Browsing by department "Department of Biosciences"
Now showing items 1-20 of 64
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(2015)Northern aapa mires are important carbon sinks. Their CO2 exchange with the atmosphere is based on plant photosynthesis and respiration, and the decomposition of peat and other dead organic matter by microbial activity. The photosynthetic efficiency of plants depends on environmental factors and the amount and development of plant leaf area and biomass. There is not much research on the leaf area index and biomass of plants on aapa mires which makes them an interesting and important subject of research. This study investigated plant community composition, development and their effect on CO2 exchange at a northern aapa mire in Finland. The purpose of this study was to survey the biomass and leaf area index of plants within mire types and determine how they, together with water level and microbial activity, and explain the CO2 exchange between the mire and atmosphere. Another aim of this study was to construct regression models for predicting biomass and leaf area index using the plants height and coverage data. The study was conducted in summer 2014 at Halssiaapa, an aapa mire in Sodankylä. The mire was divided into oligotrophic, mesotrophic and eutrophic hummocks, lawns and hollows. The plant community species, their height and coverage were determined in early June, mid-July and mid-August. Moreover, the growth of plant groups was monitored weekly at 16 inventory squares between 6th June and 18th August. Biomass and leaf area samples were collected from different mire types in mid-July, and by the use of regression analysis, two equations were modelled for each plant group, one for predicting biomass and another for predicting leaf area index. The equations were used for predicting biomass and leaf area index for plant groups and mire types based on early June and mid-August data, and the weekly inventory squares. CO2 exchange, and factors controlling it, were measured via an eddy covariance method (measures exchange at ecosystem scale), and a closed chamber method (measures exchange from 1m2). Biomass, leaf area index, species composition, water level and soil factors (indicators of decomposer activity) were used to explain CO2 fluxes. The activity of decomposers in peat was measured via a decomposition test where litter bags containing tea or straw were placed at sampling sites on the mire. The regression models based on plant sample data explained well the biomass and leaf area indices of plant groups. The mire s overall and maximum biomass and leaf area indices were 147 g m-2 and 0,73 m2 m-2 respectively for vascular plants, and 351 g m-2 and 5,5 m2 m-2 respectively for mosses. Hollows displayed the lowest leaf area index and biomass values, whilst hummocks showed the highest values. Mesotrophic hummocks had the highest values for vascular plants and eutrophic lawns showed the highest values for mosses. Despite having a considerably higher level of biomass and leaf area index than vascular plants, Sphagnum mosses had a much lower gross photosynthetic capacity. Plant communities on lawns and hummocks with an abundancy of sedges (Cyperaceae) had a higher gross photosynthetic capacity and respiration level than plant communities in hollows. Factors leading to increased respiration were increasing vascular leaf area index, release of carbon from straw (indicating decomposer activity), decreasing water level (drier peat) and carbon content in surface peat. Factors leading to increased gross photosynthetic capacity were increasing vascular leaf area index, decreasing water level and decreased straw mass in surface peat (indicating decomposer activity). Temperature and PAR (Photosynthetically Active Radiation) explained 25 % of the variation in gross primary production and 39 % in respiration. The other environmental factors explained 55 % of the residual variation in gross primary production and 78 % in respiration. The mire s gross primary production showed highest increase rate in June and reached its maximum level in early August. Mean plant biomass and leaf area index had the same growth pattern as gross primary production, reaching peak levels at the end of July. The plants Menyanthes trifoliata , Potentilla palustris, Andromeda polifolia and the functional group containing sedges and Scheuchzeria palustris, had a similar growth pattern, but no change in biomass and leaf area index of Betula nana and Vaccinium oxycoccos was detected during the growing season. The models constructed in this study for predicting biomass and leaf area index can be used at Halssiaapa and other nearby aapa mires. Plant surveying and CO2 exchange measurements will be continued at the site in future. Longterm changes in plant community species and CO2 exchange can be researched by comparing results from year 2014 with in coming summers.
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(2010)Ligniini on maakasveille hyvin tärkeä makromolekyyli. Se on olennainen osa esimerkiksi vettä ja ravinteita kuljettavien puusolukon solujen soluseinää. Ligniini on myös osallisena kasvien puolustuksessa muita organismeja vastaan. Lisäksi se on tärkeä osa metsien ekologiassa ja ihmisen hyödykkeenä. Prosessia, jossa kasvisolun seinään muodostuu ligniiniä, sanotaan puutumiseksi, eli lignifikaatioksi. Tällöin lignifikoituvan solun sisällä syntetoidut ligniinin monomeerit – monolignolit - kuljetetaan solukalvon yli soluseinään, jossa ne polymeroituvat ligniiniksi. Vähitellen kasvisolun soluseinän selluloosan, hemiselluloosien, pektiinien ja rakenne-proteiinien muodostama verkosto tukkiutuu ligniinistä. Lignifikoituessaan solu käy samalla läpi ohjelmoidun solukuoleman. Monolignolien synteesistä tiedetään suhteellisen paljon, samoin kuin ligniinin polymerisaatiosta. Monolignolien kuljetuksesta sen sijaan ei ole tietoa. Eri hypoteesien mukaan monolignolit voivat kulkeutua solukalvon yli joko diffuusion, golgin laitteen rakkuloiden tai kuljettajaproteiinien eli transportterien avulla. Teoria golgin laitteesta on osoitettu epätodennäköiseksi ja diffusiostakaan ei ole todisteita aidoilla solukalvoilla. Siksi pro gradu –tutkielmani tarkoituksena onkin testata hypoteesia transportterivälitteisestä kuljetuksesta. Tutkittavina ovat kasveissa hyvin yleiset ABC-transportterit, joiden tiedetään kuljettavan sekundaarisen aineenvaihdunnan tuotteita. Tutkielmassani testasin ABC-transportterihypoteesia kuusen (Picea abies) ligniiniä tuottavilla A3/85-liuosviljelmäsoluilla sekä bioninformatiikan keinoin. A3/85-soluille syötettiin radioaktiivista fenyylialaniinia – monolignolisynteesin raaka-ainetta – yhdessä ABC-transportteri-inhibiittoreiden (Reversin 121 ja vanadaatti) kanssa. Inhibiittoreilla ei kuitenkaan ollut vaikutusta solujen erittämien fenyylialaniinin fenolituotteiden tai näiden sokerijohdannaisten määrään. Bioinformatiikan keinoin taas yritettiin löytää kuusen (Picea spp.) ja männyn (Pinus spp.) EST-kirjastoista hyviä ABC-transportteriehdokkaita monolignolien kuljetukseen. Erityisesti lignifikoituvassa solukossa esiintyvää, ominaisuuksiltaan sopivaa ABC-transportteria ei kuitenkaan löytynyt. Tämän perusteella kuljetuksesta vastaa todennäköisesti jokin muu transportteri tai kuljetus ei tapahdu lainkaan transportterin välityksellä. Tutkielmassani oli testattavana myös usein esitetty ajatus monolignolien myrkyllisyydestä, josta ei kuitenkaan ole tehty asiaan kuuluvaa selvitystä. Monolignolien odotetulla myrkyllisyydellä saatetaan kasvitieteessä perustella erinäisiä väittämiä, joten ajatuksen paikkansapitävyys on syytä varmistaa. Hypoteesia monolignolien myrkyllisyydestä testasin lisäämällä ligniiniä tuottamattomien tupakan (Nicotiana tabacum) liuosviljelmäsolujen (BY-2) kasvatusalustaan monolignoli koniferyylialkoholia. Koniferyylialkoholi aiheutti solujen lignifikaation sekä tappoi solut korkeissa pitoisuuksissa. Näiden kokeiden perusteella voidaan myrkyllisyys-hypoteesin sanoa olevan mahdollinen, joskin koniferyylialkoholin mahdollinen osallisuus lignifikoituvassa solussa tapahtuvaan ohjelmoituun solukuolemaan täytyy ottaa huomioon.
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(2013)Pulmonary arterial hypertension (PAH) is a progressive and devastating disease with poorly understood pathogenesis. It is characterized by abnormal remodelling of pulmonary vasculature due to uncontrolled apoptosis and proliferation of endothelial (ECs) and smooth muscle cells (SMCs) in vascular wall. In severe PAH pulmonary ECs exhibit hyperproliferative and apoptosis resistant phenotype contributing to the formation of neointima and development of plexiformic lesions. Structural changes promote occlusion of vascular lumen, and thus, increase in pulmonary vascular resistance. To date we lack efficient therapy to prevent vascular remodelling and restore normal vascular function in PAH. Purinergic signalling is potential modulator of pulmonary vascular homeostasis. It comprises of extracellular nucleotides, such as ATP, which signal through their receptors on cell membrane. Ectoenzymes with nucleotide hydrolyzing activity have an essential part in controlling homeostasis and physiologic concentration of extracellular nucleotides. Ectoenzyme CD39 plays a crucial role in dephosphorylating ATP, which is a known mediator of inflammation, angiogenesis, thrombosis and vasoconstriction according to previous research. Aims of this project were to study the role of extracellular ATP in pulmonary endothelial dysfunction during PAH pathogenesis. The goal was to evaluate the significance of ATPases, such as CD39, in the disease process and to identify significant ATP receptors on pulmonary ECs. We utilized a previously unused strategy to monitor ATPase activity in vivo in pulmonary endothelium of rats with PAH. With this strategy we could identify changes in a time-line manner. Our results indicate that ATPase activity is significantly attenuated in ECs during disease process. Similar finding was also observed in human pulmonary EC isolated from PAH patients suggesting that loss of ATPase activity mediated increase of extracellular ATP could play a role in disease pathogenesis. Our in vitro experiments reveal that loss-of CD39 in human pulmonary ECs leads to an apoptosis resistant and hyperproliferative phenotype. We also identify that purinergic receptor P2Y11 is a critical mediator of ATP responses in these ECs. Suppression of ATP mediated P2Y11 response in apoptosis resistant PAH patient ECs restores normal EC phenotype and thus, suggests a novel therapeutic strategy for pulmonary occlusive vasculopathy.
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(2012)Quantitative food webs have been used since the 1990s to describe the ecology of ecosystems. Such webs describe not only 'who eats whom' but also how many individuals get eaten, giving a detailed picture of the connections in an ecosystem. This detail allows far-reaching ecological conclusions to be drawn, for all manner of questions ranging from the influence of abiotic and biotic factors on population dynamics to the effect of latitude on ecosystem structure. Currently the webs' greatest limitations are their incompleteness and lack of geographic coverage: most published webs focus on a tiny fragment of the total food web, and there are few webs from higher latitudes. In this thesis I address these limitations, by extending a parasitoid-lepidopteran web which is being collected in the High Arctic. I add data on bird and spider predation of the web's Lepidoptera, and on the lepidopterans' herbivory of their food plants. Specifically, I ask what the relative strengths of predation and parasitism are in this community, and what effect herbivory has on the plants' seed production. I measured predation rates using both live caterpillars and modelling clay dummies as bait. The former, caterpillars of Sympistis nigrita tethered to threads, gave an estimate of both spider and bird predation. The latter showed bird predation only, but gave a larger sample size than tethered caterpillars. For the herbivory rates, I measured the seed production of avens flowers (Dryas octopetala x integrifolia, one of the main lepidopteran food plants) when damaged by feeding caterpillars. I found that predation is an important cause of mortality for S. nigrita, comparable to the high rates of parasitism already observed. During the larval period, some 38% of S. nigrita caterpillars are killed by spiders, 8% by parasitoids and 8% by birds. The caterpillars ate highly variable amounts of avens flowers, with 14.4% of flowers damaged by feeding in a set of 743 flowers, and 8.3% in another set of 672 flowers. The damaged flowers produced fewer and smaller seeds than did undamaged ones, causing overall avens seed production to drop by 7%. Overall, my findings show spider predation to be a relevant addition to the current parasitoid-lepidopteran food web. Bird predation, however, is relatively light – both when compared to spider predation and also to earlier predation rates reported from warmer latitudes. It is nevertheless comparable to the (naively twice as strong) parasitism since almost half the parasites die when their host is predated. When comparing food webs in order to address large scale ecological questions, such as the effect of latitude on food web structure, broader webs may be required instead of the mere parasitoid-host webs produced to date.
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(2015)Viime vuosikymmeninä tiedonalakohtaista oppimista on tutkittu paljon käsitteellisen muutoksen näkökulmasta. Käsitteellinen muutos tarkoittaa oppijan tietorakenteiden muuttumista tai jäsentymistä virheellisistä kohti tieteellistä mallia. Käsitteellisen muutoksen teorioiden mukaan oppijoiden aikaisempien virhekäsitysten tunteminen ja huomioiminen on keskeistä oppimisen ja opetuksen onnistumisen kannalta. Tässä tutkimuksessa selvitettiin biologian yliopisto-opiskelijoiden fotosynteesiin liittyviä virhekäsityksiä, niiden muuttumista opetustekstin lukemisen seurauksena sekä biologian pääaineopiskelijoiden ja sivuaineopiskelijoiden virhekäsitysten eroja. Suomalaisten opiskelijoiden virhekäsityksiä on aiemmin tutkittu niukasti. Tämän tutkimuksen tavoitteena oli selvittää virhekäsityksiä suomalaisten opetussuunnitelmien mukaan opiskelleilta opiskelijoilta sekä selvittää virhekäsitysten yksityiskohtaista muuttumista käsitteellisen muutoksen näkökulmasta. Tutkimuksen aineistona oli yliopiston biologian peruskurssin opiskelijoiden (n=171) vastaukset fotosynteesiä ja sen ekologista merkitystä koskeviin avoimiin kysymyksiin. Opiskelijat vastasivat kysymyksiin ennen opetustekstin lukemista, heti opetustekstin lukemisen jälkeen sekä kaksi viikkoa lukemisen jälkeen. Aineisto analysoitiin laadullisin menetelmin kuvaamalla, tyypittelemällä ja tulkitsemalla havaittuja virhekäsityksiä. Virhekäsitysten tulkinnassa hyödynnettiin tiedon ja ajattelutaitojen tasojen luokittelua. Opiskelijoilla havaittiin yhteensä 58 fotosynteesiä koskevaa selkeästi virheellistä käsitystä ja 14 puutteellista tai epämääräistä käsitystä. Virhekäsitykset liittyivät fotosynteesireaktioon, kasvien rakenteisiin, kasvin energiatalouteen ja fotosynteesin ekologiseen merkitykseen. Lähes kaikki virhekäsitykset ja puutteelliset käsitykset olivat yleisempiä biologian sivuaineopiskelijoilla kuin pääaineopiskelijoilla. Virhekäsitykset vähenivät opetustekstin lukemisen seurauksena, ja monimutkaisempia tiedon ja ajattelutaitojen tasoja ilmentävät virhekäsitykset vähenivät vähintään yhtä paljon kuin muutkin. Lukuisia virhekäsityksiä kuitenkin säilyi oikeat vastaukset antaneesta opetustekstistä huolimatta. Kaksi viikkoa opetustekstin lukemisen jälkeen osa jo virhekäsityksensä korjanneista opiskelijoista vastasi jälleen virheellisesti. Tutkimus osoittaa, että suomalaisen peruskoulun ja lukion fotosynteesin oppimista koskevat tavoitteet eivät täyty edes monen biologian yliopisto-opiskelijan kohdalla. Suomalaisilla opiskelijoilla on pitkälti samoja virhekäsityksiä kuin kansainvälisesti tehdyssä tutkimuksessa on havaittu. Fotosynteesin oppimisen kannalta keskeisin tutkimuksessa havaittu ymmärryksen puute on vaikeus ymmärtää fotosynteesin merkitys Auringon valoenergian ja ravintoketjujen energian välisenä linkkinä. Opiskelijoiden vaikeudet korjata virhekäsityksiään selittyvät käsitteellisen muutoksen prosessin haastavuudella ja hitaudella. Fotosynteesiä koskevan oppimisen edistämiseksi opetuksessa tulisi aiempaa paremmin huomioida käsitteellisen muutoksen haasteet kognitiivisesta, sosiaalisesta ja motivaatioon liittyvistä näkökulmista. Tämä tutkimus tarjoaa tietoa niistä fotosynteesin oppimista koskevista erityispiirteistä, joita ilmiön tehokkaaseen oppimiseen pyrkivän opetuksen tulee huomioida.
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(2017)No comprehensive research has been conducted on matriculation examinations in biology. This kind of data are needed, however, as evaluation (e.g. challenges and contents of test assignments) direct the way students study and learn and what they consider important in the taught matter. Thus, it is impossible to say, if the tests really measure core knowledge of biology, or just fragmentary facts. The aim of this study was to provide information on the challenges and contents of matriculation examination in biology and to provide, for the first time, comprehensive information on the factors affecting competence. The results of the study can be used to evaluate the impact of the new curriculum of upper secondary school and to support the development of the new electronic matriculation examination assignments. The research questions were: 1. How do the test tasks of biology matriculation examination measure the core content and the levels of both knowledge and cognitive dimensions? 2. Are the types of test assignments, knowledge and cognitive dimensions distributed equally between all core contents? 3. Do the results vary among a) the examinations, b) the type of tasks, c) the core contents or d) the knowledge and cognitive dimensions? 4. Is the competence of a) task type or b) knowledge or cognitive dimension levels, subject to the different types of core contents? 5. How does gender explain the choice and competence of different tasks? The qualitative content analysis was used to identify the different aspects; the task types (selected response, brief constructed response or performance-based assessment), the knowledge dimensions (factual, conceptual or procedural knowledge) and the cognitive process dimensions (remember, understand, apply, analyze, evaluate or create). The content variation of matriculation exam was examined and classified using the biological core contents. The data comprised matriculation examinations in biology from spring 2011 to spring 2015 (9 exams, 108 tasks). Additionally, statistical analyses were used to examine how the test subjects (n = 28,777) succeeded in these different assignments and whether girls and boys were equally successful. The task type "selected response" was considered only in four (out of 108) tasks. All levels of knowledge dimensions were found but higher level cognitive processes, such as "analyse" or "create" were not required; the main emphasis in the tasks was in the understanding of conceptual knowledge. The majority of the tasks dealt with the core content "From the molecules to the organisms", i.e. the focus was on biological structures and processes. The analysis of scores obtained in the tests showed that the total scores were higher in the autumn examinations than in the spring examinations and that the scores had been steadily declining. The achievement in the task was most influenced by the type of task and the required level of cognitive process. The interest and knowledge varied between different core contents. Biological evolution appeared to be the most interesting topic to students and had also the highest scores. The girls mastered biology generally better than the boys, but the difference between the sexes was minimal in tasks requiring some form of applied aspect. In particular, the boys excelled the girls in tasks related to ecosystems which mostly required applications. The study also showed other factors affecting student achievement, one of the most significant being the impact of the use or non-use of verbs in the assignment. If the assignment lacked a verb describing what the task requires (e.g. distinguish, subtract, describe), the candidates managed the task poorly. Structural aspects of the task seem to have a great impact especially on the boys' level of knowledge. Future research should classify assignments, for example through assignment instruction guidance or visual interaction on knowledge.
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(2014)Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.
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(2017)Puberty is a process of physiological changes, through which an immature individual becomes sexually mature. In humans, timing of puberty is highly variable within and between sexes and populations. Timing of puberty represents a complex trait, which is controlled both genetically and environmentally. Precocious pubertal timing is associated with development of metabolic diseases later in life, such as obesity and diabetes, and other disorders as ovarian and testicular cancer. Despite the estimated high heritability (50-80%) of pubertal timing, its genetic background is still poorly understood. Recently, the genome-wide association studies (GWASs) revealed many novel pubertal timing associated loci. Nevertheless, molecular mechanisms behind these associations remain elusive. This thesis focused on gene vestigial-like family member 3 (VGLL3), which is associated with pubertal timing in humans and maturation in Atlantic salmon (Salmo salar). Since the main physical structures, such as the hypothalamus and the pituitary gland, needed in reaching puberty are evolutionary conserved and start to develop in vertebrates during embryogenesis, the aim was to study the expression pat-terns and role of vgll3 in zebrafish (Danio rerio) during this period. In order to localize expression patterns of the vgll3 gene in zebrafish embryos, a whole-mount in situ RNA hybridization (ISH) was performed. mRNA overexpression and morpholino oligonucleotide (MO) knockdown techniques were used to alter the vgll3 gene expression levels in 0-5 dpf zebrafish. The combined injections of both mRNA and MO were performed to validate MO specificity. The ISH experiment showed the expression patterns in 0-1 dpf embryos. The expression was ubiquitous up to 6 hours post fertilization becoming more localized to specific regions in the head and trunk of the embryos during the later stages. Altering vgll3 expression with high concentrations of synthetic mRNA or MO lead to phenotypical abnormalities such as shortened and curved body axis, pericardial and yolk sack edemas, deformed heads and eyes. However, it remained unclear if these malformations appear only due to the alteration of vgll3 expression levels. The results suggest that vgll3 may play an important role in the embryonic development. However, the study does not show that vgll3 has impacts on the pubertal timing in vertebrates by affecting the development of the structures required for sexual maturation.
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(2012)Genome wide linkage and association methods are used to map genes affecting traits with genetic predisposition. In this thesis, I compare the methods suitable for quantitative trait mapping in complex, extended pedigrees. As a case study, gene-mapping study of musical aptitude is performed with these methods. Linkage analysis methods are developed for family studies. However, only a few methods are suitable for extended families with a quantitative trait. Three linkage programs were successfully applied for such data in this study. These programs are the SOLAR, JPSGCS and KELVIN. All of these three programs are based on different methods and thus, the same calculations are not repeated. SOLAR is based on the variance components method, JPSGCS on a graphical method and KELVIN on the Bayesian method. Association analysis is also difficult to implement for large pedigrees, because it is best suited for case-control data. Fortunately, methods are extended also for family-based studies. Here, a genomic control method was used to correct for the familial relationships. The method evaluates the relatedness from the whole genome data and the association tests are corrected for the relatedness rates. This method was implemented from the GenAbel program. As a case study, these methods were applied to a musical aptitude study. The musical aptitude is here understood as an ability to perceive the melody, harmony and rhythm of music, and to recognize structures in set of sounds. These abilities were tested with Carl Seashore s tests for pitch and time and Kai Karma's test for auditory structuring. The data consists of 107 pedigrees and 93 sporadic subjects, comprising in total of 915 individuals. Each family includes 2 - 50 individuals. These individuals were genotyped with a SNP chip for over 700,00 SNPs. The linkage analyses revealed several promising loci for the musical aptitude. The best result was located in 4q12 and it was found with all of the three linkage programs. Most of the other results could also be identified with multiple programs, but some differences also occurred. However, none of the findings could be discovered with association analysis, probably due to a too small sample size.
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(2013)Visual working memory (VWM) maintains information for future usage. Several studies show that the cortical oscillations in the γ-frequency band (from 30 to 120 Hz) are modulated by the VWM performance. However, less is known about the cortical sources underlying the modulation of these oscillations in VWM. To address this question, we recorded human neuronal activity with magneto- and electroencephalography (M/EEG) during a delayed-matching-to-sample VWM task with three different task conditions, within which participants were instructed to focus on different object features in turn. In addition, anatomical data was acquired with magnetic resonance imaging for source modeling purposes. We then estimated the cortical amplitude dynamics across frequencies from three to 90 Hz during the VWM retention period for these three different conditions. We found that the amplitudes of the γ –frequency band oscillations were strengthened in the occipito-temporal cortical areas during the VWM for shapes but not for color or spatial locations. These data suggest that γ –band oscillations are fundamental in VWM, especially for visual stimuli requiring perceptual feature binding. Furthermore, cortical γ –band oscillations were found to be load dependently strengthened in the frontal cortex, where the central executive and attention associated processes are believed to take place. These data support the previous hypotheses stating that γ –band oscillations contribute to the maintenance of object representations in VWM.
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(2017)Trichoderma reesei, an anamorph of Hypocrea jecorina, is a filamentous fungus widely used for producing industrial enzymes. T. reesei is used for both endogenous and heterogenous protein production. The optimization of the production conditions and the effects of extracellular agents to T. reesei s production and secretion capacity are crucial for economically sustainable biotechnical production. The available carbon sources, most commonly different types of sugars, have a significant effect on the production and secretion of enzymes by T. reesei. Genetic modification of the pathways through which the fungi recognizes extracellular signals could bring advancements to industrial enzyme production. Because of T. reesei s potential and use as a production strain, the species is an interesting platform for genetic modifications that would enhance the production capacities. With the current methods the genome editing of T. reesei is however slow, and introducing multiple mutations to a single strain can take years. The aim of this study is to optimize the fairly new CRISPR/Cas9 genome editing system for use in T. reesei. In the CRISPR/Cas9 method, a catalytically active Cas9 enzyme is bound to a specific locus of the genome, guided by a guide RNA and the Watson-Crick base pairing principle. Once in the RNA-guided locus, Cas9 introduces a double stranded break in the DNA, which can be repaired by the cells endogenous non-homologous end joining pathways. This repair is error prone and produces mutations to site of the double stranded break. A donor DNA is often introduced together with the Cas9 and guide RNA. This donor DNA includes sequence homology to the site of interest and allows for the use of the cells homologous repair pathways. In this case, the mutation can be better controlled, and for example the risk of chromosomal mutations is reduced. Currently the CRISPR/Cas9 system is widely used in mammalian cell studies and up to 100% mutation frequencies have been reported in yeast cells. In this study the method is optimized for use in T. reesei. To our best knowledge, the research community has not found an organism in which CRISPR/Cas9 would not function. The question mainly lies on what type of set up and component introduction is suitable for each cell type and research purpose. In this thesis, three putative and one already published genes believed to be involved in hexose sugar sensing will be deleted from a T. reesei production strain with the help of CRISPR/Cas9. The effect of these deletions will be assessed through studying the secretion and activity of endogenous cellulases with enzymatic assays. One sugar transporter that may play a part in glucose sensing was identified in this study. The deletion of this transporter caused a decrease in cellulase production and/or secretion. The three other transporters or sensors did not have a significant effect on cellulase production in spent grain extract and lactose or glucose media. It s possible that these genes are involved in the uptake and use of other carbon sources. The continuous expression of the CRISPR/Cas9 system in T. reesei proved difficult. In the continuous expression method at least one of the CRISPR/Cas9 components, the Cas9 protein or the guide RNA, is produced in the cells in vivo. Neither was achieved in this study. Instead, a fully synthetic method in which the Cas9 is transformed into the cells as a protein along with an in vitro produced guide RNA was set up and produced up to 1000 × higher mutation frequencies when compared to the traditional transformation method used for T. reesei. This study also demonstrates a simultaneous deletion of two genes in T. reesei. To the best of our knowledge, multiple simultaneous gene modifications have never been achieved in T. reesei.
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(2011)Pro gradu tutkielmani aiheena oli tutkia koirien (Canis familiaris) saalistushalukkuutta ja persoonallisuutta sekä näiden mahdollista yhteyttä dopamiinireseptori D4 –geeniin (myöh. DRD4-geeni). DRD4-geeni ilmenee lähinnä hermosoluissa ja se vaikuttaa erityisesti aivojen limbisen järjestelmän toimintaan. Limbinen järjestelmä säätelee tunnetiloja ja tunteisiin liittyviä autonomisia toimintoja sekä motivaatiota eli samoja asioita, joihin dopamiinin tiedetään vaikuttavan. Dopamiini vaikuttaa elimistöön sitoutumalla esimerkiksi aivojen limbisen alueen dopamiinireseptoreihin. Dopamiinin on havaittu liittyvän assosiatiiviseen oppimiseen ja DRD4-geenin puolestaan mm. aktiivisuuteen ja impulsiivisuuteen sekä uteliaisuuteen mistä syystä se valittiinkin kandidaattigeeniksemme tutkimukseen. Koira puolestaan sopii erityisen hyvin mallilajiksi kandidaattigeenitutkimukseen, koska koirarodut ovat geneettisiä isolaatteja ja eroavat toisistaan niin rakenteensa kuin käyttäytymisensäkin puolesta. Kytkentäepätasapaino eli alleeliassosiaatio on koirissa huomattavasti voimakkaampaa kuin ihmisissä ja siten koiralta on merkittävästi helpompaa löytää mm. tautigeenejä. Koirilla kytkentäepätasapaino yltää noin 2 Mb laajuudelle, kun vastaava luku ihmisellä on vain noin 0,28 Mb. Tutkimuksen 405 suursnautserin ja saksanpaimenkoiran saalistushalukkuus testattiin koiran luonteenkuvauksesta eli MH-testistä tutulla viehetestillä. Viehetestissä koiran edestä yllättäen "pakenee" saalista muistuttava mutkittelevasti kulkeva kankainen viehe. Koirien suoritukset viehetestissä videoitiin ja suoritukset luokiteltiin neljään luokkaan äärimmäisen saalistushalukkaasta saalistushaluttomaan. Muita persoonallisuuspiirteitä tutkittiin koirien omistajien täyttämien luonnelomakkeiden avulla. Kaikista viehetestillä testatuista koirista otettiin verinäytteet, joista eristettiin DNA. Saalistustestissä äärimmäisen saalistushalukkaat ja täysin saalistushaluttomat saksanpaimenkoirat (N=44) valittiin DRD4-geenin alleeliassosiaatiotutkimukseen. Tutkittavaa geenialuetta monistettiin käyttämällä alleelispesifejä alukkeita PCR-reaktiossa. Tutkimuksen koirista löydettiin DRD4-geenin alleeleja 2 ja 3a. Luonnelomakeaineiston taustalta paljastui faktorianalyysin avulla neljä faktoria: sosiaalisuus ihmisiä kohtaan, leikkisyys ja aktiivisuus, aggressiivisuus koiria kohtaan sekä rohkeus. Näistä ensimmäinen assosioitui merkittävästi alleelin 3a kanssa. Koirat jotka kantoivat tätä alleelia olivat vähemmän aggressiivisia ihmisiä kohtaan kuin koirat, joilla 3a-alleelia ei ollut. Saalistushalukkuuteen kumpikaan alleeleista eikä mikään genotyypeistä assosioitunut. Saalistushalukkuutta kokonaisaineistossa selitti parhaiten leikkisyys ja aktiivisuus. Rodut erosivat toisistaan ihmisiin ja koiriin suuntautuvalta aggressiivisuudeltaan siten, että saksanpaimenkoirat olivat suursnautsereita aggressiivisempia.
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(2014)The purpose of this thesis was to examine the diversity and species composition of dung beetle (Scarabaeinae) communities in degraded rainforest landscapes in southeastern Madagascar. Several studies elsewhere in the world have revealed that forest-dwelling dung beetle communities and especially large species suffer from forest degradation and fragmentation by decreased species diversity. The most important factors affecting community structure of forest-dwelling dung beetles are habitat area, connectivity and vegetation quality i.e. microclimate. The hypothesis of this study was that the situation is the same in Madagascar. As dung beetles provide several important ecosystem services, like nutrient cycling and bioturbation, loss of dung beetle diversity imposes a secondary threat to the extraordinary nature of Madagascar by decreasing the regeneration ability of vegetation. Material for the study was collected in forest fragments of different size and quality between two areas of primary tropical rainforest – Ranomafana National Park and Vatovavy mountain – in November and December 2011 and January 2012. The sampling was conducted by transects of 30–60 fish- or carrion-baited pitfall traps which were set up in 55 localities in the study area. In each locality, several variables were measured to describe the vegetation and microclimatic conditions. The variables included temperature, humidity, estimate of vegetation quality by 6 observation-based classes, vegetation density, hights of three clearly visible vegetation layers, altitude and slope steepness. In addition, connectivities were measured for the localities using GIS and a satellite image –based vegetation classification. In order to demonstrate the differences between certain localities the study sites were divided into seven zones in terms of their distance from the Ranomafana National Park, average connectivity of the transects and elevation. Altogether 4,199 individuals belonging to 24 species were collected. Six of the species are currently under identification process in the Paris Museum of Natural History. According to the preliminary results they include two species new to science. Largest numbers of species were collected from good quality fragments between Ranomafana and Vatovavy. Also, a clear transition zone in species composition was detected a few kilometers west from Vatovavy, where altitude changes sharply. The study reveals that the species assemblages in the forest fragments and degraded forest areas are surprisingly species rich. This may, however, be partly because of extinction debt, and many of the still surviving species may soon die out due to restricted dispersal possibilities. Connectivity and vegetation quality were shown to have an effect on Canthonini species richness, with less species in less connected areas and lower vegetation quality. Vegetation quality was also shown to have an impact on the proportions of species with different body length: more small and medium-sized (< 8 mm) species were found in fragments where vegetation was more degraded. In addition to revealing how rainforest fragmentation and degradation affect local communities, the study gives interesting information about the distribution of certain species of Epilissus (Scarabaeinae: Canthonini). It has been known before that four species of Epilissus show elevational differentiation in their occurrence in Ranomafana. In this study, two more species of the same genus, E. prasinus and E. emmae obscurpennis, were shown to continue this pattern in lower elevations near Vatovavy mountain, about 50 kilometers east of Ranomafana.
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(2017)Tausta ja tavoitteet: D-vitamiinia sitova proteiini (DBP) kuljettaa D-vitamiinia ja sen aineenvaihduntatuotteita, esim. 25-hydroksi-D-vitamiinia (25(OH)D), verenkierrossa. Geneettinen muuntelu GC-geenissä, joka koodaa DBP:tä, on yhdistetty 25(OH)D-konsentraatioiden vaihteluun. Tavoitteena olikin tutkia GC:n geneettisten tekijöiden vaikutusta 25(OH)D-konsentraatioon suomalaisilla vastasyntyneillä. Lisäksi tutkittiin GC:ssä esiintyvän muuntelun aiheuttamia eroja lapsen kasvussa raskauden aikana. Menetelmät: GC:n SNP:ssä, eli yksittäisen nukleotidin vaihdoksissa, esiintyvää polymorfiaa ja raskauden aikaisen 25(OH)D-konsentraation ja napaveren 25(OH)D-konsentraation suhdetta arvioitiin 933 valkoihoisella vastasyntyneellä pojalla (468) ja tytöllä (465). 25(OH)D-konsentraatio määritettiin raskausviikoilta 6-13 ja napaverestä. Äidin D-vitamiinin saanti ravintolisistä määritettiin ruuankäytön frekvenssikyselylomakkeen avulla kahdelta viimeiseltä raskauskuukaudelta. GC:n kolme SNP:tä genotyypitettiin Taq-polymeraasin aktiivisuuteen perustuvan qPCR:n avulla (Bio-Rad, CFX384 C1000 Touch™ Real-Time PCR Detection System, USA): rs4588, rs7041 ja rs705124. Genotyyppien rs4588 ja rs7041 yhdistelmästä muodostettiin kuusi tunnettua diplotyyppiä ja kaikista kolmesta genotyypistä edelleen haplotyypit. Eroja geno-, diplo- ja haplotyyppien välillä 25(OH)D-konsentraatioissa ja kasvussa testattiin kovarianssianalyysin (ANCOVA) avulla käyttämällä relevantteja kovariaatteja. Tulokset: SNP rs4588 ja rs7041 geno- ja diplotyyppien välillä löydettiin tilastollisesti merkitsevä ero 25(OH)D-konsentraatioissa (keskiarvo (nmol/l) ± keskivirhe). Napaveren 25(OH)D-konsentraatio oli alhaisin geno- ja diplotyypillä GC 2/2 (77.8 ± 4.3, p=0.010, p=0.028, ANCOVA) ja korkein diplotyypillä GC 1F/1S (93.8 ± 2.0). Haplotyypillä ATG oli alhaisin napaveren 25(OH)D-konsentraatio (77.9 ± 3.8, p=0.016, ANCOVA). Raskauden ajan 25(OH)D-konsentraatiossa ei löytynyt eroja geno-, diplo- tai haplotyyppien välillä (ANCOVA). Genotyypin rs7041 sekä diplotyyppien ja päänympäryksen välilllä havaittiin ero (p=0.005, p=0.002, ANOVA), mutta geneettisen muuntelun ja syntymäpainon ja –pituuden välillä ei löytynyt eroja. Kun valittiin relevantit kovariaatit, niin genotyyppien rs7041 sekä diplotyyppien ja päänympäryksen välillä ei löytynyt enää tilastollisesti merkitseviä eroja. Yhteenveto: Tulokset osoittavat, että DBP:tä koodaavan geenin geneettisellä muuntelulla ja napaveren 25(OH)D-konsentraatiolla olisi vaikutusta jo vastasyntyneiden D-vitamiinitilaan ja aineenvaihduntaan. Saatujen tulosten perusteella GC:n geneettinen muuntelu ei kuitenkaan ollut yhteydessä lapsen kasvuun. Tulevaisuudessa GC:n SNP:n vaikutusta 25(OH)D-konsentraatioon voisi tarkastella syntymän jälkeisten vuosien aikana.
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(2010)Genetic variation is vital for both contemporary and long-term wellbeing of populations. Whereas heterozygosity (Ho) and allelic richness (A) are commonly used to measure the level of genetic diversity in a population, effective population size (Ne) describes the speed of loss of genetic variation. Various effective population sizes are proposed as standards for safe retention of genetic variation in a Minimum Viable Population (MVP). Since the 1940s, several types of effective population size estimators have been developed. Earlier estimators relied on demographic parameters, whereas genetic estimators are based on the analysis of either one or two genetic samples from a population. All Ne estimators have their unique sensitivities and limiting assumptions, which complicate the choice of estimator, comparison of results of different studies and the assessment of the reliability of the results. Ne estimators have recently been used e.g. in the monitoring of many aquatic populations, but their reliability and comparability has not often been tested with extensive ecological and genetic data, and it is not well established how much added value they bring to the conservation of easily observable species. I tested this with an extensive dataset on the Siberian jays (Perisoreus infaustus) living in Suupohja, Finland (62°22'N, 21°30'E). The Suupohja Siberian jays form one of the few isolates of Siberian jays in Southern Finland. I utilised three demographic and three genetic Ne estimators to estimate the Ne and the Ne/N ratio in the Suupohja Siberian jays, and compared the findings to the Ho and A estimates calculated with the same data, and to various suggested MVP standards. The results showed that the ratio of effective and census population sizes (Ne/N) is close to 0.6 in the Suupohja Siberian jays. Uneven survival of offspring and population size fluctuations are the main factors in the formation of this ratio. The average genetic Ne estimate would, then, suggest a census population size of 44 % higher than the average N in the Suupohja study area. This result is probably connected to the high proportion of breeding immigrants in the data, which would cause the Ne estimates to reflect a larger genetic neighbourhood than the study area. The genetic Ne estimates also suggest that the Suupohja Siberian jays might not be able to maintain their genetic diversity in the long term if gene flow would cease due to further isolation, especially if isolation would also cause a faster demographic decline. Conservation attempts should aim at ensuring gene flow to the remaining Siberian jay isolates in Southern Finland, in order to protect them from increasing genetic uniformity and inbreeding. It is possible that while the average dispersal distances in the Siberian jay are short, occasional long-distance dispersal events have an important role in the pretention of genetic structuring in a Siberian jay population. Ne estimation based on demographic data was laborious in the case of the Suupohja Siberian jays, whereas the genetic Ne estimates showed large variation depending on year and estimation method used. Reliable estimation of Ne with genetic methods would have required information on the large-scale genetic structure of the population. In any case, Ne estimates gave a clearer picture on the genetic viability of the Suupohja Siberian jays than the Ho and A estimates, which did not indicate any decrease of genetic diversity during the study period.
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(2015)Welander Distal Myopathy (WDM) is caused by the p.E384K mutation in the TIA1 gene. The mutation supposedly causes the disease by a gain-of-function mechanism related to the formation of stress granules (Hackman et al. 2013). Also environmental factors have been proposed to affect the development of the disease: an increased number of stress granules has been observed in cells treated with cold shock compared to cells kept in 37 °C (Hofmann et al. 2012). When patients with WDM-like symptoms have been screened for changes in TIA1, an p.N357S-change has been found enriched in these patients. The p.N357S-change has earlier been reported as a polymorphism. The change in question is located in the same prion-like domain in exon 5, in which the p.E384K-mutation also lies. Therefore, the p.N357S-change could affect the predisposition to aggregation. The pro gradu project is divided into two parts: • The effect of the p.N357S polymorphism on stress granule formation in arsenite and possibly other stress treated cells • The effect of cold shock on stress granule formation on wild type and p.E384K TIA1 The results indicate, that the p.N357S change in TIA1 causes a change in the translated protein’s behavior. Similarly to the p.E384K change, the p.N357S change also induces an increased amount of stress granules in arsenite treated cells. However, the results also show that the stress granules recover faster in fluorescence recovery after photobleaching (FRAP) studies p.N357S transfected cells as compared to TIA1 p.E384K and wild type transfected cells. The cold shock experiment indicates that there is a difference in the stress granule formation between cells transfected with p.E384K and wild type TIA1. This supports previously published results of the effect of the p.E384K change on the stress response and stress granule formation, and also the use of cold shock as a stress inducing treatment. Used methods: PCR, transformation, DNA-extraction, cell culture, transfection, induction of stress granule formation by arsenite treatment and cold shock. The cells are cultivated on well plates, imaged and the data is analyzed with an automatized high content image analysis method (the CellInsight-platform). p.N357S cells were also analyzed with FRAP.
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(2013)Adaptive radiation is an important mechanism of evolution, which can lead to emergence of sympatric species or morphotypes. Among other biological interactions, parasitic pressure can have significant evolutionary implications for host populations by reducing the fitness of the host individuals. Parasite community structure of fishes is typically strongly dependent on both host ecology (e.g. habitat and feeding behaviour) and environmental factors (e.g. water quality and temperature). However, the relative importance of these factors for parasite-mediated speciation is not known. Also, host gender-specific differences in parasite communities can have an effect on the differentiation of host morphs. In this Master's thesis, I focused on differences in parasite communities of sympatric morphs of three-spine stickleback (Gasterosteus aculeatus) in two large Icelandic lakes, Thingvallavatn and Mývatn. In these lakes, the habitats of sympatric mud and lava morphs are the same (soft/hard bottom), but the habitat water temperatures are opposite between the lakes. In this unique system, it is thus possible to compare between the effects of host ecology and water temperature on parasite community structure and strength of parasite-mediated selection. Additionally, I studied the effect of host sex on the parasitic pressure. There is also a third stickleback morph, Nitella morph, inhabiting the cold limnetic habitats in Lake Thingvallavatn. I sampled the stickleback morphs from both lakes and identified their parasite species. I discovered a total of five parasite genera: trematodes Apatemon and Diplostomum, and cestodes Diphyllobothrium, Proteocephalus and Schistocephalus. Most of the observed parasites have negative effects on health and fitness of the host. I found more parasites in sticklebacks living in higher temperature in both lakes regardless of the morph. When comparing the cold water morphs in Thingvallavatn, parasite abundance was higher in the limnetic Nitella morph than in the shallow water lava morph. Fish gender had an effect on parasitism only in Thingvallavatn as males of both lava and Nitella morphs were more heavily infected with cestodes. Similarities in parasite communities with water temperature indicate that water temperature mainly determines parasite infections in this system instead of host ecology. As similar fish morphotypes exist in different lakes under opposite parasitic pressure, parasitism has unlikely initiated host differentiation, but differences in infection probably have emerged secondary to the ecological specialization of the morphs to different habitats. These results are among the first to tackle the key question in parasite-mediated divergent selection: at which point of the speciation process parasite communities become differentiated and thus can have an effect on speciation. However, the comparison between the cold water morphs (lava and Nitella) indicates that although water temperature seems to be the main factor controlling infections in this system, its effect may still be over ridden by host ecology. Sex-depended differences in parasitic pressure, on the other hand, are likely to reflect specific characteristics of each fish population and lake. These results suggest complex interactions between host ecology and abiotic environment, such as water temperature, in determining the parasite community structure. Hence both factors have to take into consideration when studying the role of parasites in speciation processes. In future, it is necessary to pinpoint the stage of the host speciation process when parasite infections become differentiated in replicated systems to gain comprehensive understanding of the role of parasites in adaptive radiations.
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(2012)Many pathogens are able to survive and reproduce in the environment outside of host for instance by saprotrophic lifestyle. These kinds of pathogens are called opportunistic as compared to obligatory pathogens that cannot interact or reproduce in the environment outside of host. Opportunistic pathogens are subject to strong selection forces in the environment outside of host for instance while they compete for resources they share with other microbes. Ecological interactions in the environment outside of host can therefore influence on the disease dynamics and evolution of virulence of an opportunistic pathogen. No proper theoretical model that would acknowledge opportunistic reproduction and ecological interactions in the environment out side of host has been developed before. Yet it is essential to develop this kind of theoretical model so that the development and dynamics of opportunistic diseases could be predicted and prevented. In this work, an opportunistic disease model was developed that considers both the opportunistic reproduction and the influence of a superior competitor as compared to pathogenic strain on pathogen growth in the environment outside of host. Differential equations in the model represent the density changes in time in the populations of susceptible and infected host, pathogen and rival strain outside host that is not pathogenic. Evolution of virulence of the new opportunistic pathogen meaning the ability to grow from low density in presence of superior competing strain was modeled in differing circumstances. Opportunistic disease dynamics was modeled in differing circumstances, when non-pathogenic competing strain was either present or absent. Equilibrium equations were solvable to a system, where non-pathogenic competing strain was absent, but to a system where non-pathogenic competing strain was present. Analyses of the model were performed with Math Works MATLAB – program. Reproducing inside host gives an opportunity for new opportunistic pathogen to increase in density under circumstances where competition is moderate enough so that the reproduction in the environment outside host may compensate opportunistic pathogen's weaker ability to compete. Reproduction and competition in the environment outside host produce disease dynamics that differ from more traditional SI-models. Density dependence of the reproduction in the environment outside host stabilizes host-parasite system in the absence of competition in the environment outside host. Instead, in the presence of competition the competitive advantage of the non-pathogen strain destabilizes disease dynamics and prevents extinction of the susceptible host. Reproduction in the environment outside host also enables opportunistic pathogen to remain in the environment in the absence of susceptible hosts and functions thus as a potential mechanism for disease out breaks when circumstances change. However, increasing competition in the environment outside host at the expense of opportunistic pathogen may potentially prevent epidemics. Among other things, the model could be applied to biological control with the intension of removing an opportunistic pathogen naturally by weakening its survival in the environment out side of host in a competiotion situation. This kind of biological control could for example be possible in the case of saprotrophic Flavobacterium columnare –fish pathogen that is found in fish farms.
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(2016)Epidermolysis bullosa (EB) is a group of hereditary skin disorders caused by mutations in the genes that code for adhesion molecules in keratinocytes. The symptoms of the disease include blisters and erosions in the skin as well as abnormalities in the mucosal membranes, nails and tooth enamel. Depending on the causative mutation the severity of the disease ranges from mild to lethal. This master s thesis was carried out in professor Hannes Lohi s canine genetics research group. It is a case study aiming to identify the genetic cause of EB present in Central Asian shepherd dogs. Furthermore, the mode of inheritance, frequency of the mutation in the breed and the effect of the mutation on the tissue were also examined. Primary research material included blood and tissue samples from a family of Central Asian shepherd dogs. Additional samples were obtained from other Central Asian shepherd dogs as well as from dogs of closely related breeds. All dogs included in this study are owned by private persons, and participation in this study was voluntary. The research methods used in this study included both wet laboratory experiments and bioinformatic in silico procedures. The genome of one affected dog was sequenced in order to identify the gene causing EB, and data from the sequencing was filtered with multiple programs according to recessive model. The model was decided after analyzing the pedigree of the affected dogs. After finding the likely causative gene the mutation was validated in a larger cohort with Sanger sequencing. Protein expression in the tissue of affected dogs was also studied using immunofluorescence staining. As a result of this study, a new mutation causing recessive dystrophic EB was identified. The mutation is specific to Central Asian shepherd dogs. Affected dogs have a homozygous mutation in the COL7A1 gene, which codes for collagen VII α1 protein. The mutation causes a premature stop codon in the mRNA sequence, which results in abnormal protein production and separation of skin layers. The frequency of the mutation allele in the sample is approximately 18 % and more than a fourth of the dogs in the sample are carriers. Based on the pedigree analysis, the mutation is relatively new and it is only found in a small population. The disease can be prevented from becoming more common in the breed with the help of a gene test, and the test will be available in the commercial MyDogDNA gene test panel.
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(2017)Follicle-stimulating hormone (FSH) regulates mammalian reproduction. The hormonal function of FSH is exerted through its receptor, FSHR. Hormone binding leads to signal transduction by the synthesis of the second messenger (intracellular cAMP) and the activation of other downstream signaling pathways. FSH receptors are mainly found in ovaries and testes where FSH stimulates e.g. follicular growth and spermatogenesis, respectively. It has been previously shown that FSHR mutations are linked to infertility through abnormalities in the receptor function. For example, inactivating FSHR mutation (Ala189Val) leads to arrest of follicular development in females and reduced sperm counts in males. FSH action seems to be especially critical for folliculogenesis and essential for female fertility. The development of gonads and dysfunctions affecting reproduction are the focus of our research group. Studies of recent years have demonstrated extragonadal FSHR expression, including endothelial cells of female reproductive tract and developing placenta. The physiological relevance and function of this extragonadal FSHR is still not well known, especially during embryogenesis. In addition, the expression or function of the mutated FSHR has not been studied in any cells endogenously expressing the receptor. As a consequence, the purpose of this thesis was to study FSHR expression and function in human pluripotent stem cells (hPSCs) as a model for early human development. The study was conducted in a human embryonic stem cell line (hESC H9, 46, XX) and two human induced pluripotent stem cell lines (hiPSC HEL127.6 and HEL128.5; 46, XX). iPSCs were obtained from female patients carrying the A189V mutation in the FSHR gene. Cells were differentiated for 8/12 days using two in vitro cell culturing protocols for distinct differentiation attempts. The first protocol (protocol D) was designed for embryonal cell differentiation, the other (trophoblast protocol) for extraembryonal differentiation. Cells at different stages of differentiation were studied with qRT-PCR and immunocytochemistry. At d8, cells were stimulated with FSH for qPCR studies and for cAMP assay. Control cells (H9) differentiated by protocol D endogenously expressed functional FSHR. These cells responded dose-dependently to FSH stimulation by substantially increasing cAMP production, downregulating FSHR mRNA expression and altering inhibin gene expression. Patient-derived iPSCs carrying the mutation expressed FSHR as well but the receptors were non-functional as expected. Both H9 and patient-derived cells that differentiated into trophoblast-like cells with the other protocol, also expressed FSHR at low level but did not respond to FSH stimulation. Preliminary results with protocol D indicate that the cells resemble extraembryonic cell types. The study has revealed novel information and insight about extragonadal FSHR expression and function during differentiation. However, the exact identity and the biological role of these cells is yet to be confirmed.
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