Browsing by department "Bio- ja ympäristötieteellinen tiedekunta"

LCMV Lymphocytic choriomeningitis virus is a rodent-borne pathogen belongs to Arenaviridae family. Most of the studies have referred Mus musculus as the main reservoir of the LCMV. It has been detected in pet rodents, laboratory rodents, and wild mice. Humans be infected with LCMV through the ingestion or inhalation of sources contaminated with rodent feces, urine, or both. LCMV infection can be asymptomatic, present with mild symptoms, or it can cause aseptic meningoencephalitis (AME) and teratogenic effects in infants. However, clinical cases of LCMV infection have been rarely reported, and there is only fragmental knowledge on the presence and prevalence of LCMV infections around the world. Likewise, the genetic characteristics of the circulating LCMV strains and impact of LCMV on public health have remained poorly characterized. This study was performed in the Southern Iraq, due to the lack of comprehensive information about LCMV in this area. There were three main aims in this thesis. First, to assess the prevalence of LCMV among the healthy human population in the Nasiriyah region, southern Iraq. Second, to assess whether LCMV infections can be associated with neurological manifestations. Third, to characterize the genetic variation and evolutionary history of LCMV strains circulating in southern Iraq. Serum and CSF samples were collected from patients and healthy people in Nasiriyah governorate in the Southern Iraq. Serum samples were screened for LCMV using Immunofluorescence assay (IFA) to detect IgG and IgM antibodies. Real-time PCR was used to detect LCMV genome. In order to confirm the PCR positive samples, we sequenced these samples by Next-generation sequencing. The serological assay results showed 12.22% IgG prevalence of LCMV among healthy people and 7.36% IgG prevalence among patients with neurological symptoms. The IgM prevalence was 1.25% among the patients with acute infections. From symptomatic patients, we sequenced partial L-segments of two new LCMV strains. The phylogenetic tree constructed on the basis of all known LCMV strains suggested that these new LCMV strains from Iraq are genetically distant from the previously known LCMV strains and form a novel sub-cluster within LCMV species. This study is the first survey of LCMV in the Southern Iraq. LCMV appears to be a rather common infection in Iraq. I reported new strains of LCMV that are circulating in the study site and most likely is the causative agent of the central nervous system-associated clinical manifestations in these patients. For future work, I’m aiming the detection of other Arenaviruses spreading in the Southern Iraq.

The TTN gene encodes a giant muscle protein called titin that regulates the function of muscle sarcomere and interacts with several other muscle proteins. Mutations in TTN are associated with a broad range of skeletal and cardiac muscle disorders termed titinopathies. Previous studies have shown the importance of unusual TTN splicing events in patients with TTN-related cardiomyopathies and muscular dystrophies. In this project, we characterized eight TTN splicing variants to further expound on the pathogenesis of titinopathies and to enhance the diagnostic accuracy for patients with TTN mutations. In addition, we also made a comparative analysis of five different RNA/cDNA sequencing techniques to extrapolate on which approach is most suitable to study splicing variants in TTN gene. Skeletal muscle samples of six patients were analyzed in this study who were previously detected with TTN variants in a compound heterozygous state from a targeted next-generation sequencing assay. Our results from traditional Sanger sequencing methods, second-generation (Illumina RNA-Sequencing) and third-generation sequencing (Single-molecule real-time sequencing) methods showed distinct splicing events in the form of partial or complete exon skipping, intron retention, and in few instances showed multiple splicing effects rendered by a single variant. Complying with the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, the splicing variants were classified as pathogenic, likely pathogenic or variant of uncertain significance primarily on the basis of our experimental data. To address which sequencing method is most promising for analyzing TTN splicing variants, Illumina RNA Sequencing is very efficient, though, the combination of Illumina RNA Sequencing with long-read sequencing could be ideal. Our results further demonstrate that a near full-length titin is vital for survival until birth, and further studies are needed to understand the pathophysiology mechanism of congenital titinopathies.

Classical and rapid-acting antidepressant drugs have been shown to reinstate juvenile-like plasticity in the adult brain, allowing mature neuronal networks to rewire in an environmentally-driven/activity-dependent process. Indeed, antidepressant drugs gradually increase expression of brain-derived neurotrophic factor (BDNF) and can rapidly activate signaling of its high-affinity receptor TRKB. However, the exact mechanism of action underlying drug-induced restoration of juvenile-like plasticity remains poorly understood. In this study we first characterized acute effects of classical and rapid-acting antidepressant drugs on the interaction between TRKB and postsynaptic density (PSD) proteins PSD-93 and PSD-95 in vitro. PSD proteins constitute the core of synaptic complexes by anchoring receptors, ion channels, adhesion proteins and various signaling molecules, and are also involved in protein transport and cell surface localization. PSD proteins have in common their role as key regulators of synaptic structure and function, although PSD-93 and PSD-95 are associated with different functions during development and have opposing effects on the state of plasticity in individual synapses and neurons. Secondly, we investigated changes in mobility of TRKB in dendritic structures in response to treatment with antidepressant drugs in vitro. We found that antidepressant drugs decrease anchoring of TRKB with PSD-93 and PSD-95, and can rapidly increase TRKB turnover in dendritic spines. Our results contribute to the mechanistic model explaining drug-induced restoration of juvenile-like neuronal plasticity, and may provide a common basis for the effects of antidepressant drugs.

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.

Tiivistelmä – Referat – Abstract Telomeres are cap shaped structures at the very end part of each chromosome that protect DNA from degradation or unwanted chromosome-chromosome attachments. Telomere lengths show considerable heterogeneity in different cells of the same cell population. Reasons for heterogeneitiy and mechanisms inside cells causing them are not fully understood. In this study, we explored the correlation between telomere length and different gene expressions. First, using FACS technique we sorted each single cell into each well of 96-well plate. Second, we used SYBR green based qPCR for telomere length measurement. Third, we used Illumina-seq for sequencing extracted mRNAs. [6] We found a set of genes that were in strong correlation with telomere length, giving opportunity to explore the biological pathways. We compared pathways between different samples and found strong connections between genes involved in viral cycles and immune system with extracted genes that were in high correlation with telomere. We found heterogeneity of telomere lengths and transcriptomes in different cell lines. Telomere related proteins, specifically those involved in shelterin complex, are expressed highly in cancer cell lines and LPS-stimulated monocytes compared to the non-stimulated monocytes. In our study, SLC38A2, PURB, UBR3, SSR1, NCAPH2, AIMP2, PHF21A genes were highly correlated with telomere in mutual way and can therefore be considered as new biomarkers/novel candidates for telomere-related studies. The importance of these genes has been reported in aging/mortality. Concurrent with our findings, a recent report also suggested that NCAPH2 plays role in regulating telomere stability and maintenance through its interaction with TERF. [65] We found new genes in correlation with telomere regulation, and our findings are therefore of high importance in research of cancer, neurodegenerative diseases and aging. Further studies are, however, required as our data is limited by small number of samples and inability to properly validate our technique.

Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.

In addition to Chlamydiaceae, eight novel families have been discovered to belong to the phylum Chlamydiae. The eight families are Parachlamydiaceae, Waddliaceae, Criblamydiaceae, Parilichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, Clavichlamydiaceae and Piscichlamydiaceae.These families are phylogenetic relatives to Chlamydiaceae, share the intracellular developmental cycle and are widely distributed in nature and are therefore referred to as environmental Chlamydiae or Chlamydia related bacteria (CRB). CRB have a broad range of potential hosts. All families except Criblamydiaceae cause disease in animals and infect for example fish, arthropods and cattle. Families Parachlamydiaceae, Waddliaceae, Rhabdochlamydiaceae and Simkaniaceae are also shown to cause respiratory disease and adverse pregnancy outcomes in human. Free-living amoebae (FLA) are natural hosts of some CRB. CRB are able to survive and replicate inside of FLA that offers protection and nutrients for CRB. It has been suggested that CRB are transported to new environments inside of FLA. CRB DNA has previously been found on human skin (Hokynar et al. 2016, Hokynar et al. 2018, Tolkki et al. 2018) and in our water distribution system. CRB distributed to our tap- and shower water systems inside of FLA (Thomas and Ashbolt 2011) could be a potential rout of transmission of CRB DNA to human skin. As the diversity and size of the CRB group is large and CRB are very laborious to grow in vitro, it is challenging to detect CRB and to study their pathogenicity. Detection of CRB in clinical and environmental samples is mainly based on PCR methods. A non species-specific PCR method targeting Chlamydiales 16S rRNA (PanChl16S), that in theory amplifies all known CRB, has successfully been used in detection, but post PCR sequencing of the amplicon is required to identify the species. Also, more specific quantitative PCRs have been designed to detect specific families or species of Chlamydiae. However, the volume of clinical specimens available is often limited and allows only few separate analyzes. Due to the challenges identified with detection of CRB, efficient multiplex PCR assays would save time and resources and would be useful tools when detecting CRB DNA. The objective of the work was to explore the possibility of applying multiplexed analyzes to a limited specimen volume effectively. One aim of this thesis was to set up two multiplex PCR assays for detection of seven different CRB and a multiplex PCR for detection of three different FLA. Another aim of the work was to analyze the possibility of CRB to be transported to human skin from our water distribution system inside of FLA. In this thesis we set up two multiplex PCR assays for detection of CRB reference strains P. acanthamoebae, C. sequanensis, S. negevensis, Protochlamydia spp., Rhabdochlamydia spp., W. chondrophila and E. lausannensis. We also set up two PCR assays for detection of three different FLA reference strains: Acanthamoeba spp., Vahlkampfiidae spp., and V. vermiformis. We succeeded in developing two real-time multiplex PCR assays for detection of CRB DNA and two real-time PCR assay for detection of FLA DNA. Variability between replicates for each PCR target was low and the detection limit (100%) for each target ranged from 50-500 control plasmid copies per PCR reaction. The R2-value for each target was ≥0.98 and the reaction efficiency for each target ranged from 82-111%. Samples collected from showerheads (n=18) and water filters (n=2) as well as skin swabs (n=27) were studied with these newly established assays and PanChl16S PCR. The results obtained with the multiplex assays developed in this study were similar to the results obtained with the PanChl16S. CRB DNA was detected in 67% of the showerhead samples, in 100% of the water filter samples and in 31% of the skin swabs. Amoebae DNA was detected in 80% of the showerhead samples. Our results confirm earlier observation that Chlamydiae DNA is frequently observed in human skin swabs and suggest that CRB could be transported to human skin from our water distribution system inside of FLA.

Upregulation of specific helpful proteins represents a possible method for preventing or treating human diseases. Endogenous upregulation (knockup) is the increase of a gene's expression only in cells in which it is already expressed, thus avoiding physiologically abnormal spatiotemporal patterning. A gene's three prime untranslated region (3′UTR) affects protein expression through stability regulation of RNA already transcribed, which suggests 3′UTR modification as a viable route for endogenous upregulation. Mammalian model organisms can be generated in order to test the effects of different 3′UTR modifications, but at great cost of time, effort, and money. If able to predict in advance with an in vitro assay whether an in vivo modification would cause a desirable or undesirable change, these costs could be substantially reduced. In this thesis project, an in vitro assay was used to compare the protein expression influence of twenty neurodegeneration-relevant mouse genes' 3′UTRs to that of a flip-excision cassette (flex-cassette) previously used for in vivo conditional knockup. The assay used was the Promega Dual-Luciferase Reporter Assay, in which plasmids expressing Renilla and Firefly luciferase as reporter and internal control are co-transfected into in vitro cells, then each luciferase's expression measured with its respective substrate and a luminometer. Transfections were carried out in three-well replicates and on multiple days. The aims of the project were the evaluation of the assay's ability to predict in vivo results, the suggestion of 3′UTRs which could be upregulated in vivo by the conditional knockup flex-cassette, and the identification of any trends in 3′UTR-based protein expression influence according to gene function. A number of gene 3′UTRs were identified which were either candidates for flex-cassette upregulation or candidates for use in the flex-cassette to upregulate other genes. However, the flex-cassette's in vitro results were only partially consistent with its previous in vivo results. Specifically, the lox sites in the flex-cassette was observed to lower expression level to a degree not observed in vivo. Additionally, in the course of the project a number of possible workflow improvements were identified, for which suggestions have been made in the text. As such, this in vitro approach requires further study in order to determine suitability for prediction of in vivo 3′UTR behaviour.

There is a naturally reproducing Atlantic salmon population in the River Teno in northern Norway and Finland. The Teno population has a strong population structure and up to 28 subpopulations have been recognized. Estimation of effective population size is important in conservation of the subpopulations. Effective population size tells about genetic variation of a population and is among the most important concepts in conservation genetics. In this study, current and past effective population sizes of 28 subpopulations were estimated from high density SNP-data for 1137 individuals in total. The estimation was done with the linkage disequilibrium method and the effects of using different assumptions were studied. Current estimated effective population sizes in subpopulations were generally low and ranged from around nine to 272 individuals. Only four populations had a current effective population size bigger than 50 individuals. Past effective population sizes showed a clear declining trend from the most distant generations in all populations. The choice between physical and linkage map as well as female, male or average linkage map had an effect to estimates. Also, different sample size corrections resulted in different estimates. Furthermore, effective population size was estimated with temporal method in three populations. It was detected that the estimates from temporal and linkage disequilibrium method were different from each other. The results of this study suggest that Teno Atlantic salmon subpopulations have declined over the past 150 generations and are in risk of losing genetic variation due to current low effective population size. This should be taken into account in conservation plans.

Nemaline myopathy (NM) is a rare congenital disorder, the most common of congenital myopathies. It affects primarily the skeletal muscles and it is recognised by nemaline bodies in muscle tissue samples and muscle weakness. Mutation of eleven genes are known to lead to NM and the most frequent disease-causing variants are either recessive NEB variants or dominant ACTA1 variants. Variants in NEB are thought to be well tolerated and only 7% of them are hypothesized to be pathogenic. Over 200 pathogenic NEB-variants have been identified in Helsinki and the majority occurred in patients as a combination of two different variants. The missense variants were speculated to have a modifying effect on pathogenicity by affecting nebulin-actin or nebulin-tropomyosin interactions. Nebulin is a gigantic protein coded by NEB and is one of the largest proteins in vertebrates. It is located in the thin filament of the skeletal muscle sarcomere. Enclosed by terminal regions, nebulin has an extensive repetitive modular region that covers over 90% of the protein. The repetitive zone comprises of 26 modules called super repeats (SR). SRs consist of seven simple repeats. There are seven conserved SDXXYK actin-binding sites at each super repeat, one per simple repeat, and one conserved WLKGIGW tropomyosin-binding site. Due to its enormous size and highly repetitive sequence, nebulin is one of the least studied proteins in vivo, in vitro or in silico. In the NM patient database used for this study, there are 70 families with verified pathogenic mutations and in 30 of them, there were additional missense variants in NEB. These missense variants can be pathogenic modifying factors or have no impact on the phenotype. Seven missense variants were selected to study the effect of these mutations on actin-binding capacity compared to wild-type nebulin using the SR panel constructed previously by Laitila and Lehtonen. Also, due to the differences in actin-binding capacity of SRs compared to each other, one of the aims was to determine whether corresponding mutations in different SRs would have a similar or different effect on actin-binding capacity. For this aim, one missense mutation in the strongly actin-binding SR 1, and one in the weakly actin-binding SR 7 were selected from the NM database, and corresponding variants were created. Also, an in-frame deletion in SR7 found in the ExAC database and the corresponding mutation in SR1 were constructed for this study. The actin-binding strength was determined using actin co-sedimentation assay and actin affinity assay. The results for co-sedimentation assay indicate that missense variants can have an effect on nebulin-actin interactions and, therefore, can be a possible cause for NM. The corresponding mutations had no correlation in their effect on actin-binding strength, just the opposite. S1-m-2 decreased actin-binding strength of SR1 and S7-m-2 had no effect on SR7. Likewise, S7-m-1 and S7-del-1 decreased actin-binding strength of SR7 and corresponding mutations had no effect on SR1. The selected missense mutations found in NM patients in SRs 2 and 4 decreased actin-binding strength, if located at the actin-binding sites and in SR 10 increased the actin-binding strength, if located at the actin-binding site. The change in actin binding strength was defined as significant if the P-value was below 0.005. The more accurate affinity assay was performed as a trial only for S16 and S16-m-1, a variant at a tropomyosin-binding site close to an actin-binding site. It indicated a difference in actin-binding affinity missed by the actin co-sedimentation assay. The results are preliminary, but show big promise and should be optimized and implemented in the future missense mutation affinity studies. In an attempt to understand if the effect missense mutations have on nebulin-actin interaction is based on the change in nebulin structure, the 3D-structure of each produced fusion protein was predicted in silico. Considering that the variants were produced as GST-fusion proteins, the position and effect of GST in them is also a point of interest. In order to predict the structure of these large proteins, a combined approach was implemented using I-TASSER (Iterative Threading ASSEmbly Refinement) software. The software uses ab initio modeling, threading methods and atomic-level structure refinement to build an accurate 3D-model of a protein from sequence. According to the predicted 3D models of the fusion proteins, the GST-part of the proteins folds into a globular structure and acts as a core around which the nebulin fragments fold. The GST does not bind to actin and is positioned on the inside, which indicates minimal effect on nebulin-actin interaction, but may be a reason for an alternative nebulin fragment folding. The accuracy of the default set of programs in software does not give the definitive answer of the possible effect missense mutations can have on structural changes. However, I-TASSER approach for 3D-modeling is promising with further software optimization and can possibly serve as an effective bioinformatic tool in the future.