Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

Cell Model Systems in Characterizing Alpha2-Adrenoceptors as Drug Targets.

The three alpha2-adrenoceptor (alpha2-AR) subtypes belong to the G protein-coupled receptor superfamily and represent potential drug targets. These receptors have many vital physiological functions, but their actions are complex and often oppose each other. Current research is therefore driven towards discovering drugs that selectively interact with a specific subtype. Cell model systems can be used to evaluate a chemical compound's activity in complex biological systems. The aim of this thesis was to optimize and validate cell-based model systems and assays to investigate alpha2-ARs as drug targets. The use of immortalized cell lines as model systems is firmly established but poses several problems, since the protein of interest is expressed in a foreign environment, and thus essential components of receptor regulation or signaling cascades might be missing. Careful cell model validation is thus required; this was exemplified by three different approaches. In cells heterologously expressing alpha2A-ARs, it was noted that the transfection technique affected the test outcome; false negative adenylyl cyclase test results were produced unless a cell population expressing receptors in a homogenous fashion was used. Recombinant alpha2C-ARs in non-neuronal cells were retained inside the cells, and not expressed in the cell membrane, complicating investigation of this receptor subtype. Receptor expression enhancing proteins (REEPs) were found to be neuronalspecific adapter proteins that regulate the processing of the alpha2C-AR, resulting in an increased level of total receptor expression. Current trends call for the use of primary cells endogenously expressing the receptor of interest; therefore, primary human vascular smooth muscle cells (SMC) expressing alpha2-ARs were tested in a functional assay monitoring contractility with a myosin light chain phosphorylation assay. However, these cells were not compatible with this assay due to the loss of differentiation. A rat aortic SMC cell line transfected to express the human alpha2B-AR was adapted for the assay, and it was found that the alpha2-AR agonist, dexmedetomidine, evoked myosin light chain phosphorylation in this model.

3D-robottipoimintajärjestelmän kehittäminen

Lasertekniikkaa hyödyntävä 3D-kuvaustekniikka tuo uusia mahdollisuuksia robotilla suoritettavaan kasastapoimintaan. Kasasta otetun syvyyskuvan avulla tuotteista voidaan määrittää perinteisen XY-paikkatiedon lisäksi tuotteen korkeus- ja asentotieto. Näitä uusia ominaisuuksia hyödyntämällä robotilla voidaan suorittaa yksittäisen tuotteen poiminta kasasta eri korkeuksilta ja eri asennoista. Diplomityö kuuluu osana Master Automation Groupin ensimmäiseen 3D-tekniikkaan perustuvaan MAG PixCell 3D -robosoituun kappaleenkäsittelysoluun. Työn tavoitteena on kehittääsyvyyskuvan käsittelyyn algoritmeja, joiden avulla robotilla voidaan poimia yksitellen kasassa olevia metallisia saksen teriä. Algoritmien tarkoituksena on varmistaa kasasta löydettyjen terien poimittavuus sekä määrittää poimittavien terien korkeudet ja asennot. Tarkastusten jälkeen robotille välitetään terien XYZ-koordinaatti- ja asentotiedot.

Kokoonpanon kehittäminen tuulivoimalakoneikkojen valmistuksessa

Tämä diplomityö on tehty Hollming Works Oy:n Loviisan yksikölle. Työn tekovaiheessa yrityksessä oltiin aloittamassa tuulivoimalakoneikkojen sarjatyönä tapahtuvaa kokoonpanoa. Työn tavoitteena oli kehittää koneikkojen kokoonpanoverstaan toimintaa. Ennen työn alkua yritykseen oli perustettu tuulivoimalakoneikkojen kokoonpanoon tuoteverstas. Verstaalla kootaan raskaita osakokoonpanoja, jotka lopulta yhdistetään koneikoksi, jota kutsutaan myös naselliksi. Tämän jälkeen varusteluvaiheessa koneikkoon asennetaan mm. erilaisia sähköisiä ja hydraulisia järjestelmiä. Varusteluvaiheen päätteeksi koneikon toiminta testataan. Viimeisenä vaiheenakoneikon päälle asennetaan lasikuitukuori. Työn alkuosassa on käyty läpi tuotannon- ja materiaalinohjauksen perusteita, kokoonpanon kehittämistä ja layout-suunnittelua, jonka pohjalta tuulivoimalaverstaalle tehtiin kokoonpanosoluihin perustuva layoutsuunnitelma. Tuotannonohjaus suunnitelmassa perustuu kapeikko-ohjaukseen. Tuotannon kapeikon muodostava varusteluvaihe imee aiemmista vaiheista osakokoonpanot. Varusteluvaiheen ja testauksen jälkeen koneikko siirtyytyöntöohjatusti lasikuitukuoren asennukseen. Järjestelmässä pyritään tehokkaaseen tilankäyttöön, lyhyeen läpäisyaikaan ja vähäiseen keskeneräisen tuotannon määrään.

Factors Regulating Chondrogenic Differentiation

Chondrogenesis is a co-ordinated differentiation process in which mesenchymal cells condensate, differentiate into chondrocytes and begin to secrete molecules that form the extracellular matrix. It is regulated in a spatio-temporal manner by cellular interactions and growth and differentiation factors that modulate cellular signalling pathways and transcription of specific genes. Moreover, post-transcriptional regulation by microRNAs (miRNAs) has appeared to play a central role in diverse biological processes, but their role in skeletal development is not fully understood. Mesenchymal stromal cells (MSCs) are multipotent cells present in a variety of adult tissues, including bone marrow and adipose tissue. They can be isolated, expanded and, under defined conditions, induced to differentiate into multiple cell lineages including chondrocytes, osteoblasts and adipocytes in vitro and in vivo. Owing to their intrinsic capability to self-renew and differentiate into functional cell types, MSCs provide a promising source for cell-based therapeutic strategies for various degenerative diseases, such as osteoarthritis (OA). Due to the potential therapeutic applications, it is of importance to better understand the MSC biology and the regulatory mechanisms of their differentiation. In this study, an in vitro assay for chondrogenic differentiation of mouse MSCs (mMSCs) was developed for the screening of various factors for their chondrogenic potential. Conditions were optimized for pellet cultures by inducing mMSC with different bone morphogenetic proteins (BMPs) that were selected based on their known chondrogenic relevance. Characterization of the surface epitope profile, differentiation capacity and molecular signature of mMSCs illustrated the importance of cell population composition and the interaction between different populations in the cell fate determination and differentiation of MSCs. Regulation of Wnt signalling activity by Wnt antagonist sFRP-1 was elucidated as a potential modulator of lineage commitment. Delta-like 1 (dlk1), a factor regulating adipogenesis and osteogenesis, was shown to exhibit stage-specific expression during embryonic chondrogenesis and identified as a novel regulator of chondrogenesis, possibly through mediating the effect of TGF-beta1. Moreover, miRNA profiling demonstrated that MSCs differentiating into a certain lineage exhibit a specific miRNA expression profile. The complex regulatory network between miRNAs and transcription factors is suggested to play a crucial role in fine-tuning the differentiation of MSCs. These results demonstrate that commitment of mesenchymal stromal cells and further differentiation into specific lineages is regulated by interactions between MSCs, various growth and transcription factors, and miRNA-mediated translational repression of lineage-specific genes.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

Regulation of self-renewal and detection of karyotypic changes of pluripotent human embryonic stem cells

Human embryonic stem cells are pluripotent cells capable of renewing themselves and differentiating to specialized cell types. Because of their unique regenerative potential, pluripotent cells offer new opportunities for disease modeling, development of regenerative therapies, and treating diseases. Before pluripotent cells can be used in any therapeutic applications, there are numerous challenges to overcome. For instance, the key regulators of pluripotency need to be clarified. In addition, long term culture of pluripotent cells is associated with the accumulation of karyotypic abnormalities, which is a concern regarding the safe use of the cells for therapeutic purposes. The goal of the work presented in this thesis was to identify new factors involved in the maintenance of pluripotency, and to further characterize molecular mechanisms of selected candidate genes. Furthermore, we aimed to set up a new method for analyzing genomic integrity of pluripotent cells. The experimental design applied in this study involved a wide range of molecular biology, genome-wide, and computational techniques to study the pluripotency of stem cells and the functions of the target genes. In collaboration with instrument and reagent company Perkin Elmer, KaryoliteTM BoBsTM was implemented for detecting karyotypic changes of pluripotent cells. Novel genes were identified that are highly and specifically expressed in hES cells. Of these genes, L1TD1 and POLR3G were chosen for further investigation. The results revealed that both of these factors are vital for the maintenance of pluripotency and self-renewal of the hESCs. KaryoliteTM BoBsTM was validated as a novel method to detect karyotypic abnormalities in pluripotent stem cells. The results presented in this thesis offer significant new information on the regulatory networks associated with pluripotency. The results will facilitate in understanding developmental and cancer biology, as well as creating stem cell based applications. KaryoliteTM BoBsTM provides rapid, high-throughput, and cost-efficient tool for screening of human pluripotent cell cultures.

Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Alpha- and gamma-melanocyte stimulating hormones in obesity. A study of the key central areas of metabolic regulation

The melanocortin system is an important regulator of feeding, energy metabolism,and cardiovascular function and it consists of the pro-opiomelanocortin (POMC) derived melanocyte stimulating hormones (α-, β- and γ-MSH) and their endogenous melanocortin receptors, MC1R to MC5R. In the hypothalamus, α-MSH reduces food intake, and increases energy expenditure and sympathetic tone by binding to MC4R. Mutations affecting the MC4R gene lead to obesity in mammals. On the other hand, the metabolic effects of MC3R stimulation using agonists such as the endogenously expressed γ-MSH have been less extensively explored. The main objective of this study was to investigate the long-term effects of increased melanocortin tone in key areas of metabolic regulation in the central nervous system (CNS) in order to investigate the sitespecific roles of both α-MSH and γ-MSH. The aim was to stereotaxically induce local overexpression of single melanocortin peptides using lentiviral vectors expressing α-MSH (LVi-α-MSH-EGFP) and γ-MSH (LVi-γ-MSH-EGFP). The lentiviral vectors were shown to produce a long-term overexpression and biologically active peptides in cell-based assays. The LVi-α-MSHEGFP was targeted to the arcuate nucleus in the hypothalamus of diet induced obese mice where it reduced weight gain and adiposity independently of food intake. When the nucleus tractus solitarus in the brainstem was targeted, the LVi-α-MSH-EGFP treatment was shown to cause a small decrease in adiposity, which did not impact weight development. However, the α-MSH treatment increased heart rate, which was attenuated by adrenergic receptor blockade indicative of increased sympathetic activity. The LVi-γ-MSH-EGFP was targeted to the hypothalamus where it decreased fat mass in mice eating the standard diet, but the effect was abated if animals consumed a high-fat Western type diet. When the diet induced obese mice were subjected again to the standard diet, the LVi-γ-MSH-EGFP treated animals displayed increased weight loss and reduced adiposity. These results indicate that the long-term central anti-obesity effects of α-MSH are independent of food intake. In addition, overexpression of α-MSH in the brain stem efficiently blocked the development of adiposity, but increased sympathetic tone. The evidence presented in this thesis also indicates that selective MC3R agonists such as γ-MSH could be potential therapeutics in combination with low fat diets.

To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.

Fabrication and Pseudo-Analog Characteristics of Ta2O5 -Based ReRAM Cell

Memristori on yksi elektroniikan peruskomponenteista vastuksen, kondensaattorin ja kelan lisäksi. Se on passiivinen komponentti, jonka teorian kehitti Leon Chua vuonna 1971. Kesti kuitenkin yli kolmekymmentä vuotta ennen kuin teoria pystyttiin yhdistämään kokeellisiin tuloksiin. Vuonna 2008 Hewlett Packard julkaisi artikkelin, jossa he väittivät valmistaneensa ensimmäisen toimivan memristorin. Memristori eli muistivastus on resistiivinen komponentti, jonka vastusarvoa pystytään muuttamaan. Nimens mukaisesti memristori kykenee myös säilyttämään vastusarvonsa ilman jatkuvaa virtaa ja jännitettä. Tyypillisesti memristorilla on vähintään kaksi vastusarvoa, joista kumpikin pystytään valitsemaan syöttämällä komponentille jännitettä tai virtaa. Tämän vuoksi memristoreita kutsutaankin usein resistiivisiksi kytkimiksi. Resistiivisiä kytkimiä tutkitaan nykyään paljon erityisesti niiden mahdollistaman muistiteknologian takia. Resistiivisistä kytkimistä rakennettua muistia kutsutaan ReRAM-muistiksi (lyhenne sanoista resistive random access memory). ReRAM-muisti on Flash-muistin tapaan haihtumaton muisti, jota voidaan sähköisesti ohjelmoida tai tyhjentää. Flash-muistia käytetään tällä hetkellä esimerkiksi muistitikuissa. ReRAM-muisti mahdollistaa kuitenkin nopeamman ja vähävirtaiseman toiminnan Flashiin verrattuna, joten se on tulevaisuudessa varteenotettava kilpailija markkinoilla. ReRAM-muisti mahdollistaa myös useammin bitin tallentamisen yhteen muistisoluun binäärisen (”0” tai ”1”) toiminnan sijaan. Tyypillisesti ReRAM-muistisolulla on kaksi rajoittavaa vastusarvoa, mutta näiden kahden tilan välille pystytään mahdollisesti ohjelmoimaan useampia tiloja. Muistisoluja voidaan kutsua analogisiksi, jos tilojen määrää ei ole rajoitettu. Analogisilla muistisoluilla olisi mahdollista rakentaa tehokkaasti esimerkiksi neuroverkkoja. Neuroverkoilla pyritään mallintamaan aivojen toimintaa ja suorittamaan tehtäviä, jotka ovat tyypillisesti vaikeita perinteisille tietokoneohjelmille. Neuroverkkoja käytetään esimerkiksi puheentunnistuksessa tai tekoälytoteutuksissa. Tässä diplomityössä tarkastellaan Ta2O5 -perustuvan ReRAM-muistisolun analogista toimintaa pitäen mielessä soveltuvuus neuroverkkoihin. ReRAM-muistisolun valmistus ja mittaustulokset käydään läpi. Muistisolun toiminta on harvoin täysin analogista, koska kahden rajoittavan vastusarvon välillä on usein rajattu määrä tiloja. Tämän vuoksi toimintaa kutsutaan pseudoanalogiseksi. Mittaustulokset osoittavat, että yksittäinen ReRAM-muistisolu kykenee binääriseen toimintaan hyvin. Joiltain osin yksittäinen solu kykenee tallentamaan useampia tiloja, mutta vastusarvoissa on peräkkäisten ohjelmointisyklien välillä suurta vaihtelevuutta, joka hankaloittaa tulkintaa. Valmistettu ReRAM-muistisolu ei sellaisenaan kykene toimimaan pseudoanalogisena muistina, vaan se vaati rinnalleen virtaa rajoittavan komponentin. Myös valmistusprosessin kehittäminen vähentäisi yksittäisen solun toiminnassa esiintyvää varianssia, jolloin sen toiminta muistuttaisi enemmän pseudoanalogista muistia.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.