Browsing by discipline "biokemia"

Proteostasis is used by cells to maintain proteome health and understanding the biological mechanisms underpinning proteostasis is important. Despite many studies on small molecule-mediated inhibition of Sec61-dependent protein translocation, a knowledge gap exists in the quality control pathway(s) of pharmacologically-displaced secretory polypeptides. Genetic screens can be used to discover proteostasis regulators of pharmacologically-displaced secretory polypeptides. Near-haploid human HAP1 cells with an inducible Tet-on GFP reporter (reporter HAP1 cells) can be used as a cellular tool to screen for human host factors pertinent to proteostasis of secretory polypeptides. The isolation of haploid-enriched reporter HAP1 cells ensures that the inability to efficiently recover bi-allelic gene trap mutants is avoided. The use of haploid-enriched cells is a prerequisite to gene trap mutagenesis screens. Here, I present data on the isolation of haploid-enriched reporter HAP1 cells that could be used as a cellular tool in gene trap mutagenesis screens. A workflow for the isolation of haploid-enriched reporter HAP1 cells was optimised using a diploid reporter HAP1 cell line as a control. Both DNA content analysis and karyotyping showed that the isolated HAP1 reporter cell lines were haploid. In the haploid-enriched HAP1 reporter cells, the GFP-reporter compartmentalised in the ER, and a Sec61-translocon inhibitor CT8 could inhibit the GFP-mediated fluorescence. The haploid reporter HAP1 cell lines produced in this study are suitable for future gene-trap mutagenesis experiments.

Cornea is transparent layer of cells lying in front of lens. The corneal epithelium, a squamous epithelium, covers the ocular surface and ensures proper vision by preserving the integrity of the eye. Corneal epithelium is renewed continuously throughout life from a pool of stem cells (SC). There are still conflicting theories about the localization of stem cells required for the growth, renewal and maintenance of the corneal epithelium. Previous studies demonstrated that the limbus, located in the periphery of the cornea, serves as the stem cell niche (SCN) in adults. However, contrasting evidence from clonal analysis proposes that, in early postnatal life, renewal is fuelled by SCs located in the basal layer of the central cornea. There are alternate patterns of renewal in young and adult mouse cornea and that there is an important, transitional time frame called cornea maturation, when the adult patterns of gene expression, cell dynamics and tissue renewal are established. In the cornea, solid SC markers are still missing, yet studies on human limbal cells have suggested Bmi1 and C/EBPδ as limbal SC markers. There are, indeed, long-lived SCs in the central cornea and that the gene Bmi1 plays a role in these central corneal SCs. However, the physiological importance of these Bmi1+ cells remains obscure. The main aim of this project is to understand the fate and dynamics of these Bmi1+ cells and study the chronology of maturation of the cornea. In this study, I have also tried to correlate the growth of eye size with proliferation of corneal epithelial cells This study was conducted using few different kinds of transgenic mice (Mus musculus). To study the fate of Bmi1+ cells, two different mouse lines were crossed: Bmi1-CreER and ROSA26-LacZ. Mice carrying both alleles were used for lineage tracing experiments. Moreover, Hematoxylin-eosin staining was used to follow the eye morphology. Immunohistochemistry was performed to follow the chronology of maturation of the cornea, proliferation of corneal epithelial cells and the location of Bmi1+ cells in corneal epithelium. From this study, we can propose that cornea maturation is completed by the time of eyelid opening, which take place two weeks after birth. Krt19 is perfect for studying the chronology of the corneal epithelium, immunostaining of Krt19 separates the territory of limbus from central cornea enabling to distinguish limbus distinctly. Proliferating cells reside in basal layer of cornea. Bmi1+ cells found throughout the basal layer of the cornea that locally renews the corneal epithelium concluding Bmi1+ cells as the progenitor cells.

Gene therapy trials are becoming more common place with the first approved products having arrived onto markets within the last 5 years. Gene therapy trials have focused on diseases that are monogenic and curable through the reintroduction of autologous, gene-corrected hematopoietic stem cells with promising results. A popular method for gene-correction is through the integration of the wild-type gene into the patient’s genome with a class of retroviruses called lentivirus. APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) is a rare, monogenic autoimmune disease that is caused by a recessive mutation within the AIRE gene coding region. APECED has a high prevalence within the Finnish population (1/25,000) and is theoretically curable through the transplantation of autologous gene-corrected hematopoietic stem cells. Therefore, creating and studying a lentivector carrying the endogenous AIRE promoter and coding region could provide insight and a preliminary foundation on how to begin to develop a viable gene therapy for APECED. The aim of this thesis was to generate and characterise the first lentivector containing the wildtype AIRE gene, specifically the proximal AIRE promoter and the AIRE open reading frame (ORF). The lentivector was constructed and then transfected into a human cell-line, HEK293T, from which qPCR and immunohistochemistry were used to detect for AIRE mRNA and protein, respectively. The presence of significant amounts of AIRE mRNA and protein indicated that the constructed plasmid was transcriptionally functional. It is the first plasmid to have Aire transcription regulated by its endogenous promoter and can provide future insight into gene regulation. As a lentiviral plasmid, it demonstrates the integrating of the AIRE gene into an existing lentiviral system and serves as a first step and a technical proof-of-concept in developing a gene therapeutic cure for APECED.

Eph (Erythropoietin producing hepatocellular) receptors and their membrane-bound ligands, ephrins, form the largest family of receptor tyrosine kinases in mammals. They regulate functions such as cell migration and axon guidance during development, and wound healing and tissue boundary maintenance in mature tissues for example. Due to the membrane-bound nature of the ephrin ligands, Eph-ephrin signalling can proceed in two directions: forward (Eph-expressing cell) and reverse (ephrin expressing cell). In addition to its critical physiological functions in development and tissue homeostasis, the Eph-ephrin system has also been implicated in multiple diseases, including several cancers, in which the aberrant expression of Eph receptors and ephrins are often present. The EphA2 receptor for example has been identified as an oncogene with a dual mode of action as either tumor suppressor or an oncogene through distinct phosphorylation statuses of the receptor. High-grade serous ovarian cancer is the most common subtype of ovarian cancer, and the deadliest gynaecological malignancy with a dismal five-year survival rate. High-grade serous ovarian cancer does not present symptoms at an early stage, yet it quickly progresses into forming peritoneal metastases by cell shedding from the primary tumour. Small patient cohort studies have given indications of the correlation between the Eph-ephrin system and survival in ovarian cancer. This aim of this study was to investigate the impact of the Eph-ephrin system in high-grade serous ovarian cancer using a large patient cohort mRNA expression dataset to obtain survival association data of proteins of interest used with cell-based studies and the analysis of clinical samples in the form of tumor microarrays and fresh primary samples to investigate the functions of the found proteins of interest. A 428-patient The Cancer Genome Atlas high-grade serous ovarian cancer microarray mRNA dataset was analysed using the Kaplan-Meier estimator for each Eph-ephrin family member. Cell based studies were performed with recombinant ephrin treatments and ephrin knockdowns. These data were analysed using Western blotting and immunofluorescence stainings. Clinical high-grade serous ovarian cancer samples obtained from Turku University Hospital were analysed using immunohistochemistry, immunofluorescence stainings, and mRNA sequencing. EfnA5 had a significant correlation of high expression and poor survival, which is atypical to ephrins. Low EfnA3 correlated with poor survival. High levels of known oncogenes EphA2 and EphA4 also correlated with poor survival. EfnA5 treatment resulted in increased oncogenic EphA2 signaling in comparison with canonical Eph-ephrin signalling mediated by EfnA1. Knockdown of EfnA5 increased canonical, tumour suppressive EphA2 signaling, while EfnA1 knockdown increased oncogenic EphA2 signaling. Immunohistochemical analysis of tumour microarrays with multiple ovarian cancer subtypes displayed an association between the highly malignant high-grade serous subtype and EfnA5 expression. In addition to this, EfnA5 expression was increased during high-grade serous ovarian cancer progression. The Eph-ephrin system is implicated in the survival associations of multiple cancers, but the exact functions facilitated by Eph-ephrin signalling in cancer have remained relatively unknown, with the exception of EphB4-EfnB2 driven angiogenesis. This study offers insights into oncogenic Eph-ephrin signalling in ovarian cancer, displaying that oncogenic EphA2 functions can be altered by ephrins in addition to the known kinase crosstalk pathway. The noncanonical nature of EfnA5 is highlighted by its oncogenic functions in comparison to typical Eph-ephrin signalling, and the significant increase of EfnA5 expression during high-grade serous ovarian cancer progression and association with this highly malignant subtype of ovarian cancer. Although the reverse signalling effects of EfnA5 were not studied, this study highlights the importance of ephrins in Eph-ephrin signalling in cancer, presenting that the focus should not be only on the Eph receptors when studying the oncogenic signalling facilitated by the Eph-ephrin system.

Despite the advances in the management of breast cancer, discovery of novel and targeted drugs remains a challenge. It has been suggested that drug failure rates in clinical trials might be diminished by improving the predictive potential of preclinical cancer models. Three-dimensional (3D) scaffold-based cell culture has emerged as an attractive platform for mimicking tissue-like microenvironment, since it is well-known that cells respond to the cues in the extracellular matrix (ECM). The aim of this thesis was to develop fibrin-based hydrogels and evaluate their performance in 3D cell culture of breast cancer cells. The fibrin gel formulation was first optimized by testing the effect of different buffers on gel properties. Structural properties were examined with scanning electron microscopy and mechanical properties measured with oscillatory rheometry. Three different fibrin concentrations of the optimized formulation were then used as scaffolds for DU4475 breast cancer cells. After seven days of culture, the morphology, phenotype and proliferation of the resulting cell structures were assessed by using techniques such as light microscopy, immunofluorescent confocal microscopy and Western blot analysis. The desired properties for 3D cell culture were obtained by preparing fibrin gels at high pH in the absence of calcium. The main finding of the thesis was that fibrin concentration strongly affected the phenotype of DU4475 cells, with cells cultured in the softest gel retaining their original characteristics to the greatest extent. In the future, the developed scaffold could possibly be used in drug discovery and personalized medicine by culturing tumor explants from patients. However, the methods used in the study must be further optimized and the results validated with other breast cancer cell lines and with primary tissues.

Protein kinases are signaling molecules that regulate vital cellular and biological processes by phosphorylating cellular proteins. Kinases are linked to variety of diseases such as cancer, immune deficiencies and degenerative diseases. This thesis work aimed to identify direct substrates for protein kinases in the CMGC family, which consists of the cyclin-depended kinases (CDK), mitogen activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and CDC-like kinases (CLK). CMGC kinases have been identified as cancer hubs in interactome studies, but large-scale identification of direct substrates has been difficult due to the lack of efficient methods. Here, we present a heavy-labeled 18O-ATP-based kinase assay combined with LC-MS/MS analysis for direct substrate identification. In the assay, HEK and HeLa cell lysates are treated with a pan-kinase inhibitor FSBA which irreversibly blocks endogenous kinases. After the removal of FSBA, cell lysates are incubated with the kinase of interest and a heavy-labeled ATP, which contains 18O isotope at the γ-phosphate position. Resulting phosphopeptides are enriched with Ti4+- IMAC before the LC-MS/MS analysis, which distinguishes the desired phosphorylation events based on a mass shift caused by the heavy 18O. With this pipeline of methods, we managed to quantify and identify direct substrates for 26 members of CMGC kinase family. A total of 1345 substrates and 3841 interacting kinase-substrate pairs were identified in cytosolic cell lysates, from which 165 were annotated in the PhosphoSitePlus® database. To identify substrates for kinases with nuclear localization, ten kinases were tested with nuclear HEK cell lysate. We identified 194 kinase-substrate pairs, 141 of which were unique to the nuclear fraction and 27 annotated in the PhosphoSitePlus® database. Finally, kinases with outstandingly high amounts of novel substrates were subjected to gene ontology analysis. We were able to link the gene ontology classifications of novel substrates to the biological processes regulated by the kinase of interest. These results indicate that heavy-labeled 18O-ATP-based kinase assay linked LC-MS/MS is a useful tool for large-scale direct kinase substrate identification.

Type 2 diabetes mellitus (T2DM) has been shown to be associated with hyperglycemia, insulin resistance, hyperinsulinemia and impaired insulin secretion from pancreatic β-cells leading to micro- and macro-vascular complications including multiorgan failures. At the cellular level, the mechanism of insulin resistance is associated with complex PI3K-Akt mediated insulin signaling pathway. Moreover, lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) plays a vital role as a negative regulator of the insulin signaling pathway downstream of PI3K by hydrolyzing phosphatidylinositol- 3,4,5-trisphosphate (PIP3) into phosphatidylinositol- 3,4- biphosphate (PIP2). Scientific reports have shown that inhibition of SHIP2 activity might improve Akt phosphorylation and thus PI3K-Akt mediated insulin signaling pathway. Considering this, I am interested in the SHIP2 inhibitors with drug like properties such as improved solubility, pharmacokinetic and bioavailability properties with little to no contraindications. In the present thesis, I have attempted to detect indirectly the capacity of 8 novel small molecule SHIP2 inhibitors, #160, #161, #162, #163, #167B, #170A, #171, #172 for their ability to phosphorylate Akt kinase in L6 myotubes using immunoblotting as a tool and compared data using graphical representation to pick up the best candidate. Two inhibitors, #163 and #170A were further chosen for alamarBlue® cytotoxicity assay. Treatment with #163 did not display direct cytotoxic effects on the myotubes. The viability of myotubes was not affected at low concentrations of #170A, but it started to reduce at concentrations >200 µM. In my study, I came up with #163 and #170A as the best lead candidates for further analysis. In future, more trials need to be performed with these inhibitors. Moreover, there are several other novel small molecule SHIP2 inhibitors identified from chemical library that need to be tested. Briefly, in this thesis, I have first time reported 8 novel small molecule SHIP2 inhibitors which could be a significant step in the discovery of new T2DM drugs for more efficient, cost effective and safe treatment of the disease with least contraindications.

In this study 39 methicillin-resistant Staphylococcus aureus CC398 isolates from Finnish pigs and pork were sequenced by whole-genome sequencing. Also, phenotypic antimicrobial resistance of the strains was examined. The strains originated from the culture collection of Finnish Food Safety Authority (Evira) and they were isolated between the years of 2008 and 2017. MRSA strains were isolated from both asymptomatic and infected pigs and pork. The strains represented the four most common spa types in Finnish pigs (t034, t2741, t011 and t108). DNA extraction method for S. aureus was optimized by comparing automatic DNA extraction robot and manual DNA extraction method. Extracted DNA samples were sent to Germany to be sequenced with Illumina sequencing. Paired-end raw read sequences were assembled using three different algorithms: Spades, Idba and Velvet. The assembled sequences were analysed by bioinformatics tools. Possible antimicrobial resistance genes, virulence genes, plasmids and SCCmec cassettes were identified. The strains were also typed by spa typing and sequence typing. spa typing was already performed in Evira using PCR and the new spa typing results were compared to the PCR results. Using the assembled sequences, a phylogenetic tree was also constructed. Phenotypic antimicrobial susceptibility testing was performed using broth microdilution panel. The manual extraction method was slightly better since the extracted DNA was purer. The best assembly algorithm was Spades and because of that all sequences were assembled with that algorithm for further analyses. The strains carried only a few virulence genes and many genes that are important for human infection were missing. All the strains carried the same SCCmec cassette type and sequence type ST398 both of which are known to be typical for pig related MRSA. Illumina sequencing and spa typing were not working perfectly since the used program was not able to distinguish spa types t034 and t011 from each other. The problem was probably due to very similar short tandem repeats that these two spa types share. Also, Illumina sequencing is known to produce short reads that could be problematic for sequence assembly. All the strains were resistant to beta-lactams and tetracycline which is typical for MRSA CC398 strains. Many strains were also resistant to clindamycin and trimethoprim. Antimicrobial resistance seemed to be carried by plasmids in some cases because the strains carried several plasmids that are known to contain antimicrobial resistance genes. The antimicrobial resistance profiles were different between different spa types since some spa types were more resistant to certain antimicrobials than others. According to the phylogenetic tree, the strains seemed to be a very heterogenic group. The strains that belonged in the same spa type were most related to each other as expected. There were no differences between strains originating from pigs and strains originating from pork which strongly indicates that bacteria could have spread from pigs to pork. Asymptomatic pigs also carried similar strains as infected pigs. The strains that were causing infections in pigs did not contain any more virulence factors compared to the other strains. The study gave plenty of new information concerning MRSA CC398 in Finnish pigs and pork. The antimicrobial resistance and virulence patterns in addition to other qualities of the strains were very similar that was described in the literature before. The strains seemed not to be very virulent for humans, but the situation must be observed in case of increasing resistance and virulence. The best way to prevent spreading of MRSA is to decrease the amount of MRSA in pig farms since MRSA bacteria are most likely to spread from pigs to the environment and to the people who work in pig farms and from there to the rest of the population.