3-mimetics and investigate new therapeutic approaches to overcome the challenges of resistance. Aims– This study aims (i) to characterise BH3-resistant AML cell lines for uncovering the mechanisms of drug resistance, and (ii) to identify possible combination treatment options for overcoming drug-resistance. Methods– Viability assays with Cell Titer Glo® (CTG) and Drug Sensitivity and Resistance Testing (DSRT). The long-term effectiveness of venetoclax, azacitidine and talazoparib (a PARP inhibitor) as single agents, double combinations and triple combination were investigated with Time-to-Progression (TTP) assay. For the resistant cell line models, underlying resistance mechanisms were assessed by checking protein expression of pro- and/or anti-apoptotic members of the BCL-2 family members with western blot (WB). Real-time quantitative PCR (RT-qPCR) and WB were carried out for transcriptional and translational expression analyses of certain DNA damage-associated genes in PARP inhibitor-resistant cell lines. Results– Drug screening with DSRT has revealed promising results for two combination treatments of a BCL-xL inhibitor (A-1331852) (i) with an Aurora kinase A inhibitor (alisertib) and (ii) with an MCL1 inhibitor (S63845) for BCL-xL inhibitor-resistant cells. WB analyses of BCL-2 family members showed translational upregulation of un-inhibited members of the anti-apoptotic proteins in BH3-mimetic-resistant cell lines. A venetoclax-resistant AML cell line showed increased levels of the DNA damage marker P-γ-H2Ax upon treatments containing venetoclax, as well as increased levels of cleaved-PARP1, indicating induction of apoptosis. RT-qPCR analyses revealed increased mRNA expression of PARP1 in two resistant cell lines, whereas no significant expression changes in other DNA repair mechanism genes on the transcriptional level. Conclusions– In BH3-mimetic-resistant AML cell lines, apoptosis is avoided through translational upregulation of un-inhibited anti-apoptotic members of the BCL-2 family, and this resistance can be countered by combination treatment for additional inhibition of the compensatory anti-apoptotic proteins. Venetoclax is still effective on cells resistant to it, by inducing DNA damage and sensitising these cells against inhibitors of the members of DNA repair pathway. The transcriptional upregulation of PARP1 and the increase in its auto-catalytic activity suggests the DNA damage-inducing effects of the triple combination treatment [Ven + Aza + Tal].

Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.

Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.

The human cerebral cortex is characteristically large and folded, which can be majorly attributed to the high number and variety of neural progenitors during embryonic development. Radial glial cells are essential neural progenitors during neurogenesis. In addition to giving rise to new cell types, they also provide scaffold for migrating newborn neurons. Radial glia are known to portray peculiar characteristics in their cell division process, including unique migratory behavior as well as specifically regulated cleavage furrow orientation. While these processes of radial glial division have been studied extensively, the underlying molecular mechanisms are still largely unknown. ABBA (actin-bundling protein with BAIAP2 homology) and NEDD9 (neural precursor cell expressed, developmentally downregulated 9) are proteins, which are both known to be expressed in certain radial glia progenitors during embryonic development, while they are mainly absent in neurons. ABBA has a defined role of regulating plasma membrane deformation and actin polymerization in radial glia, while NEDD9 expression levels are a known factor in the correct progression from mitosis to cytokinesis. An interaction between ABBA and NEDD9 has previously been identified in a yeast two-hybrid screen done for the embryonic mouse brain. The aim of this thesis was to validate the interaction between ABBA and NEDD9 biochemically. First, their interaction was evaluated by doing co-immunoprecipitation assays on the endogenous proteins from C6 cells. The second approach was to test, whether their interaction is directly mediated by the N-terminal SH3-domain of NEDD9 and the proline-rich C-terminal portion of ABBA. This was done by doing biochemical binding assays using purified proteins and domains of interest. While co-immunoprecipitation of the two proteins gave results indicating an interaction, I could show that there is no direct binding between NEDD9 SH3-domain and ABBA, suggesting that the interaction might require other domains or be indirect. Together, these results provide valuable information that will help characterize what roles of ABBA and NEDD9 play in cortical development and beyond.

Uterine leiomyomas are benign smooth muscle tumors arising in myometrium. They are very common, and the incidence in women is up to 70% by the age of 50. Usually, leiomyomas are asymptomatic, but some patients suffer from various symptoms, including abnormal uterine bleeding, pelvic pain, urinary frequency, and constipation. Uterine leiomyomas may also cause subfertility. Genetic alterations in the known driver genes MED12, HMGA2, FH, and COL4A5-6 account for about 90 % of all leiomyomas. These initiator mutations result in distinct molecular subtypes of leiomyomas. The majority of whole-genome sequencing (WGS) studies analyzing chromosomal rearrangements have been performed using fresh frozen tissues. One aim of this study was to examine the feasibility of detecting chromosomal rearrangements from WGS data of formalin-fixed paraffin embedded (FFPE) tissue samples. Previous results from 3’RNA-sequencing data revealed a subset of uterine leiomyoma samples that displayed similar gene expression patterns with HMGA2-positive leiomyomas but were previously classified as HMGA2-negative by immunohistochemistry. According to 3’RNA-sequencing, all these tumors overexpressed PLAG1, and some of them overexpressed HMGA2 or HMGA1. Thus, the second aim of this study was to identify driver mutations in these leiomyoma samples using WGS. In this study, WGS was performed for 16 leiomyoma and 4 normal myometrium FFPE samples. The following bioinformatic tools were used to detect somatic alterations at multiple levels: Delly for chromosomal rearrangements, CNVkit for copy-number alterations, and Mutect for point mutations and small insertions and deletions. Sanger sequencing was used to validate findings. The quality of WGS data obtained from FFPE samples was sufficient for detecting chromosomal rearrangements, although the number of calls were quite high. We identified recurrent chromosomal rearrangements affecting HMGA2, HMGA1, and PLAG1, mutually exclusively. One sample did not harbor any of these rearrangements, but a deletion in COL4A5-6 was found. Biallelic loss of DEPDC5 was seen in one sample with an HMGA2 rearrangement and in another sample with an HMGA1 rearrangement. HMGA2 and HMGA1 encode architectural chromatin proteins regulating several transcription factors. It is well-known that HMGA2 upregulates PLAG1 expression. The structure and functionality of HMGA2 and HMGA1 are very similar and conserved, so it might be that HMGA1 may also regulate PLAG1 expression. The results of this study suggest that HMGA2 and HMGA1 drive tumorigenesis by regulating PLAG1, and thus, PLAG1 rearrangements resulting in PLAG1 overexpression can also drive tumorigenesis. A few samples, previously classified as HMGA2-negative by immunohistochemistry, revealed to harbor HMGA2 rearrangements, suggesting that the proportion of HMGA2-positive leiomyomas might be underestimated in previous studies using immunohistochemistry. Only one study has previously reported biallelic inactivation of DEPDC5 in leiomyomas, and the results of this study support the idea that biallelic loss of DEPDC5 is a secondary driver event in uterine leiomyomas.

Tiivistelmä – Referat – Abstract ROS or Reactive Oxygen Species can be found throughout all living organisms on the planet. Without ROS, processes, which are essential for the sustainment of most living organisms, such as respiration would not be possible. On the other hand, uncontrolled ROS generation can cause severe damage to the cellular structure. The family of ROS includes multiple compounds, which share a common trait of high chemical activity. ROS can be produced on demand by specific enzymes which are localized within cellular structures, such as membranes. One group of enzymes is called NADPH (Nicotinamide adenine dinucleotide phosphate) oxidases. These enzymes possess common structure which is composed of transmembrane region with multiple loop helixes and usually two or more terminal motifs, which are devised into regulatory EF-hand motifs and catalytic motifs. NADPH oxidases are essential ROS producers and can be found throughout most clades of living organism and are widely represented in different cellular compartments and distributed across different tissues in multicellular organisms. As an example, Nox family of NADPH oxidases can be found in human tissues and immune cells. Another common group of NADPH oxidases is respiratory burst oxidase homologues (RBOH) can be found in plants. Members of this group play important role in plant immune defense against pathogens. One example is AtRBOHD, which is expressed in Arabidopsis genus of plants. Upon activation, these enzymes are known to produce hydrogen peroxide (H2O2) as mean of antibacterial defense. These host defense mechanisms are known to be driven by different signaling molecules. It has been determined that in some examples of NADPH Oxidases, including Nox5 and RBOHD, the state of activation can be induced through the effects of Ca2+ ions. Moreover, it has been determined, that ROS-producing state of these NADPH oxidases is achieved through change of conformation. This change in conformation is attributed to the different modes of interaction of motifs of oxidases, which are dependent on concentration of bivalent cation Ca2+. Previous research regarding intramolecular interactions within specific NADPH oxidase- Nox5β has been performed by multiple research teams and different sources appear to contradict each other on the exact mode of interaction of Nox5β EF-hand upon presence of Ca2+. Therefore the exact interaction model of terminals of Nox5β is unclear. In addition, the effect of presence of Ca2+ on the interaction terminals in another representative of NADPH oxidases- AtRBOHD, which possess highly analogous molecular structure of catalytic C-terminus to Nox5β, has never been thoroughly studied, as well as interactive cross-compatibility of the C and N terminals from these two distinct species of NADPH oxidases. The objectives of this research are to analyze intramolecular interactions of N- and C- terminals in Arabidopsis RBOHD and Human Nox5β upon presence of ionic calcium, compare Ca2+-induced terminals interactions in said oxidases and to establish possible cross-compatibility of terminals in these two distinct NADPH oxidase species. Practical aspects of this research included cloning the C- and N- cytoplasmic regions of Nox5β and AtRBOHD into bacterial expression vectors utilizing the PIPE cloning method, heterologous production of epitope-tagged tails of NOX5β and RBOHD in E. Coli BL21 and finally in-vitro pull-down assays to analyse the interactions of the tails upon the presence of Ca2+ as well as interactive cross-compatibility of these tails. By utilizing methods mentioned above, this research has demonstrated that interactions of terminals motifs both in Nox5β and AtRBOHD are possible even in calcium-deprived environment, which was achieved through use calcium-binding agent (EDTA) and the effect of calcium on interactions of terminals both in RBOHD and Nox5β is very limited if not insignificant. This research has also demonstrated that the cross-compatible interactions between terminals of Nox5β and AtRBOHD are possible. Results of this research indicate a strong structural conservation within NADPH oxidases, which indicates similar intramolecular interaction mechanisms within two highly diverged species. These findings may prove to be useful as a background for the future research regarding ROS producing enzymes and evolutional conservation in structures of oxidases.

The protein kinase C (PKC) enzyme is a type of peripheral membrane protein that is classified as a member of the Ser/Thr kinases superfamily. Its function is to add phosphate groups to serine or threonine residues in other proteins. The multitude of functions that PKC performs, and consequently its involvement in a diverse array of diseases, is attributed to the complicated nature of the enzyme: it consists of 12 distinct isoforms, each exhibiting minor variations in catalytic activity. Out of them, eight have therapeutic potential and utilize diacylglycerol (DAG) as a secondary signalling molecule. The research in this thesis is to perform computational modelling that assists an experimental research program searching for selective activators for PKC. The particular project involves combining molecular modelling with insight from structural biology. In concrete terms, three pieces of scientific work are involved: 1) The investigation of the behaviour in the membrane of drug candidate molecules that emerge as new scaffolds is developed by the experimental medicinal chemistry team. 2) Predicting the structure of PKCα, PKCδ, and PKCϵ using AlphaFold2 and evaluating their stability using molecular dynamic simulation. 3) Combining the predicted structures with DAG-containing membranes to examine predicted protein binding to the membrane. According to the results, 15 candidates out of 21 exhibited comparable behaviour to the positive control in terms of their angle of penetration into the membrane leaflets. Turning to regulatory domain prediction, all three proteins’ C1 and C2 domains were predicted with high confidence scores. All predicted structures showed root mean square deviation (RMSD) and root mean square fluctuation (RMSF) within the normal range, and most of their secondary structures were stable during simulation. When combined with the membrane, the C2 domains showed stable interactions with the membrane, and C2 binding to the membrane completely triggers C1 to bind to the membrane; however, there were some instabilities between the C1 domain binding and the membrane. This study highlights promising PKC activators, demonstrating the utility of computational modelling for identifying potential therapeutic agents.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant disease that leads to cognitive impairment, vascular dementia and ischemic strokes. In CADASIL, vascular smooth muscle cells (VSMCs) degrade gradually and are replaced by connective tissue in the small and mid-sized arteries in the brain. Extracellular granular osmiophilic material (GOM) that surround the VSMCs are a unique feature in CADASIL. The causal gene behind CADASIL is Notch3, which encodes a transmembrane protein with a signaling function. There are over 200 cysteine-altering mutations that cause CADASIL in Notch3. The potential pathology causing mechanism is still unclear, but most likely the mechanism is linked to the aggregation of GOM deposits that are potentially toxic to VSMCs. This thesis project aimed to correct CADASIL causing c.475C>T mutation in Notch3 in different CADASIL cell lines with different CRISPR base editor systems. Another aim was to create induced pluripotent stem cell (iPSC) lines from a CADASIL patient-derived skin biopsy sample to be used in the creation of an in vitro disease model for CADASIL. RNA-based ABEmax base editor system was used to correct immortalized- and primary- CADASIL cell lines. DNA-based ABEmax base editor system was used as a positive control. Simultaneous pluripotent reprogramming and pathogenic CADASIL mutation correction were done in the same transfection during this project. The editing efficiencies were evaluated by Sanger sequencing the genomic target region before and after the transfection. The editing efficiencies were good in general compared to literature. They ranged from 27 % to 73 % target base editing efficiency depending on the editing system-, guide-RNAs - and electroporation parameters used. Confirmed proximal off-target effects were not detected, and distal off-target effects were not evaluated.