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(2020)In this study, the goal was to determine which nutrient, phosphorus or nitrogen, limits the phytoplankton growth at the Vanajavesi freshwater site. The aims were to detect spatial and temporal changes and find out if the wastewater treatment plant (hereafter, WWTP) located by the study site affects the nutrient concentrations and the limiting nutrient. The reliability of determining limiting nutrient by bioassays and measuring the phytoplankton response to different treatments as fluorescence was also evaluated. The study was conducted because knowledge of nutrient limitation is essential when allocating resources to reduce nutrient loading and planning other remediation practices in eutrophicated waterbodies. According to the EU Water Framework Directive, all waterbodies in the EU must be in a good ecological status by the year 2027. This goal is yet to be achieved in Vanajavesi; the ecological status of the river Vanajanreitti is poor and that of lake Vanajanselkä is moderate. The samples for bioassays were taken from five different locations. Three sampling sites were in the river and two by the lake. Based on the direction which the water flows, one of the sampling sites was before the outlet from the WWTP and the rest after it. The bioassays were carried out with the water and natural phytoplankton community taken from the study site. The experiment was conducted five times: in November, March, May, July and August. The temperature and light conditions in the incubation room were set to mimic those in Vanajavesi at each given time. Part of the preparations was to filter out the zooplankton using 50 μm plankton net. There were four different treatments: control without nutrient additions, nitrogen addition, phosphorus addition and nitrogen and phosphorus additions. Fluorescence from the 2 litre incubation bottles was measured every 1-3 days during each experiment. Chlorophyll a was determined in laboratory before and after the experiments. Nutrient concentrations were also determined before each experiment. Small seasonal and temporal changes were observed in the nutrient concentrations and the limiting nutrient. These changes were most likely due to changing seasons, effluent from the WWTP and denitrification at lake Vanajanselkä. Phosphorus limited phytoplankton growth year around at all places. At the end of the summer also nitrogen was limiting. In July co-limitation was detected in all sampling sites. In situations of co-limitation there was either no secondary limiting nutrient, or it was phosphorus. Only once, in August at the sampling point before the outlet from the WWTP, was the secondary limiting nutrient nitrogen. On average the nutrient concentrations were higher in the river than in the lake. Chlorophyll a concentrations and some nutrient concentrations were higher after the WWTP. However, no significant negative impact due to WWTP could be detected, especially at lake Vanajanselkä and the WWTP did not result in a change from phosphorus limitation to nitrogen limitation. Bioassays and the phytoplankton yield measured with a fluorometer was a reliable way of determining the limiting nutrient. Chlorophyll a concentrations verified the fluorescence results. The probe used in this study measured only the fluorescence of chlorophyll a. Even more accurate result of the phytoplankton biomass would have been obtained with a probe that measures also the fluorescence of phycocyanin, the photosynthetic pigment in cyanobacteria, because cyanobacteria has less chlorophyll a than other phytoplankton groups. As Vanajavesi is phosphorus limited or co-limited by phosphorus and nitrogen year around, reductions in phosphorus loading will likely improve the water quality. The main source of phosphorus to Vanajavesi is the nutrient loading from agricultural practises on the drainage basin. Efficient management of this diffuse loading will cause the phytoplankton biomass, especially the biomass of harmful cyanobacteria, to decrease. Nitrogen-fixing cyanobacteria is not dependent on the nitrogen concentrations in the water column, but the concentration of phosphorus. Significantly reducing the phosphorus loading is a prerequisite for the Vanajanreitti and Vanajavesi to be in a good ecological status by the year 2027.
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(2020)Hypolimnetic withdrawal and purification is a new method developed to restore eutrophic lakes, which enables the removal and capture of nutrients such as phosphorus from hypolimnetic water. The method is currently being studied in a pilot-scale project at Lake Kymijärvi, Lahti, Finland. The project employs a closed-circuit system, in which the hypolimnetic water is pumped into a filtering system onshore, where the dissolved phosphorus is first precipitated. There are two possible methods for precipitation: aeration of water, in which dissolved phosphorus is precipitated by iron hydroxides as the water aerates, and addition of calcium hydroxide (Ca(OH)2) (hereafter “chemical treatment”), in which dissolved phosphorus is precipitated as hydroxyapatite. The water then flows through a nutrient filter, which traps the precipitate. The nutrient filter consists of two parallel filters, which enables the simultaneous comparison of two different filter materials. Finally, the water is returned to the lake via a wetland. A closed hypolimnetic withdrawal and purification system like this has not been previously studied as a restoration method. The aim of this master's thesis is to study the operation of the hypolimnetic withdrawal and purification system of Lake Kymijärvi by comparing the phosphorus retention capacity with 1) two different filter materials and 2) two different precipitation methods. The compared filter materials were fine gravel (manufactured by Rudus) and calcitic waste rock (manufactured by Nordkalk). Additionally, the thesis investigates the ratio of phosphorus fractions (Ca-P and Fe-P) of the phosphorus trapped in the filters, the distribution of trapped phosphorus between different filter layers and the effect of the filter materials on the concentrations of calcium, iron, manganese and sulfur of the water flowing through the filters. The operation of the hypolimnetic withdrawal and purification system was tested during four hypolimnetic water test pumping periods in summer 2019. The material for this thesis consisted of water and filter material samples collected from the system during the test pumping periods. The water samples were used to determine the capacity of the filter materials to capture phosphorus and other elements. The filter material samples were used to study the ratio of different phosphorus fractions and the distribution of trapped phosphorus between different filter layers. Both filter materials captured phosphorus efficiently via both precipitation methods. At their highest efficiency, both materials captured over 90 % of dissolved phosphorus and over 75 % of total phosphorus. No statistically significant difference was found in the phosphorus retention capacity between the filter materials, nor the precipitation methods. Of other elements, both filter materials captured iron very efficiently during both precipitation methods, and manganese during the chemical treatment. The phosphorus trapped in the filters was not evenly distributed between different filter layers and most of the phosphorus was trapped in the bottom-most layer in both filters. The phosphorus fractions between the two precipitation methods differed less than expected. After water aeration, all the phosphorus trapped in the filters was bound to iron. Even after the chemical treatment, less than half of the trapped phosphorus was bound to calcium, with the remainder bound to iron. According to the results, it is possible to remove phosphorus efficiently from hypolimnetic water with the system, using whichever of the studied precipitation methods or the filter materials.
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Framing of the Amazon rainforest fires in the Colombian news media El Tiempo: Populism vs. science (2022)The topic of forest fires has gathered a lot of media attention in recent years as it relates closely to climate change and other sustainability issues. The media has an important role in communicating these issues as it affects, how the public percieves them, how different sustainability problems are defined and what kind of solutions are seen plausible. I became interested in how the media represents the issue of forest fires. My aim in this thesis is to find out, how the Colombian news media has framed the Amazon rainforest fires. As my material I used the news articles from the Colombian news media El Tiempo. I definined my timeframe from July 2019 to september 2019 because at that time the news subject was of high interest. After the initial search I went through the articles and left out any irrelevant ones. I was left with 24 news articles. As an analytic tool I utilized qualitative frame analysis guided by Robert Entman´s definition of frames and used ATLAS.ti to make an initial thematic coding. After that I mapped out all the actors that were cited in the news articles and divided them into groups. Using these actor groups, I searched for the frames. There were eight actor groups, politicians and political organizations being the most prominent one. Looking at how these actors talked about the forest fires, two main frames came out: populist and scientific. The populist frame concentrated mainly on Jair Bolsonaro. In this frame there was a lot of nationalist and economic arguments and responsability was directed away from him. There seemed to be a lot of enemies also. The second frame concentrated in deforestation, climate change and the forest fires being a global issue. In this frame international actors used economic and political pressure in order to affect Bolsonaro´s policy solutions. The research showed, how difficult it can be to solve shared global problem and made visible a historical political division between authoritarianism and democracy. An other important notion is the lack of marginalized groups in the news media.
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(2023)Urban areas have a central role in human’s impacts on the planet. A persistent, fundamental and systemic transformation of urban areas to be more sustainable is a widely recognized pursuit. Involving a variety of stakeholders in decision-making and discussing how, why, and to whose benefit urban areas should be changed is central for governing urban transformations. The study elaborates which features and negotiations key stakeholders relate to sustainable urban transformation. This is done through a frame analysis, and a serious game is used in data collection to facilitate discussion between participants. The results of this study show how urban sustainability and transformation can be framed in many ways that highlight different aspects. Role of private businesses, a competitive setting between cities, trust between different groups and accountability to citizens are elaborated in the negotiations on sustainable urban areas. Urban transformation is discussed especially related to low-carbon traffic, greening urban areas, preventing climate-change related flooding, adding possibilities to participate decision-making and more adaptive city planning. The study concludes that open communication between stakeholders of urban transformation is crucial to build trust and understanding between groups, but demand for openness may contradict with the interest for urban areas to appear in good light to and desirable for businesses and new residents.
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(2016)Parasitoid host interactions are intimately associated with food web dynamics as well as with evolutionary change. The parasitoid s possibility to expand its host range or switch to different hosts is affected by parasitic strategy. Strong specialization to a narrow host repertoire can arise e.g. if the larva spends considerable time in close contact with an active, developing host (koinobiosis). In contrast, an ectoparasitic larva, growing rapidly on a sessile host, does not have to be equally tightly adapted to host physiology. This may permit a wider host range. However, there are other restricting factors, such as egg production capacity. Extant parasitoids show a staggering species diversity, most notably among the Hymenoptera. Theories concerning speciation in association with host range have been developed, but draw from knowledge of merely a small part of this diversity. This study adds information by showing a number of radical host shifts in the evolutionary history of the ichneumonid wasp genus Gelis. Species of this genus exploit either insect prepupae or spider eggs. These are similar in being rather defenceless, aside from a protective silk covering. Nonetheless, differences are large enough to restrict Gelis species to each utilise only one of the two groups. In order to trace ancient host group shifts, host data was mapped onto a phylogeny constructed through bayesian analysis of sequence data. Sequences from the COI (mitochondrial) and ITS2 (nuclear, noncoding) regions were used. The results suggested at least four host group shifts within the genus. In addition, wing reduction was found to have occurred in two different lineages. Through COI barcode sequence clustering supported by morphological traits, a previously undescribed species from the G. bicolor-species complex was detected. The phylogenetic analyses also indicated the existence of further cryptic species, and the genus Thaumatogelis Schwarz was found to be nested within Gelis.
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(2022)Urban energy transitions play a key role in achieving climate targets and keeping the climate crisis from escalating. The district heating system of Helsinki has been characterised as a path-dependent and locked in system that will face difficulties in transitioning away from fossil fuels. In 2021 the city owned energy company Helen announced that it will quit coal burning in 2024, which is five years ahead of the national coal ban prohibiting coal use for energy in 2029. The coal phase out of Helsinki is a concrete example of a demanding coal phase out in a northern city with high energy demand. This thesis aims at answering the research question on how the multilevel policy mix, consisting of policy instruments on the municipal level, the national level, and the international level, contributed to the coal phase out of Helsinki. Through a case study approach relying on ten expert and stakeholder interviews as well as complementary material consisting of key strategy documents, this thesis aims to widen the understanding of the role of policy and politics in sustainability transitions and urban energy transitions. This study covers both policymaking and implementation processes as well as system impacts of the policy mix contributing to the coal phase out of Helsinki. Through empirical reviews the thesis contributes to the conceptualisation of policy mixes on multiple governance levels by studying the combined impact of policy instruments formed at the local, national, and international level. Regulatory policies on the national and international level (emissions trading, national ban on coal, taxation etc.), policies supporting low-carbon solutions on the national level (tax-exemptions) and climate target setting as well as support for low-carbon solutions on the municipal level (deregulation, innovation competitions) altogether contributed to the coal phase out. The findings of this study are in line with previous research emphasising the destabilisation of fossil fuel regimes to achieve transitions towards sustainability. Incorporating the elements of policy processes and strategies as well as policy effects and feedbacks into the concept of policy mixes is important to assess the efficacy and long-term impacts of policy mixes. The coherence of policies on multiple governance levels and the balance between regime destabilising and niche creating policies is also important in ensuring transitions towards sustainability. The results of this study support previous research findings on cities being important arenas and actors for sustainability transitions. Policies from different governance levels intersect on the urban level and decisions on infrastructure transformation are made on the municipal level.
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(2020)Leaf senescence is a developmental and physiological phase in plants to end leaf development. Environment factors such as drought stress, extreme temperature, and pathogen threat and internal factors including age and reactive oxygen species induce leaf senescence. Some phytohormones such as jasmonic acid and salicylic acid play a key function in cell death in plants. WRKY transcription factors is known as one of the largest transcription factor family in plants which regulates a variety of plants processes. WRKY75 which belong to WRKY transcription factors has shown multiple functions in plant development like regulation of Pi starvation responses and root development and flowering. In my thesis, I focused on the role of WRKY75 in senescence and stress responses. WRKY75 was identified as a positive regulator of cell death in Arabidopsis. WRKY75 can promote salicylic acid biosynthesis by promote transcript levels of SID2 and also cause hydrogen peroxide accumulation by suppressing the transcription of CAT2. Hydrogen peroxide and salicylic acid can promote WRKY75 transcription at the same time. To evaluate the function of WRKY75 transcription factor in SA signalling and cell death, three lesion mimic mutants acd5, cat2, dnd1 and their corresponding wrky75 double mutant were used. Interestingly, no different phenotypes were found between acd5, cat2, dnd1 and their corresponding wrky75 double mutants in cell death and hydrogen peroxide accumulation detection in Arabidopsis leaves. Meanwhile, marker genes transcription levels were not different in both short day and long day growth condition. However, different phenotypes were observed in botrytis infection. Based on these results, we formed a hypothesis that gene redundancy could influence genetic characterization of WRKY75. To overcome this problem, SRDX-WRKY75 chimeric repressor transgenic lines were generated. The SRDX domain act as a dominant negative regulator to suppress WRKY75 target genes. In future research, these new lines can be used to test transcript levels for putative WRKY75 target genes.
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(2019)Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
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(2023)Lynch syndrome (LS) is the most common cancer predisposition disease caused by dominantly inherited pathogenic variant (PV) of a mismatch repair (MMR) gene leading to a defective gene allele. The four major MMR genes encode MMR proteins – MSH2, MSH6, MLH1 ja PMS2 – that participate in the proofreading and repairing of the daughter strand for mismatches after every replication. The inherited PVs predispose to cancer development as only one somatic allele loss is required for biallelic loss according to the Knudson’s “two-hit” hypothesis. The biallelic loss of an MMR-gene leads to disrupted protein function altering the MMR process. When mismatches are left unrepaired, genomic instability is caused, which can eventually lead to tumorigenesis. Especially, the risk of colorectal cancer (CRC) and endometrial cancer (EC) is increased in LS. The predisposition syndrome, LS, is important to detect as early as possible to decrease the risk of cancer by prevention and surveillance. The MMR genes and their defects vary in their consequences to the repair process considerably, and thus, it is crucial to know the different characteristics and functional effects of them when estimating the level of cancer risk. Variants of uncertain significance (VUS) are especially prevalent among LS variants. More information about their impact to the disease can be acquired by in vitro and in silico methods, for instance. The main goal of the efforts for early detection and prevention is to reduce cancer morbidity and mortality. In this thesis, the pathogenicities of MSH2 and MSH6 variants were studied with DiagMMR assay, which has been developed for studying the protein function of these genes. In addition to the traditional agarose gel electrophoresis (AGE), the samples were also analyzed by a fragment analyzer, Labchip, that bases its function on capillary electrophoresis. This way the MMR detection efficiency of the methods could be compared. Samples were collected as skin biopsies from controls and LS patients with known MMR gene variants by Helsinki University Central Hospital (HUCH). InSiGHT database, that collects the different MMR-gene variants and their pathogenicity classification, was used to ensure that different kinds of variations, both pathogenic (class 5) and currently internationally unlisted variants, were analysed. The skin samples were cultured to acquire primary fibroblasts for nuclear protein extraction. The level of pathogenicity was revealed by MMR-protein activity when substrate DNA with a mismatch was added to the extract. Then, restriction enzymes were used for producing fragments of different lengths, depending on the repair action, and the MMR efficiency was visualized by both electrophoretic methods. Additionally, MAPP-MMR tool was used for studying the MSH2 mismatch variants in silico. By comparing the results from these two methods, we show that the more quantitative Labchip brings diagnostic value to DiagMMR suggesting 100% specificity (n=10) and 90,9% (n=11) sensitivity in reference to the variant information. For example, MSH6 c.3103C>T, which is listed as pathogenic in InSiGHT, was more consistent in giving a MMR deficient (dMMR) result with Labchip. Difference in the functional detection could be seen particularly with the MSH6 variants, but the differences were less notable when Labchip results were compared to the previous interpretations of the samples made based on the validated DiagMMR protocol. With the unlisted MSH6 variants, c.3139dupT was detected as dMMR by Labchip which was in unison with the previous interpretation. Another one, MSH6 c.551delA, was seen as MMR proficient (pMMR) in all the results by both the methods, and with the previous interpretation being unclear, which highlights the importance of further testing of this variant. There was also one unlisted variant (c.1805T>C) among MSH2 for which we got uniform dMMR results in two patients. The high MAPP-MMR score (25.150) for the MSH2 p.Leu602Pro amino acid change also supported the evidence gained of the pathogenic nature of this variant. As a conclusion, DiagMMR can be used reliably for MMR efficiency analysis, especially when performed together with a more quantitative analysis method.
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(2019)Tree shoot architecture research is important due to its significance in fields such as timber production, fruit and nut production and aesthetics of common areas. Also, research on genetic factors that regulate shoot and root system architecture might provide novel methods to store more carbon in forests and, hence, mitigate global warming in the future. LAZY1 is one of the major genes that affects branch and tiller angle in herbaceous and woody species such as Arabidopsis, rice and peach tree. LAZY1 has been under scrutiny over a decade but its molecular function remains unknown. However, it is known that lazy1 mutation affects polar auxin transport. Here it is studied how LAZY1 affects initial branch angle, fiber length and reaction wood development in silver birch (Betula pendula). Also, transcript levels of few shoot architecture related genes were analyzed. LAZY phylogenetic analysis provided evidence of a duplication of LAZY1 in three studied tree species (Betula pendula, Prunus persica, Populus trichocarpa), duplicated genes are here named LAZY1a and LAZY1b. Plant material employed in this study was a segregating population (50:50) of back-cross 1 of weeping birch (B. pendula ´Youngii´) which has a truncated lazy1a. Histological samples of branches were prepared by cryo-sectioning, stained with carbohydrate binding Alcian Blue and lignin binding Safranin dyes to reveal patterns of tension wood development. Due to the large size of branch sections, samples were imaged with a microscope and the images were merged together in a Photoshop application. Branch angles were measured manually with a protractor (angle) tool from stem to the middle of a branch. The data was analyzed using mixed linear models due to the nature of used plant material. We could not use clones because of major issues in in vitro propagation. Branch samples were macerated, fibers imaged and measured by ImageJ software. LAZY1a gene expression levels were analyzed by RT-qPCR method. RNA-sequence analysis indicated that the expression pattern of LAZY1a and LAZY1b is similar in B. pendula. However, one should construct a promoter-reporter line to study with better resolution if their expression is spatially analogous. Initial branch angle was significantly different in wild type compared to lazy1a mutant. For future, one could generate single and double knock out lines of lazy1a/b to study if they have cumulative effect on the branch angle, an important factor in timber quality. Tension wood formation was difficult to quantify with the employed method, due to issues in segregating G-layered tension wood from thick-walled reaction wood. A chemical analysis of cellulose content might provide a more objective method to observe tension wood in branches. RT-qPCR method indicated that LAZY1a transcript levels are higher in wild type compared to mutant. A complementation or knock down experiment would provide sound evidence that lazy1a induces the weeping phenotype. X-ray diffraction method could be employed to study the orientation of cellulose microfibril angle in branches of the wild type vs. mutant. Generation of effective tensional stress requires a cellulose microfibril angle less than 10 and this angle is affected by auxin concentration. It is possible, that this angle is larger in lazy1a due to defect in polar auxin transport.
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(2022)The analysis of gaze behaviour is nowadays commonly employed to help with the diagnosis and exclusion of differential neurological conditions as well as to help researchers better understand cognition in the early stages of life. However, its application in the developmental evaluation and follow-up of children with early-onset epilepsy has not been profoundly studied yet. Therefore, the current study aimed to investigate the association between the gaze behaviour of infants with early-onset epilepsy and their future neurodevelopmental outcome. To study the association and its predictive ability, three models were created. Sixty-three infants with epileptic seizure onset before 12 months of age participated in the study with the voluntary consent of their parents. Infants’ gaze behaviour was recorded with Tobii Pro-X3-120 at two measure points. The results showed infants’ initial ability to fixate their gaze, changes in their gaze shift probability in the first 12 months of life, and structural aetiology to be significantly associated with the infants' developmental outcome at 24 months of age. Where the structural aetiology was significantly associated with poorer developmental outcome, good initial fixation ability and improvements in the infants’ gaze shift probability during their first year of life were significantly associated with more positive outcome. These findings suggest that gaze behaviour at an early age is an essential predictor of later development in infants with early-onset epilepsy. Hence, eye-tracking could provide means to evaluate the later neurocognitive outcome of infants with early-onset epilepsy at an early age.
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(2023)Women have exhibited higher levels of math anxiety (MA) compared to men. Despite the relationship between MA and sex differences being topic of interest for over twenty years, the focal point has mostly been on the psychological causes. This review investigates the biological underpinnings of the social phobia and identifies numerous possible correlates which could inform future efforts to support students experiencing math anxiety and encourage more females to pursue STEM related careers. Articles that investigated sex differences in the context of MA were selected for the study using PRISMA guidelines. Females were found to experience higher levels of math anxiety compared to males. Age was shown to influence the prevalence of MA between sexes, with sex differences in MA being more infrequent in children and increasing towards adulthood. The findings suggest that a diverse set of biological sex differences related to to brain function, cognition and sex hormones can, in part, explain the higher prevalence of MA among females and age-related deviations. By shedding light on possible biological factors contributing to sex differences in MA, this review represents a valuable step toward a more comprehensive understanding of this complex issue.
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(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.
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(2023)Nuclear receptor subfamily 5 group A member 1 (NR5A1) is a master regulator of both steroidogenesis and gonadal development. Disruptions of NR5A1 can result in differences in sexual development (DSD). With proven interspecies differences in NR5A1 functioning and human material not being available, human stem cells are one of the most achievable, ethical, and accurate models to study the earliest developmental stages of foetal life. However, in currently existing human stem cell-derived gonadal models the expression of NR5A1 has been insufficient without artificial induction due to the lack of knowledge of its distinct biological mechanisms, endogenous ligands, and co-factors. A functional reporter cell line would enable high throughput microscope screening of differentiation protocols with expressed NR5A1. The aim of this thesis was to generate a functional monoclonal human embryonic stem cell (hESC) reporter line for the gene NR5A1 with Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) complex. Firstly, an efficient guide RNA was determined for NR5A1 by T7 assay, and a homology-directed repair (HDR) donor plasmid was designed based on it. Secondly, monoclonal hESC lines were generated with the Alt-R CRISPR-Cas9 RNP complex knock-in method and HDR donor plasmid via electroporation and single-cell sorting. Finally, monoclonal hESC reporter lines were screened with Touchdown PCR and a functionality analysis based on fluorescence and mRNA expression was performed. Two monoclonal hESC reporter lines H9-NR5A1-eGFP cl. 1 and dual-inducible H9-NR5A1-DDdCas9VP192-eGFP cl. 28 were established by using Alt-R CRISPR-Cas9 RNP complex. However, a functional validation performed on H9-NR5A1-DDdCas9VP192-eGFP cl. 28 cells showed the cell line to be non-functional upon NR5A1 upregulation regardless of the expressed eGFP mRNA detected with RT-qPCR.
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(2022)Lifespan is a key fitness trait, together with fecundity, dispersal, and growth. In addition to environmental factors shaping variation in lifespan, it is also influenced by genetic components. Based on theory, genetic variation in lifespan is expected to be reduced due to its high relevance to fitness. However, due to trade-offs between different life-history traits and the variable or unstable environmental conditions organisms face in nature, life-history traits are also expected to sustain higher genetic variation. From studies in model organisms, such as the fruit fly and the roundworm, researchers have uncovered key insights into the genetic basis of lifespan. Some genes have been shown to contribute more to lifespan than others and different species seem to share homologous genes influencing lifespan that have been conserved. Many of these genes relate to the insulin receptors and insulin signaling processes. The allelic variation and over- or under-expression of these genes have been shown to be associated with changes in lifespan. However, regardless of our accumulating knowledge of these genes in impacting lifespan under laboratory conditions, we have little understanding of the role of these genes impacting variation in lifespan under more natural conditions. In general, assessment of genes affecting variation in lifespan in natural populations is rare, even under circumstances where we know that the lifespan has a heritable component. The Glanville fritillary (Melitaea cinxia) is a butterfly that inhabits most of Europe. It is used as a model species in ecology and evolution in relation to metapopulation dynamics and spatially structured habitats. It has been studied extensively both under experimental conditions and via observational studies in the field. The Glanville fritillary butterfly works as a good model organism for assessments of genetic components of life-history variation, as vast amounts of genomic and ecological data are already available. In this thesis, I aim to shed light on the genetic background of lifespan by using the Glanville fritillary as a model organism. More specifically, I will test the association of some well-known lifespan-related candidate genes with a phenotypic variation on the butterfly’s adult lifespan based on previously obtained experimental data on individuals collected from the natural metapopulation during the larval stage.
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(2021)Skeletal dysplasias are a group of rare monogenic bone disorders affecting joints and the skeleton. An increasing number of gene defects have been associated with skeletal dysplasias, but many cases remain without a known cause or a clear diagnosis. Exome sequencing data of the family with two siblings affected with an undiagnosed type of bone dysplasia was examined in this study with the aim of determining the genetic cause behind the phenotype. The causal variant was assumed to be in a novel disease-causing gene, since a previously performed gene panel of skeletal disease-causing genes had not revealed any positive results. The search for potential rare pathogenic variants in genes linked to the skeleton was done with VarAFT filtering software. The search revealed a short list of candidate variants confirmed first with Broad Institute’s Integrative Genomics Viewer (IGV) and then with targeted Sanger sequencing. Conservation analysis on the affected amino acids, in silico functional analysis on the variants and a comprehensive literature review on all candidate genes were performed to evaluate the likelihood of them being the variant behind the phenotype. A shortlist of three genes were obtained with the analyses, with one of them seeming to be the most likely candidate. However, to assuredly identify the disease-causing variant, further testing should be performed. Functional analyses should be done to test the functions of the proteins encoded by the candidate genes and the consequences of the pathogenic variants.
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(2021)The European rabbit (Oryctolagus cuniculus) is a small mammal native to the Iberian Peninsula, but introduced by humans to all continents except Antarctica. The rabbit has been a remarkably successful invasive species due to its generalist nature and fast reproduction. Its spreading has mostly been destructive to the local nature, and humans have used fatal rabbit diseases such as rabbit haemorrhagic disease (RHD) to control harmful populations. The rabbit population in Helsinki is one of the most northern annually surviving rabbit populations in the world. It is believed to have originated from escaped pet rabbits in the late 1980s, and in the early 2000s, the rabbits spread rapidly around the Helsinki area. RHD spread unintentionally to Finland in 2016, and the disease caused a significant reduction in the Helsinki rabbit population. Rabbit population genetics has previously been studied in several countries, but never before in Finland. The aim of the thesis was to examine the genetic diversity and population structure of the Helsinki rabbit population before and after the RHD epidemic, and to compare the results to similar preceding rabbit population genetic studies. Rabbit populations have previously been found to recover from major population crashes without a notable loss in genetic diversity using DNA microsatellite markers. The recent RHD epidemic in Helsinki provided an opportunity to study, whether a rabbit population can recover from a population crash even in a harsher environment without losing genetic diversity. To conduct genetic analysis, fourteen DNA microsatellite loci were genotyped from individuals caught during two distinct time periods, in 2008-2009 (n=130) and in 2019-2020 (n=59). Population structure was observed in both temporal rabbit populations with small but significant FST values. The 2019-2020 population was more diverse than the 2008-2009 population in terms of allele numbers and expected heterozygosity. This result was unexpected considering the recent RHD-epidemic but could be explained by gene flow from new escaped rabbits. Compared to other wild rabbit populations around the world, the Helsinki area rabbits exhibit significantly lower genetic diversity. Bottleneck tests showed a significant signal separately in both temporal populations, but the RHD bottleneck cannot be distinguished based on the tests. The results could be biased by new gene flow, or the initial bottleneck caused by the founder effect of only a few pet rabbits. The rabbits have demonstrated their adaptation and survival skills in the cold climate of Helsinki. The population has significantly lower genetic diversity compared to other wild populations, yet recovered from a major RHD epidemic without reduction in genetic diversity under these more extreme environmental conditions. It has been proven again; the rabbit is a thriving invasive species.
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(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.
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(2023)Progressive retinal atrophy or PRA is a collective term for a group of hereditary degenerative retinal diseases in dogs. PRA affects the photoreceptor cells of the eye ultimately progressing into complete vision loss. Documented in over 100 breeds, it is the most common type of canine retinal diseases. PRA is considered a homologous disease to human retinitis pigmentosa, thus providing a large animal model for studying retinal biology and genetic aetiology of its diseases. The objective of this thesis was to study the genetic cause of a novel form of PRA in young Finnish Lapphunds. Analysis built upon a combination of gene mapping methods and analysis of next generation sequencing data. Gene mapping was performed with two analysis methods, genome-wide association study and homozygosity mapping, utilising single nucleotide polymorphism microarray based genotype data. Identifying a clinical phenotype from the canine biobank at the University of Helsinki resulted in a study cohort of six case and 10 control dogs. Combined with pedigree information, this early-onset PRA was most likely a new autosomal recessive condition in the breed. Genome-wide analyses resulted in the discovery of a disease-associated locus on chromosome 27. Findings of single nucleotide variant filtering of one whole-genome sequenced affected dog led to the prioritisation of an intronic substitution variant (T > C) in SOX5 gene as a potential cause of PRA. Genetic validation of the variant with 23 dogs showed promising results. Four out of five affected dogs were homozygous for the variant, while controls were either wild-type or heterozygotes. As a result, a previously unknown disease locus was successfully identified, suggesting a possible new spontaneous canine model of retinitis pigmentosa. By better understanding the pathophysiological processes of disease, improved diagnostics and marker-based testing as well as novel therapies can be developed for both dog and man. However, further studies are needed to understand the underlying molecular mechanism of the candidate disease variant.
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