Browsing by study line "none"
Now showing items 1-20 of 34
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(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.
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(2018)The arylhydrocarbon receptor (AHR) is known for its xenobiotic role. In the last decades we have realized it has an important role even in normal physiology. Earlier studies have shown different circadian behavior in mice and rats when AHR is activated with the environmental toxoid TCDD. Also, AHR knock-out (AHRKO) mice have shown to adapt quicker to new lighting conditions. The aim of this study was to chart AHRs role on the circadian behavior in rats, by comparing daily eating and drinking habits under normal lighting condition for 7 days and for 7 days after a 12-hour light shift. Tissue samples to be used in continuing studies were taken after the 14 days long follow up. These studies will chart how the circadian timekeeping genes are expressed in the central (suprachiasmatic nucleus) and periphery (liver) cells in AHRKO rats after an adaptation to phase shift compared to wild type rats. This way the study will provide information that will help us understand the role of AHR in different species regarding behavior and in continuing studies gene expression. In our study no differences in drinking and eating activity could be seen between AHRKO and wild type rats. Both groups adapted to new lighting conditions equally fast.
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(2018)This master’s thesis addresses the role of intermediary organizations in sustainability transitions, specifically in the field of energy. The thesis discusses how intermediary organizations can diffuse and support the development of novel sustainable socio-technical niche-innovations through experiments. Theoretically, this thesis draws mostly upon the sustainability transition literature, particularly strategic niche management theory. Empirically, this thesis focuses on a case study of joint procurement of solar power plants that was implemented as a part of “New and innovative low-carbon business generates competitive advantage for companies and municipalities” (Välke) project in South-western Päijänne during 2016. Välke is a sub-project of Carbon-neutral municipalities (HINKU) and therefore the case study of this thesis links to other similar experiments in the HINKU network. The material of the thesis was collected through 9 semi-structured interviews with different stakeholders involved in the experiment and by using pre-existing secondary material. The material was analysed by qualitative content analysis using an analytical framework adopted from a previous study. The findings show that intermediaries support niche development by aggregating, circulating and applying lessons learned between and within joint procurement experiments. This was done by producing and disseminating documents, but more importantly through personal contact between intermediaries. The network of intermediaries showed hierarchical features as the coordinator of HINKU, the Finnish Environment Institute (SYKE), maintained the repositories of the learned lessons and also formed a link between municipal intermediaries involved with the experiments. Also, during the experiments the more experienced higher profile intermediaries – mostly SYKE – provided assistance to lower profile municipal intermediaries. This support included providing knowledge, but also raising the confidence of lower profile intermediaries. Following, the lower profile intermediaries provided similar assistance to the participants of the experiments. However, the intermediary roles were not stable as lower level intermediaries were adopting some of the roles of the higher profile intermediaries after gaining experience during the experimentation. In order to support the Finnish solar niche, the intermediaries went beyond mere aggregating, circulating and applying lessons between and within experiments. They were actively initiating new experiments in adjusted formsin new geographical locations and advocating the niche. This was partially linked to the strategic nature of the intermediaries, as they were established to catalyse activities that would lead to mitigation of greenhouse gas emissions. However, this active support for a particular socio-technical innovation contradicted the pursuit of intermediaries to be viewed as neutral and credible actors. Partially as a consequence – but also because of the phase of solar niche development in Finland and lack of resource and interest – SYKE chose to withdraw from future joint procurements of solar power plants for private actors. However, SYKE was planning to utilize the governance innovation of joint procurements in order to support other niche-innovations with sustainability gains. The findings show that intermediaries can accelerate energy transitions, at least on a regional scale. They emphasize the importance of cooperation and personal contact between intermediaries and the ability of intermediaries to utilize governance innovations. Also, the findings support stronger inclusion of intermediaries in governance frameworks to hasten energy transition and achieve wider sustainability goals. However, the thesis shows that particularly public intermediary organizations have to work under unclear mandates.
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(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.
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(2018)Staphylococcus aureus is a commensal bacterium in humans and approximately 30% of healthy people carry it as part of their microbiome, in the nasal cavity and skin, without any harm. However, it is an opportunistic pathogen that causes severe infections in immunocompromised and hospitalized patients. Typical infections caused by S. aureus are wound and skin infections, pneumonia and urinary tract infections in people with a medical implanted device such as for example a catheter. S. aureus has gained resistance to virtually all antibiotics over the years of excessive antibiotic consumption, making treatment nearly impossible in some cases. MRSA, methicillin resistant S. aureus, is a worldwide problem in hospitals and the mortality rate is still rising. One of the most common MRSA lineages is USA300, a community-acquired MRSA, which is notorious not only for its antibiotic resistance but also for its ability to form prolific biofilms. Biofilm production combined with antibiotic resistance complicates treatment of S. aureus even further. A detailed understanding the molecular mechanisms of biofilm formation might bring us closer to a cure for infections caused by MRSA biofilms. The study comprised two parts. First, characterize the phenotype of the mutants under static and dynamic conditions, test the minimal inhibitory concentrations (MIC’s) for antibiotics and verify the gene knockout by real-time RT-PCR. Second, study gene function by transduction to the parental strain USA300-UAS391 EryS and a MRSA strain TCH1516 EryS to study the gene function in a different bacterial background. The methods used were cell culturing for static and dynamic biofilm as well as growth curve, fluorescence microscopy, antibiotic susceptibility testing and real-time RT-PCR. In total seven strains were selected for characterization. The chosen seven knockouts were ΔHAD (HAD-superfamily hydrolase, subfamily IA, variant 1), non-coding region, ΔausA (non-ribosomal peptide synthetase), ΔoppA (Oligopeptide ABC transporter substrate-binding protein), ΔclfB (clumping factor B), ΔampA (cytosol aminopeptidase), and ΔpgsA (CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase). General characterization showed a few changes in biofilm formation for the genes ΔoppA, ΔausA, ΔHAD and ΔpgsA. Especially ΔpgsA is interesting because of increased ciprofloxacin resistance. The real-time RT-PCR showed some altered gene expression patterns, but no connection to poor biofilm formation. With fluorescence microscopy the growth patterns of USA300 transposon mutant strain biofilms could be described. To verify the results of the characterization, further experimentation is needed, such as RNA sequencing and complementation. Also expanding the studies to other gene hits of the screening is recommended.
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(2019)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.
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(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.
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(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.
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(2019)This thesis examines the strengths and weaknesses of transport sector planning documents in reaching post-carbon future in Helsinki. The three last governmentally produced planning documents, regarding the future of transport in Helsinki, are being analyzed with the use of a framework created by Wiseman etc. (2013), pathways by Rosenbloom (2017) and semiotics. As the planning of the city’s transport is continuous by na-ture, knowledge of how the previous documents would succeed in reaching a post- carbon future is essential in order to form the new set of actions needed, even thou different set of aims were present at the time the documents were created. Helsinki aims to be carbon neutral by 2035. The goal was first published in 2017. Previously there have been less ambitious carbon reduction targets regarding Helsinki and its transport sector. The role of traffic is essential, as in 2017 the traffic was accountable for 23 % of the overall carbon emissions in Helsinki. This thesis uses a framework created by Wiseman etc. (2013) initially to analyze large- scale transfor-mations regarding the society as a whole. As the traffic sector is facing similar challenges, the path to post-carbon future is possible through the same identified set of means. As with Helsinki’s transport planning documents it is possible to reduce emissions by using a slow evolu-tionary approach, according to Wiseman etc. (2013) the approach is insufficient in making a leap to a post-carbon future in a short timeframe. The planning needs to include actions that make more rapid and trans-formational changes to the society as a whole. This thesis argues that the frames that need to be present in order to successfully reach a post- carbon future are already present in the transport planning documents, but they only need strengthening and precision. According to Wiseman etc. (2013) the success in reaching the target dependents on the social and physical transformations happening at the same speed and magni-tude. This is something to pay a special attention to in the future planning of reaching a carbon free traffic.
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(2018)Plants control the exchange of gases through the stomatal pores. Stomata are formed by guard cells and the closure of stomata are regulated via a complex signaling network in response to various biotic and abiotic stimuli, such as pathogens, elevated levels of CO2 and darkness. The leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) is part of the network regulating stomatal closure. GHR1 is an inaktive pseudokinase that can activate SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), an anion channel that is crucial for stomatal closure, via interacting proteins. The exact role of GHR1 is still partly unknown, however, it has been suggested that GHR1 could function as a scaffold or as an allosteric regulator of additional components required for stomatal closure. The aim of this study was to identify novel interactors of GHR1. First stable plant lines expressing fusion proteins GHR1-YFP, GHR1W799*-YFP and plain YFP as a negative control were generated and from these lines fusion protein expression levels and the subcellular localization were studied. Next the plant lines were used for purifying GHR1 interacting proteins with the use of co-immunoprecipitation and identification of the proteins with mass spectrometry. The unlikely GHR1 interactor candidates were then filtered from the mass spectrometry data. The subcellular localization and the protein expression of the interacting proteins were studied with the use of internet databases. Literature of the GHR1 interacting proteins were studied in order to make possible connections with GHR1 and stomatal closure. In this study 38 GHR1 interactors were identified. Literature search revealed that many of the identified interactors had a known role in stomatal movements. These included proteins such as PLASMA MEMBRANE INTRINSIC PROTEIN2-1 (PIP2-1) and BETA CARBONIC ANHYDRASE 4 (BCA4), that are known to have a role in stomatal closure. Future work includes confirming the interactions with independent methods and studying the molecular mechanisms related to stomatal movements. The GHR1 interactome identified here for the first time reveals novel parts of the network regulating stomatal movements and thus increases our understanding of molecular mechanisms behind stomatal functions.
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(2013)Intellectual disability (ID) is a clinically diverse and genetically heterogeneous disorder characterized by central nervous system defects of varying severity resulting in substantial impairment of intellectual and adaptive functioning as expressed in conceptual (IQ<70), social and practical adaptive skills diagnosed before 18 years of age. The condition is referred to as non-syndromic when ID is the only clinical feature and syndromic when ID is accompanied by specific other features, for example, Down syndrome. Intellectual disability is one of the largest unsolved problems of health care with a prevalence of 2-3% in the population. There is a 30-40% excess of male versus female patients in ID which refers to over-representation of X chromosomal defects causing ID. In this study, exome sequencing of the X chromosome was applied in order to identify genes and their mutations in two Finnish families with intellectual disability of unknown cause. The mutations were identified using Agilent Sure select array that covers almost 93% of the coding region of the chromosome. Exome sequencing resulted in 11 variations in total. Segregation of these variants was studied using PCR, ExoSAP-IT purification protocol and BigDye® Terminator v3.1 Cycle Sequencing Kit. Eventually, two novel mutations were identified: one for each family. Both mutations reside in genes that have previously been shown to cause X-linked intellectual disability. Both of the mutations were absent in over 120 control DNA samples. In one family with three affected males, a novel splice mutation was identified in discs large homolog 3 (DLG3), which encodes synapse-associated protein 102 (SAP102). The mutation is located at the splice site in intron 1 (500+1 G>C) and its effect on protein function needs to be analyzed at the RNA-level using cDNA-sequencing. The clinical phenotype of the three affected brothers is mild to moderate intellectual disability. In the other family with three severely affected male patients, a novel mutation in exon 12 was identified on glutamate receptor, ionotropic, AMPA 3 (GRIA3) resulting in amino acid glycine (GGG) changing to arginine (CGG) at codon 630 (G630R). GRIA3 belongs to AMPA receptors implicated in the regulation of several biological processes. Our findings elucidate the power of exome sequencing in the diagnosis of rare, genetically heterogeneous disorders like intellectual disability. The results obtained will help in assessing the prognosis of the disease, in estimating the risk of the disorder to other family members, and in facilitating the development of future therapies for these devastating disorders. The results also further confirm the role of DLG3 and GRIA3 in human cognitive development.
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(2018)Limasienet (Myxomycota) ovat aitoameeboihin (Amoebozoa) kuuluvia, aitotumaisia eliöitä, joiden elinkierrossa vuorottelevat yksisoluinen, yleensä haploidi ameeba- ja kenosyyttinen, diploidi limakkovaihe. Limasieniä tunnetaan noin 1000 lajia, joista Suomessa on tavattu reilut 200. Lajimonimuotoisuus on suurinta metsissä mm. karikkeessa, lahopuussa ja elävien puiden kaarnalla. Suurimman osan elämästään ne viettävät joko lepovaiheina (esim. itiöinä tai sklerootioina) tai ameeboina. Limakko kehittyy tavallisesti ameebojen pariutuessa, ja se kykenee liikkumaan hitaasti elinalustallaan etsien ravinnokseen mm. bakteereja ja sieni-itiöitä. Sopivissa olosuhteissa limakko muuttuu kokonaisuudessaan itiöpesäkkeiksi, joissa syntyvät meioottiset itiöt. Kaarnalimasienet ovat sopeutuneet viemään koko elinkiertonsa läpi elävien puiden kaarnalla. Niiden lepoasteet kestävät kasvualustalleen tyypillisiä ääreviä ja nopeasti vaihtuvia kosteus- ja lämpöoloja. Elinkierto ja limakkovaihe ovat yleensä nopeita ja limakot sekä itiöpesäkkeet pienikokoisia, tuskin silmin havaittavia. Ehdottomien kaarnalajien lisäksi kaarnalla elää myös joukko laaja-alaisempia ja opportunistisia lajeja. Tässä tutkimuksessa kartoitettiin kaarnalimasienten lajistoa 15 yleisellä, Suomessa luonnonvaraisena esiintyvällä puulajilla ja neljällä eri kasvillisuusvyöhykkeellä (boreaalisen vyöhykkeen alavyöhykkeellä). Tavoitteena oli selvittää lajiston lisäksi, onko eri isäntäpuulajien tai vyöhykkeiden välillä eroja limasienilajistossa. Kaarnanäytteitä kerättiin yhteensä 196 puuyksilöstä (yksi näyte/yksilö) hemi-, etelä- ja pohjoisboreaaliselta sekä orohemiarktiselta vyöhykkeeltä. Näytteistä kasvatettiin limasieniä kosteakammioviljelmissä, ja itiöpesäkkeitä tuottaneet lajit tunnistettiin ja valokuvattiin. Puulajien ja vyöhykkeiden eroja tutkittiin tilastollisin menetelmin. Lisäksi mitattiin ja tutkittiin kaarnan happamuuden ja vedenpidätyskyvyn vaikutusta limasienilajistoon. Kaarnanäytteistä 65 % tuotti limasienten itiöpesäkkeitä. Tutkituilta 15 puulajilta löytyi yhteensä 23 tunnistettua limasienilajia, joista kaksi hapsista (suku Echinostelium) havaittiin nyt Suomessa ensi kertaa. Yhden puulajin limasienilajien lukumäärä vaihteli välillä 1-9 ja vyöhykkeittäin 1-19. Sekä puulajien että vyöhykkeiden välillä havaittiin merkitseviä eroja, eli ainakin osa limasienilajeista vaikuttaa suosivan tiettyjä isäntäpuulajeja tai lajiryhmiä (esim. havu- tai lehtipuita) ja painottuvan levinneisyydeltään maan etelä- tai pohjoisosiin. Toisaalta muutamaa limasienilajia esiintyi hyvin laajalti eri puilla ja alueilla. Kasvualustan happamuuden ja vedenpidätyskyvyn vaikutukset lajistoon eivät olleet yksiselitteisiä. Tutkimustulosten perusteella limasienet ovat sienten ja jäkälien tavoin hyvin yleinen osa kaarnan mikrobistoa, eikä niiden esiintyminen eri puulajeilla tai kasvillisuusvyöhykkeillä ole täysin satunnaista. Tulokset tukivat useiden aiempien tutkimusten tuloksia kaarnalimasienten puulaji- ja aluespesifisyydestä. Edellä kuvattu tutkimus oli puulajien osalta laajin tähän mennessä tehty selvitys Suomen kaarnalimasienistä.
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(2018)Recently, our group reported that mutations in KCNQ1, a potassium channel gene usually linked to long QT syndrome, cause growth hormone deficiency and maternally inherited gingival fibromatosis. Expression of the mutated KCNQ1 with KCNE2 subunit was shown to reduce pituitary hormone secretion in functional experiments in the original study. The aim of this thesis was to investigate if germline mutations in KCNQ1 and KCNE2, a gene encoding an auxiliary potassium channel subunit, could also play a role in the opposite phenomenon, growth hormone excess. Growth hormone (GH) excess causes acromegaly, a condition that is typically due to a GH secreting pituitary adenoma. I screened KCNQ1 and KCNE2 for germline mutations in 45 acromegaly patients by Sanger sequencing and predicted effects of the mutations on protein function by in silico tools. Only deep intronic and synonymous polymorphisms were detected in KCNQ1. These findings were likely insignificant based on the in silico predictions and the variants’ frequencies in the general population. In KCNE2, a heterozygous c.22A>G, p.(Thr8Ala) mutation with an unknown significance was found in two patients. It was present in the general population with a frequency of 0.0038. In conclusion, no evidence of KCNQ1 or KCNE2 mutations being associated with growth hormone excess was found. Mutation screenings of larger patient series and additional functional experiments are needed to shed more light on the roles of KCNQ1 related genes in growth hormone secretion.
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(2018)There are about 30 000 dogs in the city of Helsinki, the capital city of Finland. Dog excrements are destined eather on the communal waste system or in the city environment: in the streets, parks, beaches or forests. Dog droppings cause both a visible and smelly problem especially in the springtime when the snow melts. Nutrients and microbiological harmful substances run to the runoff waters, and the growing amount of dog excrements is engaging the communal waste management by collecting the manure and handling it. In this study the dog droppings were studied in four parks in the city of Helsinki. The droppings that were collected in the trash bins by the dog owners and the droppings that were left in the ground near the bins were weighted daily for twenty days in November- December 2017. The study was a quantitative field study, in which the weight of the droppings were weighted and the material of the excrement bag was registered. The study was divided in two parts. In the first part the amount of droppings that were left uncollected in to the intended bins was measured. In the other part the effect of dropping bag was estimated from the sewage handling and waste disposal point of view. The theoretical frame of reference was the analysis of environmental impacts of dogs, which was considered by using the amount of dogs in Helsinki and the results of the measurements, and reflecting them in the international scientific studies. The results were also compared with a survey ”Koirat kaupungissa” that was published by the city of Helsinki in 2017. The results of the measurements show that the dog population of Helsinki put out approximately 1 358 tons of manure per year, and 148 tons of that amount end up in the streets and the park environment despite of the sanitation law. Slight differences between parks were discovered: in relation the largest amount of excrements were left uncollected in the Runar Schildt park, in the suburban area in the northwest of the city of Helsinki. The dog excrement bag materials variated in the trash bins. The waste management could theoretically benefit of standardization of the bags, and it could be beneficial to get all the manure collected in bags that compost easily and would be composted. The nutrients of the excrements could be then collected and taken advantage of. This was anyhow found difficult, because even the best of the bag materials do not compost properly, and are being burned as mixed garbage with the dog excrements. The effects of dog excrements in the urban areas are also considered in this study.
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(2018)Kokonaisvaltainen maatalousympäristön vesienhallinta pitää sisällään perus- ja paikallis-kuivatuksen sekä valumavesienhallinnan toimenpidekokonaisuuden huomioiden luonnon monimuotoisuuden ja kalatalouden tarpeet. Tämän tutkielman tavoitteena on ollut kirjallisuuden sekä käytännön toimijoiden raporttien avulla laatia toimintatapamalli maatalousympäristön tuotantotalouden kehittämiseen sekä vesistökuormituksen vähentämiseen. Kokonaisvaltaisella vesienhallinnalla tähdätään tulvien ja märkyysongelmien poistoon sekä maanrakenteen ja kasvukunnon parantamiseen ja tätä kautta kiintoaine- ja ravinnekuormituksen vähenemiseen. Kokonaisvaltainen toimintatapa tavoittelee yhdenaikaista tuottavuuden kasvua sekä vesien- ja luonnonhoidon turvaamista sekä tuotantotalouden ja ympäristönhoidon yhteensovittamista. Peruskuivatuksia koskevan kunnostustarpeen on todettu olevan huomattava ja vesienhallinnan rakenteiden korjausvelan olevan suuri. Maatalouden toimintaympäristössä tapahtuneiden muutosten myötä toimenpiteiden toteuttaminen on hankaloitunut ja monimutkaistunut. Toimenpiteiden järjestelmälliseen ja laajamittaiseen toteuttamiseen tarvitaan valuma-aluekohtainen toimintatapamalli. Valuma-aluekohtainen toimintatapamalli kokoaa yhteen ne toimenpiteet, joilla maatalous-alueiden tuotantotaloutta sekä vesien- ja ympäristönhoitoa voidaan valtakunnallisesti edistää ja toteuttaa yhdenaikaisesti. Tarvitaan laajamittainen riskialuekartoitus ongelma-alueiden selvittämiseksi ja tiedotuksen kohdentamiseksi. Ojitusyh-teisöt tulisi palauttaa ojitusisännöitsijöiden kanssa vastaamaan ojien huollon- ja kunnossapidon järjestämisestä yhdessä suunnittelijoiden kanssa. Vesienhallinta tulisi myös saada osaksi tilakohtaista viljelysuunnittelua. Valuma-aluekohtaisen toimintatapamallin mukainen järjestelmällinen toimintaketjun sisältävä vaiheittainen toteutus valuma-alueelta vesistöön takaa todennäköisesti laadukkaimman lopputuloksen kuivatustilan parantamiseksi ja vesistökuormituksen vähentämiseksi. Korjausvelan ollessa suuri tulisi pyrkiä pois toimenpiteiden epäsuunnitelmallisesta ja epäsystemaattisesta toteuttamisesta. Laajamittaisessa kansallisessa toteuttamisessa ei ole oikein toteutettuna kyse pelkästään vesienhallinnasta ja ympäristönhoidosta vaan myös maanarvon ja bruttokansantuotteen kasvamisesta sekä infrastruktuurin kunnossapidosta.
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(2019)Tiivistelmä – Referat – Abstract Variable electricity production poses challenges for the electricity grid, where demand and production must be balanced at all times. Transition to variable electricity production requires new solutions for grid flexibility. Electricity consumption has traditionally been an inflexible component in the electricity system but technological development enables demand side flexibility. Demand response (DR) is demand side measure, where energy consumption is shifted due to an external incentive. DR has multiple benefits such as improving reliability with high integration of variable energy production and cutting emissions during peak production. Despite years of modelling and analysing DR, there is lack of experience with commercial end-users in real-life context. In recent years, transmission system operator Fingrid has conducted experiments with stakeholders to find new demand response resources outside the traditional industrial end-users. The market models and services have not yet matured and therefore actors experiment to find solutions to resolve demand response barriers. The difficulty of scaling up sustainable innovations is a well-known challenge in energy transition research. In strategic niche management theory experiments are seen as tools for sustainable transition. This qualitative case study examines how piloting demand response in grocery store promotes energy transition. I chose the case of Virtual Service Environment (VIRPA-B) experiment, where participants tested DR in two grocery stores. The data were gathered in eight interviews with stakeholders and experts and through literature review. With theoretical framework I analysed, how the experiment contributes to implementation of demand response through expectations, learning and the ways pilot was scaled up after the experiment. Thesis sheds light to stakeholders’ role in implementing new technology and business model in real-life context. The results indicate that DR does not disturb the functions of the grocery store. The technology is matured, but the instalment practises have not been standardized. The greatest barrier for upscaling seems to be the regulations of the electricity markets, as they do not encourage end-users to invest in DR. VIRPA-B experiment did not lead to a rapid upscaling. However, lessons scaled up through other projects that support the niche development. For actors experiments are a platform to develop expertise and influence the new business models. To overcome the barriers, more attention should be directed at the synergies between the technologies. In VIRPA-B pilot actors noted benefits with solar panels, energy efficiency and DR. Combining technologies can lead to significant electricity savings. Promoting DR as a part of intelligent building automation system could also help overcome DR barriers. The results of thesis indicate that experiments can produce capabilities that promote energy transition.
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(2018)Laxpopulationer både globalt och i Finland är hotade som följd av överfiske och förstörda livsmiljöer. För att bevara och återuppliva naturliga laxpopulationer har man länge planterat ut laxar odlade i fiskodlingsanstalter. Dock är det endast en liten andel av de utplanterade laxarna som överlever till fortplantningsålder vilket gör utplanteringen både ekonomiskt och etiskt ohållbar. Orsaken till att så få av de utplanterade fiskarna överlever i naturen anses vara att de är dåligt anpassade till de naturliga förhållandena, eftersom miljöförhållandena i odlingsbassänger och naturen skiljer sig starkt från varandra. Målet med denna studie var att ta reda på ifall berikade odlingsbassänger med mer varierande och naturenliga förhållanden, vore ett alternativ att förbättra de utplanterade fiskarnas duglighet genom bättre anpassade morfologiska egenskaper. Jag undersökte morfologiska skillnader mellan laxar med olika härstamning; fiskar med vilt ursprung och odlat ursprung, vilka härstammade från fiskar som levt i naturen ända fram till fortplantningsålder och fiskar som under flera generationer levt i odlingsförhållanden. Fiskarna växte upp i olika miljöer, vanliga bara odlingsbassänger och berikade bassänger, och släpptes därefter ut i konstgjorda strömmar för att efterlikna en utplantering. Jag förväntade mig att fiskar som växt upp i berikade bassänger skulle ha lättare att anpassa sig till sin miljö, eftersom en varierande uppväxtmiljö anses bidra till fiskens förmåga att anpassa sig till miljöförändringar, och även att fiskarna med vilt ursprung skulle anpassa sig bättre, eftersom odlade fiskar anses mista genetisk diversitet till följd av bl.a. domesticering. Jag undersökte ifall det fanns morfologiska skillnader mellan fiskarna före de släpptes ut i de konstgjorda strömmarna då de växt upp i olika miljöer, och efter att de levt i samma miljöförhållanden i de konstgjorda strömmarna samt hurdana skillnader som uppkommit, genom att analysera fotografier. Fiskar med odlat ursprung uppvisade större morfologisk variation mellan de olika uppväxtförhållandena än fiskarna med vilt ursprung innan utplanteringen, medan fiskar med vilt ursprung uppvisade större morfologiska skillnader efter att de levt i de konstgjorda strömmarna. Både före och efter den efterliknade utplanteringen hade fiskar som växt upp i berikade bassänger en högre kropp. Fiskar med odlat ursprung hade både högre huvud och bakkropp än fiskarna med vilt ursprung efter utplanteringen, vilket indikerar en jämntjock kroppsform. Jag undersökte även fiskarnas specifika vikttillväxthastighet. Fiskarna från berikade uppväxtförhållanden, speciellt de med vilt ursprung, ökade i vikt, medan fiskar med vilt ursprung som växt upp i standard förhållanden minskade i vikt under den tid de levde i de konstgjorda strömmarna. Fiskarnas morfologiska variation påverkades mest av uppväxtförhållandena. Fiskar som växt upp i berikade bassänger hade störst tillväxt vilket tyder på att de kunde anpassa sig bättre till förhållandena i de konstgjorda strömmarna och var bättre på att hitta föda än fiskar från standard bassänger. Fiskar som växt upp i berikade bassänger hade även högre kropp, vilket indikerar att de anpassat sig till strömmande vatten. Även fiskarnas ursprung påverkade deras morfologiska egenskaper. Fiskar med vilt ursprung var mer spolformade och strömlinjeformade, vilket anses vara fördelaktigt vid simning, än fiskar med odlat ursprung som hade en mer jämntjock kroppsform. De morfologiska skillnaderna mellan fiskar med olika ursprung, kunde tyda på skillnader i fiskarnas fenotypiska plasticitet, d.v.s. förmågan hos en genotyp att anpassa sin fenotyp som gensvar på miljön. Resultaten tyder på att berikade uppväxtbassänger påverkar laxarnas morfologiska egenskaper så att de lättare kan anpassa sig till ett liv i naturen, och kunde således vara ett alternativ för att producera mer dugliga fiskar för utplantering i framtiden
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(2018)nxiety disorders are the most common psychiatric disorders. Iiris Hovatta’s group (Neurogenomics lab) uses inbred mice to study effects of psychosocial stress. Mice can be divided to susceptible and resilient phenotypes, based on their behavioral response to psychosocial stress. Differences between phenotypes can be examined for example on brain structural or transcriptional levels. These studies have shown changes in brain myelination and expression levels of myelin-related genes between phenotypes. Transcriptomic studies have also revealed differences in microRNA (miRNA) expression profiles. Especially miR-219a, a known regulator of myelination, appears to be differentially expressed. Bioinformatic studies also revealed that many predicted target genes of miR-219a are connected to Wnt/β-catenin signaling pathway, which is also known to regulate myelination. This led to a hypothesis that miR-219a regulates myelination through Wnt/β-catenin signaling pathway in adult mouse brain. Prediction of miRNA target genes is based on algorithms using varying criteria, and their biological significance is uncertain. Assumptions about the biological role of specific miRNA require experimental validation of the silencing interaction between the miRNA and the target gene mRNA. The purpose of this study was to validate five Wnt-signaling related mouse genes (Gsk3b, Esr1, Tcf7l2, Fkbp5 and Acvr1b) as targets for miR-219a-5p or miR-219a-1-3p. For validation a luciferase-reporter assay was used, in which the target sequence of the gene’s mRNA is cloned to a reporter vector, then cotransfected to cultured cells (human HEK293 cells) with a miRNA-mimic. The light emitting luciferase activity is measured to examine the possible silencing effect of the miRNA. Results of this study showed that miR-219a mimics did silence the expression of the target genes. Although some variation was observed in the efficiency of silencing between the genes and based on the mimic concentration. The most efficient silencing was observed between miR-219a-5p and Gsk3b construct. These results implicate that miR-219a regulates several genes related to Wnt/β-catenin pathway, and that stress-related differences in its expression may be associated to differential myelination observed between phenotypes that react differentially to psychosocial stress.
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(2018)PURPOSE AND GOALS Microalgae are unicellular eukaryotic organisms capable of photosynthesis. They harvest sunlight and efficiently take up carbon dioxide and nutrients such as nitrogen and phosphorus from their environment and use them for their growth. Due to these properties, their rapid growth and ability to survive in a variety of environments, microalgae have potential in biotechnological applications that promote nutrient recovery and recycling, water purification and the carbon neutral production of biochemicals and possibly biofuels. The purpose of this study was to investigate the suitability of a side stream water originating from the production of baker’s yeast (yeastwater) for the cultivation of a species of microalga called Euglena gracilis. The study aimed to determine the capacity of this water to support growth and protein production of E. gracilis as well as the capacity of E. gracilis to remove nutrients from the water. The effect of filtration of the water on these parameters was also studied. Yeastwater contains an organic molecule called betaine in relatively high concentrations. Betaine has previously been shown to boost the production of the important vitamin cobalamin in bacteria. The study aimed to determine the effect of betaine on the growth of E. gracilis and on the production of cobalamin in the algal-bacterial symbiosis. METHODS E. gracilis was cultured in laboratory scale photobioreactors. Its growth, protein production and nutrient uptake capacity was determined. Baker’s yeast production side stream water diluted with MQ-water was used as the growth medium either in filtered or unfiltered form. A control treatment was prepared where no microalgal inoculate was added to the photobioreactor. The same microalga was also grown in a synthetic nutrient medium with and without betaine. The uptake of betaine and biomass concentrations of cobalamin were determined. For the determination of microalgal growth, dry weight determination and flow cytometry analysis were used. Protein production was determined on the basis of total nitrogen concentration in the biomass. Spectrophotometric measuring kits were used for the determination of nutrient concentrations. Liquid chromatography techniques were used for the determination of betaine and cobalamin concentrations. RESULTS Significant microalgal growth was observed in filtered yeastwater, while growth in unfiltered yeastwater was very low. Nitrogen removal was higher in presence of E. gracilis compared to the control treatment. Protein production in yeastwater was comparable to that of microalgae grown in synthetic medium. E. gracilis grew much better in the synthetic media supplemented with betaine than without the addition. Betaine enrichment had no effect on cobalamin production. Cobalamin was produced in unfiltered yeastwater both with and without the presence of E. gracilis. CONCLUSIONS Unfiltered yeastwater does not support growth of E. gracilis possibly due to its high turbidity. Filtered yeastwater, on the other hand can support the production of E. gracilis biomass. E. gracilis can be used to reduce nitrogen concentrations in yeastwater. Yeastwater can support cobalamin production by bacteria, but this phenomenon did not benefit from the presence of the microalga. The effect of betaine on microalgal growth warrants further study to determine whether it is related to the accumulation of intracellular nutrients, storage compounds or to some other phenomenon. Yeastwater is a promising nutrient feedstock for microalgal biomass production. However, the role of filtration and possibility of using other methods for turbidity reduction needs to be further studied.
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