Browsing by Subject "Biotechnology"

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  • Kylväjä, Riikka; Kankainen, Matti; Holm, Liisa; Westerlund-Wikström, Benita (2011)
  • Elf, Sonja (Helsingin yliopisto, 2019)
    Despite recent advances in understanding, diagnosis and treatment of cancer, this complex and versatile disease remains one of the leading causes of death worldwide. New and rapid diagnostic methods are needed to detect cancers at their early stages of development, thus enabling earlier prognosis, better risk assessment and more efficient treatment of the disease. There has been an increasing interest in specific molecular biomarkers as the hallmark for cancer research, and the detection of these markers from liquid biopsies using advanced molecular diagnostics methods provides major advantages over the conventional imaging methods currently used in oncology. The aims of this thesis were to examine the applicability of a novel molecular method, SIBA® (Strand Invasion Based Amplification), for the detection of cancer biomarkers, and to develop an assay targeting androgen receptor splice variant 7 (AR-V7) mRNA. The AR-V7 is proposed as a treatment-response biomarker in patients with castration-resistant metastatic prostate cancer (mCRPC). The expression of this variant can indicate resistance to hormonal therapies used for the treatment of advanced prostate cancer. Prostate cancer is the most common cancer after lung cancer in men worldwide and can gradually develop into a highly advanced lethal form, mCRPC, that is not responsive to androgen deprivation therapies. Positive AR-V7 status is suggested to represent the phenotype of this advanced stage of prostate cancer, and its detection can assist in treatment selection for the mCRPC patients. SIBA is a novel isothermal method for the amplification and detection of nucleic acids. The technology offers significant advantages over the more conventional molecular detection method, polymerase chain reaction (PCR), since the amplification reaction occurs at constant temperature and does not require sophisticated laboratory equipment for the thermal cycling. Reverse transcription SIBA (RT-SIBA) enables reverse transcription of RNA to cDNA as well as the simultaneous amplification and detection of the cDNA in one-step reaction under isothermal conditions. The method displays both high analytical sensitivity and specificity to the target nucleic acids. The RT-SIBA technology has not formerly been applied for the detection of human DNA or RNA. The main finding of this thesis was, that the RT-SIBA technology can be applied for rapid detection of specific molecular cancer biomarkers such as the AR-V7 mRNA. In this study, two RT-SIBA assays targeting the full-length androgen receptor (AR-FL) mRNA and the AR splice variant 7 mRNA were developed and optimized. Performance of the assays were evaluated by testing RNA isolates from AR-V7 positive and negative prostate cancer cell lines in the presence of human whole blood and plasma in the reaction. The developed RT-SIBA assays provided high analytical sensitivity and specificity: low copies of the target mRNA were amplified within 20 minutes without the production of non-intended amplicons. The results suggest that the RT-SIBA technology can be utilized for easy and rapid detection of AR-V7 and AR-FL mRNA directly from liquid sample material without a need for time-consuming sample treatment. Further performance evaluation using real AR-V7 positive clinical samples from mCRPC patients is necessary for the reliable validation of the developed assays.
  • Hyvärinen, Kati Susanna (2001)
    Gene technology is a rapidly growing area in the field of biotechnology. People's lives are pervaded by a flow of biomedical knowledge showing the associations between biology and identity. Gene testing has already improved lives of a number of people. Some reliable, low cost tests are used to clarify a diagnosis and direct a physician toward appropriate treatments, while others allow families to avoid having children with devastating diseases or identify people at high risk for conditions that may be preventable. The goal of this paper is to study the kind of attitudes Finnish general population has toward gene tests. Rapidly developing gene technology gives people the reason to think of how to act in a situation where the hereditary make up of the future generations can easily be altered to please ones ideals. People were asked to describe in their own words what benefits and drawbacks do they find in prenatal gene tests. The basic finding of my study is that people people are concerned about the new innovations, yet raising hopes for the new discoveries. Respondents show more positive attitudes toward prenatal gene tests than negative. The most crucial finding, however, is that the tests are able to provide information about the health of a baby. Everyone is wishing for healthy offspring and hopes to avoid unnecessary pain and worries. The most active respondents seem to be females in the most acute child bearing age. Marital status, nor ones education level do not seem to explain my findings. Income level, however, does indicate that people with more money seem to have stronger attitudes toward the issue. Prenatal gene tests raise a lot of feelings because they clearly have two different sides to an issue. The ones against the testing are afraid of its possible effects to eugenics, the ones in favor of the test plead to the decrease of human suffering. The question is difficult do to the fact that it has no either or answer. Public needs more information about the issue as well as clear laws and regulations to follow the correct use of those tests.
  • Oghabian, Ali (Helsingin yliopisto, 2018)
    In the Eukaryotes, DNA sequences in genes are often interrupted by non-coding sequences called introns. These sequences are removed from the transcripts via a process known as splicing either while the genes are being transcribed (co-transcriptionally) or after transcription (posttranscriptionally). In higher eukaryotes two separate pre-mRNA splicing machineries have been described: the U12-dependent spliceosome which is responsible for splicing of approximately 700-800 unique introns (known as the U12-type introns), and the U2-dependent spliceosome responsible for splicing all other introns (known as the U2-type introns). The two intron types show divergent sequence elements in their 5' splice site and branch point sequences. In addition, earlier reports have indicated that U12-type introns are spliced with a slower rate comparing to the U2-type introns, suggesting that the splicing of U12-type introns is rate-limiting to the expression of the U12-type intron containing genes. This slower splicing is manifested as unspliced or retained U12-type introns in the otherwise fully processed mRNA products. In this work I developed a novel computational tool called the intron-exon retention estimator (IntEREst) which allows accurate detection, quantification and differential analysis of the intron retention levels from RNAseq data. Additional features of IntEREst include a tool for identification of U12-type introns, and a number of tools to compare the retention levels of userdefined subclasses of introns across several samples. An already published RNAseq dataset (available under accession GSE63816 in NCBI Gene expression Omnibus database) from patients and control subjects of myelodysplastic syndrome (MDS) was used to assess the functionality by benchmarking IntEREst. This dataset included RNAseq data from MDS patients featuring mutations in the ZRSRS2 gene that functions in the recognition of U12-type introns, and from control subjects that were either healthy or MDS patients without ZRSR2 mutations. Additionally, I used a Maize dataset consisting of samples with mutated and wild-type RGH3 gene, which is an ortholog of human ZRSR2. My results indicate that IntEREst is a reliable tool for analyzing intron retention events from RNAseq data producing comparable or better results than the other similar methods. I used IntEREst to globally compare the retention of the U12-type introns to that of U2-type introns. I found that U12-type introns show on average a 2-fold higher retention levels compared to that of U2-type introns both in human and plant cells. This result recapitulates the findings from earlier studies using a small set of selected genes and generalizes the increased intron retention of U12-type introns to a genome-wide scale. Furthermore, the results of this work provide evidence that transcripts containing unspliced U12-type introns are degraded in the nucleus by the nuclear exosome. Together, these results support the hypothesis that U12-type introns are globally spliced less efficiently than the U2-type introns and can thus regulate the rate of mature mRNA formation with the genes containing U12-type introns. Additionally, intron retention analysis of human/plant cells containing mutations in the U12-dependent spliceosome showed that such defects lead to a further increase in the levels of unspliced U12-type introns. In conclusion, this thesis extends current knowledge concerning the significance of the correct splicing of U12-type introns and the consequences of their abnormal splicing. Furthermore, it describes a combination of available tools together with a novel software tool (i.e. IntEREst) that can be used to measure and compare the efficiency and accuracy of RNA splicing across multiple samples. We show that these tools can reveal valuable information about the molecular mechanisms involved in various conditions, e.g. diseases caused by defective spliceosome.
  • Jha, Sawan (Helsingfors universitet, 2014)
    Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.
  • Zaki, Urfa (Helsingin yliopisto, 2019)
    Cerebral dopamine neurotrophic factor (CDNF) belongs to the the family of neurotrophic factors that are evolutionary conserved, having a unique structure, with two domains: C-terminal domain and the N-terminal domain, and a cysteine bridge. It is known to be involved in the repair of the dopaminergic neurons when studied in the animal models of PD, which shows their different mode of action as compared to other neurotrophic factors, highlighting their therapeutic potential. Analysis of the crystal structure shows that CDNF and MANF consist of two domains: the saposin-like N-terminal domain with five α-helices stabilized by three disulphide bridges, and presumably unstructured C-terminal domain with a disulphide bridge. Characteristic feature of saposin-like proteins is their ability to interact with membranes or lipids. The lipid interaction may be crucial for the activity of CDNF and MANF proteins. In the first part of this project, the binding of CDNF was tested with several oxidized lipids, using two methods; Co-sedementation assay and lipid fluorescence assay;with two different types of probes. According to the results, CDNF seemed to show binding with POVPC. The second part of the project involved testing the binding and internalization of CDNF to mouse myoblast cells in the presence of oxidized lipid; POVPC. It was observed that CDNF seemed to show binding to the cell surface of the mouse myoblast cells (C2C12) and is also observed to be internalized to the cells as well. However, as these are the preliminary results, so we need to further test the binding between the protein and other lipids and devise more precise protocols for the testing the internalization to the cells.
  • Banerjee, Rishi (Helsingin yliopisto, 2019)
    After birth, stem cells act as the source of reparative and regenerative potential in various tissues. Among different tissues and organs in human body, tooth is one of the organs which does not undergo continuous regeneration. Therefore, tooth regeneration must be studied in a different animal, which possesses continuously growing teeth. In mouse, the incisor undergoes continuous growth which is fueled by the interaction between epithelial and mesenchymal stem cell compartments located at its apical end. The inferior alveolar nerve, which supports mandibular dentition, and its surrounding blood vessels (combinedly known as neurovascular bundle or NVB) were previously shown to act as a source of the mesenchymal stem cells during incisor growth and regeneration. However, the regulation of the cells in the NVB is not well understood. The primary aim of my master’s thesis was to characterize the effect of the Hh pathway modification on cellular properties of the NVB and the MSCs within it. The Ptch2 KO mouse model used in this study demonstrated increase in the number of blood vessel in the NVB. Additionally, analysis of the structure of skin in the mouse model was the second aim of my project, which showed significant increase in the thickness of the dermis at the postnatal day 1. Collectively, the change in structure of skin and NVB showed that Ptch2 might regulates the cellular properties of tooth mesenchyme and dermis by modulating the structural components of the NVB of continuously growing mice incisor and skin, respectively.
  • Sultana, Nasrin (Helsingin yliopisto, 2020)
    Tiivistelmä – Referat – Abstract Plant lives and grows in variable environment and climate conditions. Everyday plants can be confronted with a variety of abiotic (temperature, light, salt, water availability) and biotic stress (pathogens, insects etc). These abiotic and biotic stress can halt plant growth and influence crop productivity. Plant has evolved signaling mechanism and different responses to adapt or respond with these unfavorable environmental conditions. Our group’s previous research identified a new mutant in the model plant Arabidopsis thaliana with a striking phenotype – when the plants ages it progressively becomes yellow and eventually the entire plant is white. The mutant was named “white” after its striking appearance. The phenotype is associated with increased accumulation of mRNA transcript for stress and senescence regulated genes. Mapping of the mutation identified a 4 bp deletion in a gene EGY1 that encodes a metalloprotease located in the chloroplast. To identify molecular mechanisms that regulate this unusual type of premature senescence, a suppressor mutants screen was performed in the white mutant, and three suppressors that restore normal appearance to the plant was identified. Mapping of one of these suppressors, identified a mutation in STAY GREEN1 (SGR1) as a likely candidate. SGR1 encodes the protein that catalyze the first step in chlorophyll breakdown, removal of Mg2+ from chlorophyll. The overall aim of my master thesis was to understand the molecular mechanisms behind the development of the age and chlorophyll related phenotypes in the white mutant and its two suppressors S1 and S2. Furthermore, with gene expression analysis, plant stress and senescence responses were studied in white, S1 and S2. By complementation method I proved that mutations in SGR1 gene caused the development of suppressor mutant phenotype and restoration of wild type allele of SGR1 gene restore white phenotype in suppressor mutant. Measurements of chlorophyll concentration provided further evidence that the mutation in SGR1 stabilizes the suppressor mutant phenotype, stops chlorophyll breakdown and keep the leaves green. Gene expression study using qPCR with marker genes provided insight of molecular changes within these phenotypes.
  • Böcker, Sebastian; Mäkinen, Veli (IEEE/ACM, 2008)
    Mass spectrometry has become one of the most popular analysis techniques in Proteomics and Systems Biology. With the creation of larger datasets, the automated recalibration of mass spectra becomes important to ensure that very peak in the sample spectrum is correctly assigned to some peptide and protein. Algorithms for recalibrating mass spectra have to be robust with respect to wrongly assigned peaks, as well as efficient due to the amount of mass spectrometry data. The recalibration of mass spectra leads us to the problem of finding an optimal matching between mass spectra under measurement errors. We have developed two deterministic methods that allow robust computation of such a matching: The first approach uses a computational geometry interpretation of the problem, and tries to find two parallel lines with constant distance that stab a maximal number of points in the plane. The second approach is based on finding a maximal common approximate subsequence, and improves existing algorithms by one order of magnitude exploiting the sequential nature of the matching problem. We compare our results to a computational geometry algorithm using a topological line-sweep.
  • Oikkonen, Jaana (Helsingfors universitet, 2012)
    Genome wide linkage and association methods are used to map genes affecting traits with genetic predisposition. In this thesis, I compare the methods suitable for quantitative trait mapping in complex, extended pedigrees. As a case study, gene-mapping study of musical aptitude is performed with these methods. Linkage analysis methods are developed for family studies. However, only a few methods are suitable for extended families with a quantitative trait. Three linkage programs were successfully applied for such data in this study. These programs are the SOLAR, JPSGCS and KELVIN. All of these three programs are based on different methods and thus, the same calculations are not repeated. SOLAR is based on the variance components method, JPSGCS on a graphical method and KELVIN on the Bayesian method. Association analysis is also difficult to implement for large pedigrees, because it is best suited for case-control data. Fortunately, methods are extended also for family-based studies. Here, a genomic control method was used to correct for the familial relationships. The method evaluates the relatedness from the whole genome data and the association tests are corrected for the relatedness rates. This method was implemented from the GenAbel program. As a case study, these methods were applied to a musical aptitude study. The musical aptitude is here understood as an ability to perceive the melody, harmony and rhythm of music, and to recognize structures in set of sounds. These abilities were tested with Carl Seashore s tests for pitch and time and Kai Karma's test for auditory structuring. The data consists of 107 pedigrees and 93 sporadic subjects, comprising in total of 915 individuals. Each family includes 2 - 50 individuals. These individuals were genotyped with a SNP chip for over 700,00 SNPs. The linkage analyses revealed several promising loci for the musical aptitude. The best result was located in 4q12 and it was found with all of the three linkage programs. Most of the other results could also be identified with multiple programs, but some differences also occurred. However, none of the findings could be discovered with association analysis, probably due to a too small sample size.
  • Ravikumar, Balaguru (Helsingin yliopisto, 2020)
    Rational approaches to the traditional drug discovery process rely on high-throughput and lowthroughput bioactivity or phenotypic screening studies. Such profiling strategies have been the foremost step utilized when identifying or curating lead molecules, and more recently, when evaluating compound repositioning/repurposing opportunities. Although promising, such systematic approaches are inherently limited by their cost and labor constraints, and the necessity of specific screening equipment’s renders them outside the scope of many academic laboratories. Further, elucidation of any novel associations through such experimental techniques require exhaustive searches of compound libraries. Hence, many positive drug indications revealed, though rational, are often by the virtue of serendipity. The accumulation and standardization of existing compound profiling datasets has paved the way to the field of chemoinformatics. Wherein, data-driven computational approaches are employed as a pragmatic solution to challenge the inherent notion of serendipity in screening studies. Such in silico models serve as an efficient and cost-effective augmentation to the experimental screening approaches, circumventing the intrinsic limitations of current drug discovery methods. This study therefore was motivated towards identifying niches and limitations prevalent in the current pharmacological paradigm, where an effective computational framework could be utilized to complement and expedite the conventional drug discovery process. The various computational approaches proposed in this article-based thesis include exploratory web-tools, new data resources, and prediction models, that are introduced as supplements to extend the current computer-aided drug discovery process (CADD). Firstly, I developed a web-application, termed C-SPADE, a novel compound-centric chemoinformatic tool that facilitates interactive analysis and visualization of compound screening experiments using Compound-SPecific bioActivity DEndrograms. The tool employs compound-compound similarity metrics to estimate the diversity amidst compounds profiled in the screening panel and intuitively represents the chemical similarity space and the observed bioactivity values. The web-tool provides users an exploratory framework to perform pharmacological analysis and investigate novel compound associations employing diverse compound similarity clusters. Secondly, to address the heterogeneous and non-standardized bioactivity data in existing data resources, a comprehensive open-data platform, called Drug Target Commons (DTC), was developed. DTC feature tools for data annotation, standardization, curation to address intra-resource heterogeneity and provide users a one-stop resource for drug discovery and in silico model development endeavours. User-specific applications of both the above-mentioned resources have been demonstrated through several case studies and experimental validations. In addition to the tools and databases, I have also designed and implemented diverse machine learning models primarily to predict potent compound-kinase interactions and to fill the current experimental gaps in large-scale activity profiling studies. Firstly, through collaborative efforts, we employed the Kronecker kernel-based regularized least square regression (KronRLS) algorithm under different crossvalidation settings to predict both the uncharacterised binding measures in large-scale profiling studies and novel compound-target associations. As a case study, the off-target profile of an investigational VEGF receptor inhibitor tivozanib was predicted and experimentally validated. Secondly, I designed and implemented an efficient statistical model utilizing an ensemble SVM classifier to prioritize potent compound-kinase association for biochemical testing. The developed computational framework was termed Virtual Kinome Profiler (VKP) and was efficiently used in compound repositioning and lead identification studies, wherein 19 novel kinase-compound interactions spanning across different kinases were predicted and experimentally validated. Apart from elucidating the chemogenomic similarities prevalent among distinct kinase proteins, VKP with a positive prediction value (PPV) of 84% was shown to reduce the time and cost constraints related to traditional experimental screening process. Most of the computational frameworks proposed in this thesis are designed and deployed with an accompanying web-based graphic user interface (GUI). This in turn aids in the translatability of the platforms, overcoming the current prerequisites required when utilizing CADD models, including prior expertise in data analysis and scripting languages. These studies together exemplify new applications of computational models in diverse areas of the drug discovery process, subsequently making invaluable augmentations to a chemical biologist’s toolbox.
  • Österman, Janina (Helsingfors universitet, 2009)
    Pseudomonas syringae pv. tomato DC3000 is a Gram-negative plant pathogen that causes bacterial speck disease on tomato. The virulence of this bacterium is based on the type III secretion system (T3SS). Similar systems are also used by many other plant and animal pathogens, as well as symbiotic bacteria. The T3SS enables the transfer of specific bacterial virulence proteins from the bacterial cytoplasm into the host cell. This secretion is mediated by a needle-like structure that penetrates the plant cell wall. Once inside the host cells, the effector proteins are capable of shutting down the host’s immune system. However, what happens at the plant cell membrane is not well understood. One of the first bacterial proteins that come into interaction with a host protein during P. syringae pv. tomato DC3000 infection is the HrpZ1 protein that is believed to participate in a membrane interaction, facilitating the transfer of effector proteins. Previous research has shown that HrpZ1 binds to a peptide and using an antiserum raised against this peptide in immunoblotting tests of tomato proteins separated by SDS-PAGE and isoelectric focusing, the target tomato protein of HrpZ1 has been found to be small and acidic. However, this specific protein has not yet been fully characterized. This is why the goal of the work for this thesis was to identify and characterize the target protein of HrpZ1 in tomato. For this purpose, a lambda cDNA expression library from tomato leaves was constructed, followed by immunoscreening of the library with the abovementioned antiserum, and analysing candidate clones by sequencing. Even though a good cDNA library was obtained, the immunoscreenings did not yield satisfactory results. Thus, the pursuit of the HrpZ1 target protein needs to be continued.
  • Deb, Debashish (Helsingin yliopisto, 2019)
    There is significant reduction in number of approved drugs for acute myeloid leukemia in recent years. Partially it may be due to the failure of discovery and validation approach to new drugs as well as the complexity of the disease. Ex vivo functional drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). In ideal condition, an effective drug should eradicate the immature AML blasts, but spare non-malignant hematopoietic cells. However, current strategies like conventional cell viability assay fail to measure cell population-specific drug responses. Hence, development of more advanced approaches is needed. Using multiparameter, high-content flow cytometry (FC), we simultaneously evaluated the ex vivo sensitivity of different cell populations in multiple (10) primary AML samples to 7 FDA/EMA-approved drugs and 8 drug combinations. Amongst the 7 tested drugs, venetoclax, cytarabine and dasatinib were very cytotoxic with venetoclax had the highest blast-specific toxicity, and combining cytarabine with JAK inhibitor ruxolitinib effectively targeted all leukemic blasts but spared non-malignant hematopoietic cells. Taken together, we show that the ex vivo efficacy of targeted agents for specific AML cell population can be assessed with a cell phenotype, FC-based approach. Furthermore, we put an effort to analyze the potential of this assay and biomarkers to predict the clinical outcome of individual patients and future perspectives.
  • Korppoo, Annakarin (Helsingfors universitet, 2017)
    Trichoderma reesei, an anamorph of Hypocrea jecorina, is a filamentous fungus widely used for producing industrial enzymes. T. reesei is used for both endogenous and heterogenous protein production. The optimization of the production conditions and the effects of extracellular agents to T. reesei s production and secretion capacity are crucial for economically sustainable biotechnical production. The available carbon sources, most commonly different types of sugars, have a significant effect on the production and secretion of enzymes by T. reesei. Genetic modification of the pathways through which the fungi recognizes extracellular signals could bring advancements to industrial enzyme production. Because of T. reesei s potential and use as a production strain, the species is an interesting platform for genetic modifications that would enhance the production capacities. With the current methods the genome editing of T. reesei is however slow, and introducing multiple mutations to a single strain can take years. The aim of this study is to optimize the fairly new CRISPR/Cas9 genome editing system for use in T. reesei. In the CRISPR/Cas9 method, a catalytically active Cas9 enzyme is bound to a specific locus of the genome, guided by a guide RNA and the Watson-Crick base pairing principle. Once in the RNA-guided locus, Cas9 introduces a double stranded break in the DNA, which can be repaired by the cells endogenous non-homologous end joining pathways. This repair is error prone and produces mutations to site of the double stranded break. A donor DNA is often introduced together with the Cas9 and guide RNA. This donor DNA includes sequence homology to the site of interest and allows for the use of the cells homologous repair pathways. In this case, the mutation can be better controlled, and for example the risk of chromosomal mutations is reduced. Currently the CRISPR/Cas9 system is widely used in mammalian cell studies and up to 100% mutation frequencies have been reported in yeast cells. In this study the method is optimized for use in T. reesei. To our best knowledge, the research community has not found an organism in which CRISPR/Cas9 would not function. The question mainly lies on what type of set up and component introduction is suitable for each cell type and research purpose. In this thesis, three putative and one already published genes believed to be involved in hexose sugar sensing will be deleted from a T. reesei production strain with the help of CRISPR/Cas9. The effect of these deletions will be assessed through studying the secretion and activity of endogenous cellulases with enzymatic assays. One sugar transporter that may play a part in glucose sensing was identified in this study. The deletion of this transporter caused a decrease in cellulase production and/or secretion. The three other transporters or sensors did not have a significant effect on cellulase production in spent grain extract and lactose or glucose media. It s possible that these genes are involved in the uptake and use of other carbon sources. The continuous expression of the CRISPR/Cas9 system in T. reesei proved difficult. In the continuous expression method at least one of the CRISPR/Cas9 components, the Cas9 protein or the guide RNA, is produced in the cells in vivo. Neither was achieved in this study. Instead, a fully synthetic method in which the Cas9 is transformed into the cells as a protein along with an in vitro produced guide RNA was set up and produced up to 1000 × higher mutation frequencies when compared to the traditional transformation method used for T. reesei. This study also demonstrates a simultaneous deletion of two genes in T. reesei. To the best of our knowledge, multiple simultaneous gene modifications have never been achieved in T. reesei.
  • Dilokpimol, Adiphol; Mäkelä, Miia R; Aguilar-Pontes, Maria V; Benoit-Gelber, Isabelle; Hildén, Kristiina S.; de Vries, Ronald P (BioMed Central, 2016)
    Abstract Feruloyl esterases (FAEs) represent a diverse group of carboxyl esterases that specifically catalyze the hydrolysis of ester bonds between ferulic (hydroxycinnamic) acid and plant cell wall polysaccharides. Therefore, FAEs act as accessory enzymes to assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass conversion. Their ability to release ferulic acid and other hydroxycinnamic acids from plant biomass makes FAEs potential biocatalysts in a wide variety of applications such as in biofuel, food and feed, pulp and paper, cosmetics, and pharmaceutical industries. This review provides an updated overview of the knowledge on fungal FAEs, in particular describing their role in plant biomass degradation, diversity of their biochemical properties and substrate specificities, their regulation and conditions needed for their induction. Furthermore, the discovery of new FAEs using genome mining and phylogenetic analysis of current publicly accessible fungal genomes will also be presented. This has led to a new subfamily classification of fungal FAEs that takes into account both phylogeny and substrate specificity.
  • Auvinen, Pauliina (Helsingin yliopisto, 2018)
    Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.
  • Kaya, Meryem Ecem (Helsingin yliopisto, 2019)
    Synthetic biology is an emerging interdisciplinary field of biology that aims to system-atically design artificial biological systems. As synthetic biologists seek increasingly complex control over cellular processes to achieve robust and predictable systems. A new frontier in synthetic biology is engineering synthetic microbial consortia. This ap-proach employs the concept of division of labor, instead of introducing large genetic cir-cuitry to homogenous cell populations. In this approach, different cell types are assigned to execute a portion of the overall circuit. Each cell type communicates with their co-worker subpopulations to complete the circuit. The main advantage of this strategy is the reduced metabolic burden on each cell type. Thus, leading to more reliable and stable overall performance. In this work, to simplify cellular communication between the mem-bers of the consortium, we used the simple architecture of quorum sensing machinery. We constructed a toolbox that contains promoter, receptor and quorum sensing signal synthase genes along with fluorescent reporters. Using this toolbox, we constructed dif-ferent cell types that can be used in synthetic consortia forming various communication topologies. We characterized the constructed cell types individually and in co-cultures.
  • Partti, Edvard (Helsingin yliopisto, 2018)
    Kaurapohjaiset elintarvikkeet ovat terveellisiä. Monet niiden terveyshyödyt johtuvat kauran liukoisen ravintokuidun suuresta β-glukaanipitoisuudesta. β-glukaanin terveysvaikutukset ovat riippuvaisia sen molekyylipainosta ja viskositeetista. Viskositeetilla on myös muuta merkitystä kaurapohjaisissa elintarvikkeissa kuten kaurajugurteissa ja kauramaidoissa. Aiemmassa Folafibre-tutkimusprojektissa oli tutkittu kaurakuidun folaattipitoisuuden (B9 vitamiini) kasvattamista fermentoimalla sitä eri mikrobeilla. Hyvin folaattia tuottaneet mikrobit myös alensivat kaurakuituvalmisteen viskositeettia, ja erittivät glykosyylihydrolaaseja ja/tai proteaasia. Oli kuitenkin jäänyt epäselväksi, olivatko entsyymit ainut syy viskositeetin alenemiseen, ja kuinka paljon kullakin niistä oli vaikutusta viskositeettiin. Lisäksi tämän alan kirjallisuudessa on pidetty epäselvänä, onko esim. tärkkelyksen ja β-glukaanin välillä jotain interaktioita jotka mm. nostavat niiden viskositeettia yli yksittäisten polymeerien viskositeetin summan. Näitä voitaisiin selvittää puhdistamalla viskositeettia alentaneista mikrobikannoista kyseiset entsyymit ja tutkimalla niiden vaikutusta kaurakuidun viskositeettiin. Yhdeksi mikrobiksi valittiin Exiguobacterium sp. RB3 kanta, koska em. syiden lisäksi tässä bakteerisuvussa esiintyy psykrofiilejä ja mikrobeja jotka voivat kasvaa korkeassa pH:ssa, jolloin sen erittämät entsyymit saattaisivat olla aktiivisia matalissa lämpötiloissa ja korkeassa pH:ssa, ja olla siten teollisesti kiinnostavia. Toiseksi mikrobiksi valittiin Bacillus sp. ABM5119, koska sitä oli käytetty monissa Folafibre-projektin tutkimuksissa. Työn tavoitteena oli puhdistaa Bacillus sp. ABM5119:n endo-β-1,4-glukanaasi, ja Exiguobacterium sp. RB3:n α-amylaasi ainakin siinä määrin, että ne ovat muista endoaktiivisista glykosyylihydrolaaseista ja proteaaseista puhtaita, ja sitten mitata puhdistettuja entsyymejä ja proteaasia käyttäen niiden vaikutus yhdessä ja erikseen keitetyn kaurakuituvalmisteen viskositeettiin. Lisäksi tavoitteena oli karakterisoida Exiguobacterium sp. RB3 amylaasin olennaisimmat biokemialliset ominaisuudet, sekvensoida sen geenin amylaasia koodaava alue, ja selittää sen ominaisuuksia myös sekvenssistä johdettavissa olevan tiedon perusteella. Keitetyn kaurakuitupreparaatin viskositeettia alentavat eniten α-amylaasi ja endo-β-1,4-glukanaasi. Proteaasi ei vaikuta kaurakuitupreparaatin viskositeettiin, kun kuitupreparaatti on keitetty. Endo-β-1,4-glukanaasi alentaa viskositeettia yhtä hyvin kuin β-1,3-1,4-glukanaasi. Synergiaa viskositeetin alentamisessa α-amylaasilla ja endo-β-1,4-glukanaasilla ei havaittu, mutta havaittiin että β-glukaani saattaa estää tärkkelyksen retrogradaatiota. Hyvin pienetkin entsyymiaktiivisuudet vaikuttavat viskositeettiin. Jos halutaan β-glukaanin viskositeetin ja siten terveysvaikutusten säilyvän, täytyy esim. kauramaidon valmistuksessa käytettävien entsyymivalmisteiden olla β-glukanaaseista hyvin puhtaita. Mikrobifermentaatioissa tapahtuvat viskositeetin alenemat johtuvat lähinnä mikrobien erittämistä glykosyylihydrolaaseista, ei niinkään esim. niiden metabolian sivutuotteista kuten happiradikaaleista. Exiguobacterium sp. RB3 α-amylaasi on rakenteeltaan Bacillus licheniformis α-amylaasin kaltainen glykosyylihydrolaasiryhmän 13 entsyymi. Se sitoo rakenteeseensa kolme kalsiumiatomia, ja kalsiumpitoisuus vaikuttaa sen aktiivisuuteen. Se on aktiivisimmillan pH alueella 5,0 – 7,5. Se sietää detergenttejä, toisin kuin eräs aiemmin karakterisoitu Exiguobacterium-α-amylaasi. RB3 α-amylaasin turnover number oli korkea, 29000 1/s. Exiguobacterium-suvun psykrofiilisessä haarassa esiintyy kahta eri α-amylaasia, joista yksi on tässä karakterisoitu, ja toinen on selvästi erilainen rakenteeltaan ja biokemiallisilta ominaisuuksiltaan.
  • Martins, Beatriz (Helsingin yliopisto, 2020)
    According to the latest estimations, cancer is the second leading cause of death worldwide. Despite the significant advances in the range of drugs and treatment modalities to treat cancer, the number of deaths is estimated to continue rising, posing serious challenges for the patients, their families, and the healthcare systems. Conventional treatments tend to be associated with severe adverse side effects and treatment resistance. Consequently, safer and more efficient therapy options are urgently needed, especially for the treatment of metastatic tumors refractory to conventional treatments. A new and revolutionizing field in oncology is immunotherapy, in which oncolytic viruses are included. Oncolytic viruses have an inherent or acquired selectivity to replicate exclusively in tumor cells, ultimately destroying them. Simultaneously, they also activate the dormant host’s immune system to fight against the tumor. Adenoviruses, particularly, have shown to be safe, inducing only mild adverse side effects in clinical trials, making them a great candidate for further clinical development. Adenoviruses can be genetically modified to increase their infectivity or improve the anti-cancer immune responses induced by the virus, e.g., through the expression of immunostimulatory molecules. The focus of this thesis was to develop and characterize several genetically modified oncolytic adenoviruses expressing either OX40L alone or OX40L and CD40L, two co-stimulatory molecules capable of engaging both the innate and adaptive arms of the immune system to fight the tumor. The insertion of the transgenes into the E3B-14.7k region of the Ad5/3-∆24 adenovector plasmid was performed using Gibson Assembly® cloning approach. After successful cloning, the recombinant viral genomes were transfected into A549 cells for viral amplification, followed by CsCl purification to produce a high titer viral preparation. The expression of the transgenes was studied in vitro by ELISA and functional assays, showing promising expression levels of functional OX40L and CD40L. However, when the infectivity and virus killing potency were analyzed, in vitro by immunocytochemistry and MTS assay; and in vivo using an immunodeficient mouse model, the data showed that the cloned viruses performed sub-optimally when compared to the control unarmed virus (Ad5/3-∆24). These findings suggest that the insertion of the two transgenes in place of the E3-14.7k gene was detrimental to the fitness of the virus.
  • Kerminen, Sini (Helsingfors universitet, 2015)
    Studies of population structure are motivated by the need to understand population history and to have well-characterised groups of individuals in studies of genetics of diseases and traits. A standard method to analyse genetic population structure is principal component analysis (PCA). A disadvantage of PCA is that it can reliably handle only independent genetic markers. This means that the genetic markers that are correlated with other genetic markers have to be excluded from the data. This leads to a loss of information. In 2012, Lawson et al. published a chromosome painting method that can utilise haplotype information, i.e. information from correlated markers, and thus it can detect more subtle differences in populations than the standard PCA. This thesis studies two questions. The first question is whether the chromosome painting method can provide more precise genetic clustering of geographically defined Finnish groups than the standard PCA method. The second question is whether the chromosome painting method can reveal new details of population structure in Finland. The data used in this study are from the FINRISK Study survey of 1997. This cohort includes the genotype data of about 4,000 individuals and the information about individuals and their parents birthplaces. 345 Individuals were randomly chosen from the cohort in such a way that both of their parents were originated from the same province. Ten provinces of Finland were used as study groups for the method comparison. First, the data were analysed with SmartPCA (a standard PCA method) and ChromoPainter (the chromosome painting method) and the results were compared both visually and quantitatively. Finally, the individuals were assigned to populations based on the ChromoPainter result using FineSTRUCTURE program and these genetic populations were compared to the geographic origin of the individuals. The results showed that the chromosome painting method clustered seven out of ten groups significantly tighter than the standard PCA. Nevertheless, SmartPCA was faster and easier to use than ChromoPainter. The main population genetic division was found between the eastern and western parts of Finland, which was consistent with earlier studies. All in all, 15 populations were detected and the results revealed that they were geographically clustered. The genetic populations correlated well with the borders of Finnish provinces and counties. As the first conclusion, the chromosome painting method was able to give more precise results than the standard PCA but the standard PCA is still more suitable for quick preliminary analyses of genetic data. As the second conclusion, the chromosome painting method was able to detect detailed subpopulation structure in Finland and these populations are geographically clustered. Results provide an excellent basis for the future studies of population structure and genetic diseases in Finland.