Browsing by discipline "Genetics"

The Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein (Cas9) (CRISPR-Cas9) system is a widely used gene editing technology due to its potential to alter the genome precisely in desired locations. Due to the potential of the CRISPR-Cas9 system, the objective of the thesis is to improve the precise editing of genes by modifying the CRISPR-Cas9 platform. Ultimately, the aim is to develop a platform that can edit any mutation and repair it to a normal, functional gene in patient cells. In general, CRISPR-Cas9 provides opportunities in treating monogenic diseases, for example by modifying long-term hematopoietic stem cells in immunodeficiencies. CRISPR-Cas9 can target disease-causing mutation sites and introduce double-strand breaks. Afterwards, the native DNA repair machinery of a cell repairs the cut site either by more efficient, error-prone non-homologous end joining (NHEJ) or precise homology-directed recombination (HDR). In most clinically oriented genome editing studies, the desired repair outcome is the latter because it allows precise repair of the mutation according to the exogenous repair template. Despite all its positive features, the optimization of CRISPR-based editing system is crucial before medical use; CRISPR-Cas9 induces a p53-mediated DNA damage response, which leads to a transient G1 cell cycle arrest and hampers HDR-based precision genome editing. Other problems include the repair pathway depending on the cell cycle phase, repair template proximity, and off-target activity. This thesis demonstrates that Cas9 fusions allow addressing the problems mentioned above. Cas9 fusions with DNA repair proteins ensure improved editing efficiency at the close proximity to the target site in HEK293T, BJ5-ta and RPE reporter cell lines. In addition, Cas9 coupled with the engineered cell cycle timer, AcrⅡA2-cdt1, favors the editing at the S/G2 cell cycle phases avoiding the p53-mediated response. AcrⅡA2-cdt1 is a reversible, phage-derived CRISPR inhibitor that selectively inhibit CRISPR-Cas9 at the G1 cell cycle phase and releasing it at the S phase. This thesis provides extensive look on the CRISPR-Cas9 editing and its challenges in immortalized cell lines and primary cells. In the thesis, the generation of reporter cell lines is prior to the validation of the novel Cas9-fusions. Furthermore, the optimization of primary T cell and CD34+ hematopoietic stem cell electroporation with different electroporation systems brings the study closer to clinical applications. The thesis provides insights about the effect of the target site and the cell type for genome editing outcomes. The editing efficiencies depend on the Cas9 fusion protein, cell type and its proliferation rate. The editing efficiency in primary T cells and CD34+ hematopoietic stem cells can significantly improve by optimizing transfection and culturing conditions, such as concentration of the CRISPR-Cas9 complex, cell culturing time and electroporation program. Cas9 fusions improve the safety and efficiency of the CRISPR-Cas9 system depending the cell type and the proliferation rate of the cell. Timing the induction of double-strand breaks also improves the editing efficiency. Overall, the methods used in the thesis give useful tools for eventual translational applications.

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a plant pathogen which causes bacterial speck in the host plants, especially tomato. The genome sequence of Pst DC3000 was resolved in 2003 by Buell et al. and since then it has served as an important model organism in plant pathology. Pst DC3000 secretes proteins through so-called type III pathway (T3SS). The secreted proteins, called Hrp (Hypersensitive response and pathogenicity) or Hop (Hrp outer protein), are considered as main virulence determinants of the bacterium. Aim of the study was to find out the suitability of the phage Mu DNA transposition complex to generate a transposon insertion mutant library for Gram-negative phytopathogen. Then further screen the mutant library for Hop effector protein knock-out mutants and evaluate their virulence in Lycopersicon esculentum cv. Moneymaker tomato plants. The thesis was part of a research project that characterized virulence determinants of Gram-negative phytopathogenic bacteria, including their gene regulation and effects on the plant-bacterium interaction. Pst DC3000 transposon insertion mutant library was generated by electroporation of phage Mu DNA transposition complex and a nested PCR method was used for screening the library. Three hop transposon insertion mutants were found, and those strains were extracted from the library. The three mutant strains where then used to infect tomato plants. Virulence was evaluated through phenotypic lesions and bacterial endophytic growth. Some of the results were different from the previous studies. The ability of HopM1 mutant to form lesions was weakened and it´s endophytic growth was slowed down. In previous studies the ability to form lesions was also weakened, but the endophytic growth was not affected until avrE gene was also deleted. HopM1 deletion alone did not have an effect to bacterial growth. In HopAO1 mutant the virulence was not affected. In other studies, HopAO1 deletion weakened bacterial growth in Arabidopsis and tobacco plants, but in tomato it has not been tested. HopD1 mutant was avirulent in this study. In previous studies HopD1 was essential for Pst DC3000 bacteria in defeating plants effector-triggered immunity (ETI), but hopD1 deletion alone did not make the bacteria avirulent. Pst DC3000 has 28 different effector proteins so usually one effector protein mutation does not affect its virulence. Hence the next step of the study was to make double mutants for Hop proteins, and this included deletion of the antibiotic marker by Cre-lox mechanism, which was started in this study.

Darwinian selection can be measured and investigated from gene sequences. A certain gene form favored by positive selection will become more common in the population. Detecting strong positive selection is rare, but it has been found to affect genes involved in immune defense and perception of odorants. Genes under positive selection have a possible role in speciation or adaptation. This is why chemical communication, being based on the sense of smell, is an interesting topic for measuring natural selection and positive selection in particular. Social insects, such as ants, are model organisms for chemical communication. They use chemical communication not only for finding nutrition and detecting intruders, but also in coordinating the activities of several thousands of colony members. Insects perceive odorant signal molecules with their antennae. Odorant binding proteins (OBP) and chemosensory proteins (CSP) bind and transport odorant molecules through the sensillar lymph. OBPs and CSPs are also suspected to have a role in selecting odorants. This work focuses on two OBP genes and two CSP genes to study natural selection. All of these genes are conserved among all ant species. Three of these genes, OBP1, CSP1 and CSP7, are strongly expressed in the antennae suggesting that they function in chemical communication. CSP7 also has a known function in nest mate recognition in ants and OBP1 is known to bind a queen pheromone in the honeybee. The data includes gene sequences from 7 Formica ant species (270 sequences in total). The main goals of the research were to find out 1) the extent of variation between and within closely related ant species, 2) which evolutionary forces, natural selection or random drift, are behind the variation and 3) are there systematic differences between the two social forms of ants suggesting that these genes would affect the social structure of an ant colony. The variation in the sequence data was visualized by phylogenetic, principal coordinate and fixed differences analyses. Differences between populations were studied by FST values. The evolutionary forces shaping chemical communication genes within the species and populations, were studied by McDonald-Kreitman test, Tajima s D, Fu and Li test and MFDM test. The data shows that two of the species, F. cinerea and F. exsecta, significantly differ from the other five ant species, the F. rufa group species, and that the F. rufa group species don t significantly differ from each other based on these genes and this data. This could be due to their recent speciation or crossing between the species leading to hybrids in the data. The results of the evolutionary analyses are inconsistent. However, CSP7 has the strongest indication of selection based on all of the tests. Possible selection and sequence variation was detected at a predicted transcription factor binding site in F. cinerea. This indicates that selection might affect the regulation of CSP7. In the future it would be interesting to check the true transcription factor binding sites experimentally.

Cancer-associated fibroblasts (CAF) form a heterogenous stromal cell population of a solid tumor. They are known to promote tumor growth and survival through metabolic reprogramming and inflammation. It is unclear though whether CAF are crucial component of tumor initiation and whether CAFs are dispensable altogether from the fully developed neoplasm. Tumor suppressor LKB1 regulates AMPK and AMPK-related kinases (ARK), and its function is compromised in familial disorder Peutz-Jeghers syndrome (PJS). Fibroblast specific haploinsufficiency of LKB1 alone is sufficient of initiating gastrointestinal polyposis but the mechanism through which LKB1 mediates this is only partially understood. We provide evidence that LKB1 is downregulated in multiple human malignancies including high grave serous ovarian cancer (HGSOC). Human ovarian cancer is the most lethal gynecological disease, characterized by metastasis of omentum. Loss-of LKB1 in ovarian fibroblasts was accompanied with metabolic changes associated with CAF-transformation. We screened down critical LKB1 substrates through transcriptomic and functional assays revealing AMPKa1 and MARK3 as potential downstream effectors of oxidative phosphorylation. AMPKa1, MARK1 and SIK-family were the glycolytic counterparts. We also took an initiative of cataloguing published human cancer stroma data in order to gain more comprehensive look of tumor heterogeneity. Metabolic rewiring was also observable in published cancer-stroma datasets. Human cancer-stroma divided into metabolically active and highly inflamed subtypes. These results highlights LKB1’s role as a conserved metabolic caretaker in fibroblasts. Our data also support mechanistic model in which LKB1 and ARKs regulate mitochondrial metabolism, essential for CAF transformation.

Ihmistoiminnan vaikutukset ovat muuttaneet voimakkaasti ekosysteemien rakennetta ja toimintaa maailmanlaajuisesti viimeisten vuosikymmenten aikana. Ekologisen tilan heikentyminen aiheuttaa vakavan uhan virtavesien eliöyhteisöjen monimuotoisuudelle. Tulevaisuudessa tarve kustannustehokkaille, luotettaville ja nopeille keinoille arvioida vesistöjen ekologista tilaa kasvaa. Biomonitorointi, eli eliöiden käyttö ympäristömuutosten seurantaan ja mittaamiseen, pyrkii tunnistamaan, seuraamaan ja arvioimaan ekosysteemien toimintaa uhkaavia ympäristön stressitekijöitä. Tulevaisuudessa yksi mahdollinen biomonitoroinnin työkalu ja ekologisen tilan mittari yhdessä muiden biologisten (esim. piilevät, pohjaeläimet, vesikasvit ja kalat) ja fysikaalis-kemiallisten mittarien kanssa voi olla mikrobit. Uuden polven sekvensointimenetelmien kehitys ja sekvensointikustannusten aleneminen viime vuosina on mahdollistanut yhä useamman ympäristömikrobiaineiston analysoinnin uuden polven sekvensointimenetelmillä. Tämän pro gradu -tutkielman tarkoitus on tuoda lisätietoa siitä, mitkä eri ympäristötekijät vaikuttavat virtaavien vesien biofilmibakteerien yhteisörakenteeseen, yhteisö-ympäristösuhteen voimakkuuteen, yhteisöjen koostumusta selittävien muuttujien identiteettiin ja miten harvinaisten bakteerisukujen poisto aineistosta vaikuttaa erilaisiin ympäristömuuttujiin. Tutkimusaineistoa analysoidaan redundanssianalyysin (redundancy analysis, RDA) ja monimuuttujaregressiopuiden (Multivariate regression trees, MRT) avulla. Tutkielman tavoitteena on myös kuvata länsisuomalaisten virtavesien koskipaikkojen kivien pinnalla elävien biofilmibakteerien ominaispiirteitä pääjakso-, luokka-, lahko-, heimo- ja sukutasoille tehtävien BLAST-analyysien (basic local alignment search tool) avulla. Pro gradu tutkielmani bakteeriaineiston näytteet on otettu 21 suomalaiselta kolmannen jakovaiheen jokivaluma-alueelta, joilta eristettiin erillisten purojen koskipaikoista bakteerinäytteitä yhteensä 84 kappaletta. Samaan aikaan bakteerinäytteiden ottamisen kanssa tutkimuskohteilla toteutettiin vesinäytteenotto, ja kohteista määritettiin myös piilevä- ja pohjaeläinlajisto. Näytteet sekvensoitiin Ion Torrent Semiconductor -sekvensaattoria käyttäen ja valmis sekvenssiaineisto analysoitiin QIIME (Quantitative Insights Into Microbial Ecology) –ohjelmalla. Sekvenssit klusteroitiin toiminnallisiksi taksonomisiksi yksiköiksi (OTUiksi) 97 % samankaltaisuusastetta käyttäen. OTUille haettiin BLASTia (basic local alignment search tool) käyttäen paras mahdollinen pääjakso-, luokka-, lahko-, heimo- ja sukutason vastaavuus. Yleisimmät havaitut luokat olivat Alphaproteobacteria (28.9%), Betaproteobacteria (27.7%), Gammaproteobacteria (7.5%) Cytophagia (7.3%), Saprospirae (6.5%) ja Flavobacteriia (5.1%). Tuloksista havaitaan, että yhteisön ympäristösuhteisiin vaikuttavat erityisesti yleiset lajit. Harvinaisten lajien poistolla ei tässä tutkimuksessa siten havaita olevan suurta merkitystä, vaan merkitsevät muuttujat pysyvät lähes samoina huolimatta siitä, poistetaanko aineistosta ne lajit, jotka esiintyvät vain yhdellä paikalla vai kaikki ne lajit, jotka esiintyvät vain ≤ 40 paikalla. Myös selitysasteet jäävät alhaisiksi ja satunnaisten eli stokastisten tekijöiden vaikutus yhteisön ympäristöolosuhteisiin luultavasti korostuu. Jatkossa eri bakteeritaksonien roolista muuttuvissa ympäristöolosuhteissa tarvitaan tarkempaa tietoa ennen kuin virtavesien biofilmibakteereja voidaan laajamittaisesti käyttää biomonitoroinnin täysimittaisena työkaluna ja ympäristön tilan kuvaajana. Tämän tutkielman redundanssianalyysin, vaihtelun osituksen ja monimuuttujaregressiopuiden avulla saatujen tulosten perusteella voidaan todeta virtaavien vesien mikrobiyhteisöjen yhteisö-ympäristösuhteiden olevan heikkoja. Ympäristön tilan seurannassa tulisikin jatkossa tulosten perusteella käyttää mikrobien osalta muita vastemuuttujia kuin yhteisörakennetta.

nxiety disorders are the most common psychiatric disorders. Iiris Hovatta’s group (Neurogenomics lab) uses inbred mice to study effects of psychosocial stress. Mice can be divided to susceptible and resilient phenotypes, based on their behavioral response to psychosocial stress. Differences between phenotypes can be examined for example on brain structural or transcriptional levels. These studies have shown changes in brain myelination and expression levels of myelin-related genes between phenotypes. Transcriptomic studies have also revealed differences in microRNA (miRNA) expression profiles. Especially miR-219a, a known regulator of myelination, appears to be differentially expressed. Bioinformatic studies also revealed that many predicted target genes of miR-219a are connected to Wnt/β-catenin signaling pathway, which is also known to regulate myelination. This led to a hypothesis that miR-219a regulates myelination through Wnt/β-catenin signaling pathway in adult mouse brain. Prediction of miRNA target genes is based on algorithms using varying criteria, and their biological significance is uncertain. Assumptions about the biological role of specific miRNA require experimental validation of the silencing interaction between the miRNA and the target gene mRNA. The purpose of this study was to validate five Wnt-signaling related mouse genes (Gsk3b, Esr1, Tcf7l2, Fkbp5 and Acvr1b) as targets for miR-219a-5p or miR-219a-1-3p. For validation a luciferase-reporter assay was used, in which the target sequence of the gene’s mRNA is cloned to a reporter vector, then cotransfected to cultured cells (human HEK293 cells) with a miRNA-mimic. The light emitting luciferase activity is measured to examine the possible silencing effect of the miRNA. Results of this study showed that miR-219a mimics did silence the expression of the target genes. Although some variation was observed in the efficiency of silencing between the genes and based on the mimic concentration. The most efficient silencing was observed between miR-219a-5p and Gsk3b construct. These results implicate that miR-219a regulates several genes related to Wnt/β-catenin pathway, and that stress-related differences in its expression may be associated to differential myelination observed between phenotypes that react differentially to psychosocial stress.

Nucleosides are one of the fundamental building blocks of the cell and are precursors of DNA and RNA and serve as universal signalling molecules for cell surface receptors. These nucleosides play vital roles in myriad of physiological processes, such as cardioprotection, platelet aggregation and coronary vasodilation. In clinical settings, synthetic analogs of nucleosides are used as therapeutic against cancer and other viral diseases. In human, uptake and regulation of nucleosides and their analog drugs is facilitated by the two families solute carrier membrane transporter proteins (SLC); SLC28 and SLC29. SLC29 consists of four human equilibrative nucleoside transporter members (hENT1-4) with different sub-cellular localization, tissue distribution and substrate selectivities. Human equilibrative nucleoside transporter 1 (hENT1) is the major plasma membrane nucleoside transporter and has shown to play an important role in adenosine signalling and anticancer therapies. Despite the significant physiological and pathophysiological role, little is known about the structure and molecular function of ENT homologs. Structural studies of hENT1 and other nucleoside transporter is hampered by their low expression and hydrophobic nature. The aim of this thesis work is to find suitable nanobodies (Nbs) that may serve as crystallization chaperone to facilitate the structural studies of hENT1 transporter. Camelid heavy chain only antibodies (Nanobodies) raised against recombinant hENT1 were screened for their suitability for future structural investigation of hENT1. Selected nanobodies were expressed and purified from E.coli cell in milligram quantities. Affinity of nanobodies with hENT1 was assessed using co-elution on size exclusion chromatography. High affinity binders were further screened for their ability to conformationally stabilize hENT1. In future, high affinity nanobodies will be explored for x-ray crystallization and single particle cryo-electron microscopy of hENT1. For cryo-EM it's important to convert the selected Nbs into megabody which constitute large scaffold proteins. 3D structure determination of hENT1 will be significant in understanding its molecular function and to accelerate rational drug designing against cancer, HIV and other viral infections.

Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.

Tiivistelmä – Referat – Abstract Celiac disease (CD) is a serious lifelong condition, in which the immune system attacks an individual’s own tissue when eating gluten. This leads to inflammation and damage to the small intestine. Celiac disease often goes undiagnosed because many of its symptoms are nonspecific. The prevalence of combined undiagnosed and diagnosed CD is estimated to affect 1 in 100 people throughout Europe and USA. CD is a polygenic disease, it is known that the human leukocyte antigen (HLA) system plays a crucial role. HLA-DQ2/DQ8 risk allele genotyping screening test from a whole blood sample (B -HLAKeli) is routinely used to estimate the genetic risk of a patient having CD. HLA genotyping test result is routinely used to rule out celiac disease rather than confirming it; if an individual does not have celiac disease related risk alleles, it is very unlikely that he or she has celiac disease. The Celiac disease diagnosis decision making process is based on the classic triple combination of serological antibody tests, the HLA-DQ2/DQ8 genotyping test and duodenal biopsies. The aim of this master’s thesis was to study evaluate how the two different risk classification praxis for HLA-DQx.5 allele used for celiac disease diagnostics in SYNLAB Finland and Estonia central laboratory and in SYNLAB Suomi central laboratory might influence the clinical process and final diagnosis. In SYNLAB Suomi central laboratory HLA-DQx.5 is classified and interpreted as a risk allele predisposing to celiac disease. In SYNLAB Finland and Estonia central laboratory this allele is classified as CD-non-risk-allele based on recommendations in international guideline. In addition, the aim was to get a general understanding of celiac disease prevalence and risk allele distributions among the study population. From the study population of 196 celiac disease suspect patients, 9% had a celiac disease positive laboratory result and the HLA risk genotype distribution among positive cases was well aligned with the expected values described in the literature. Study results indicated that there’s no additional clinical value if HLA-DQx.5 is classified as a celiac disease predisposing risk allele; the study data implies that it is very unlikely to find celiac disease positive cases from laboratory test perspective among HLA-DQx.5 carriers. Based on the study, approximately 7% of the celiac disease suspects carry the allele HLA-DQx.5 and therefore probably go through additional celiac disease related laboratory testing if this allele is interpreted as a risk allele. According to the study findings and general recommendations based on international guideline of celiac disease diagnosis, it seems that there is no clear clinical benefit if HLA-DQx.5 is classified as a CD risk allele.

Cells in tissues have only three serious options in life; they can grow and divide, remain static, or die by apoptosis. Upon growth factor stimulation a cell enters the so called cell cycle which will eventually lead to the division of the cell. Cell cycle can be divided into four phases; G1, S, G2 and M. The current model of the cell cycle control holds that the transitions between different cell cycle states are regulated by cyclin dependent kinases (CDK) with their activator subunits, the cyclins. CDK regulation can be separated into four distinct mechanisms, one of which being phosphorylation on the so called T-loop leading to complete activation. This phosphorylating activity is mediated by apparently a single enzymatic activity termed the CDK activating kinase, CAK. CAK activity was originally isolated as a biochemical purification extract and the enzyme was surprisingly noticed to be structurally related to CDKs. Since a novel cyclin was identified to be associated to it, the enzyme exercising CAK activity was named CDK7 and the cyclin was designated cyclin H. An entirely new perspective on CDK7 function was opened when CDK7 was identified as a subunit of transcription factor IIH (TFIIH) and shown to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). CDK7 has also been suggested to be involved in irradiation sensitivity pathways and nucleotide excision repair functions. To elucidate the intriguing in vivo role of CDK7, proteins interacting with CDK7 were screened for using the yeast two-hybrid method as part of previous studies of the laboratory. The results showed that 15 out of 144 (10,4%) positive clones were identified to encode a peptide sequence of a protein previously known as the inhibitor/interactor of protein kinase C (PKCI). These yeast colonies had an unexpected phenotype; contradictory to a dark blue color of the colonies, indicating strong interaction, the size of the PKCI colonies was small compared to others, indicating a possible growth inhibition effect. Several DNA open reading frames (ORF) coding for proteins related to human PKCI have been identified in a broad range of species representing mammalian, plant, fungal and bacterial kingdoms, all these forming a HIT (conserved triad of histidines) protein family. Another human member, part of this now super family, named FHIT (fragile triad of histidines) was identified with a dinucleoside 5’,5’’’-P1,P3-triphosphate hydrolase activity. These molecules; substrates of FHIT and related enzymes have been proposed to have various intracellular functions, including signalling stress responses. The aim of this study was to extend the investigation of the interaction between CDK7 and PKCI observed in yeast two-hybrid by means of several genetic and biochemical approaches to determine if this observed interaction and growth phenotype has any physiological significance. Investigations included performing yeast two-hybrid screening for PKCI, developing yeast three-hybrid system and carrying out growth rate assays for yeast liquid cultures. These studies also included performing biochemical purifications of over-expressed proteins, immunoprecipitations, western blot analysis and kinase activity assays. Protein extracts originated from transformed yeast cells, transfected mammalian cells or from in vitro transcription and translation reactions. On basis of growth rate assays it can be concluded that PKCI has an inhibitory growth effect in yeast. The preliminary finding of a specific PKCI-CDK7 interaction in yeast two-hybrid, however could not be conclusively verified by the other methods that were used in this study. Studies of PKCI characterisation also included examination of the subcellular localisation of PKCI in mammalian cells by immunofluorescence labelling of HA-PKCI. Results showed PKCI to localize both in the nucleus and in the cytoplasm. Also, studies to elucidate the function of PKCI were performed; whether it possesses enzymatic activity related to that of FHIT. By NMR spectroscopy using bacterially produced GST-PKCI, hydrolase activity towards ADP was indeed observed. Future studies will include elucidation of possible links between growth inhibition and hydrolase activity, in the form of stress signalling functions. The main focus of our future studies will be the generation of mice with targeted PKCI alleles offering powerful means to reveal the function of PKCI through observing phenotypes and through detailed analysis of these mice harbouring wild type, hypomorphic or null alleles.