Browsing by Subject "qRT-PCR"
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(2013)Filamentous cyanobacteria taxa Nostocales and Stigonematales cells can differentiate into heterocysts nitrogen fixing cells when nitrogen is limiting the growth and into resting cells akinetes when nutrients decrease or the growth conditions become unfavorable for growth. Akinetes overwinter in the water sediments during the unfavorable growth time. When the growing condition improves akinetes germinate and can start a new cyanobacterial bloom. Akinete differentiation remains unclear. It is known from the literature that only a few akinete specific genes exist. First described akinete specific gene was avak. The morphological changes of akinete differentiation are known but the changes at molecular genetics level in regulation and differentiation remains unclear. The aim of this study was to design a method for akinete differentiation-related genes, avak, hepA and hap, for an Anabaena 1TU33s10 strain and to monitor the gene expression changes in a seven-week growth experiment. Primers for the differentiation related genes were designed based on the known whole genome sequence of the Anabaena 1TU3310 strain. In this study it was managed to design a quantitative reverse transcriptase polymerase chain reaction, qRT-PCR method, based on the genes involved in the akinete differentiation process. It was observed that gene expression changed when akinetes began to differentiate into the filaments. In the growth experiment II avaK-gene expression was increased 2-fold between the 14. and 30. days, and hap-gene showed 1.5 fold growth between 14. and 30. days. The number of akinetes was also increasing at the same time. In the growth experiment I heap-gene showed 1-8 fold growth between the days 21. and 27. –30. days when the number of heterocysts were also increasing. The number of akinetes was relatively low compared to number of vegetative cells which also explains the small expression fold-differences in the cultures during the experiment time when compared to expression fold-differences described in the literature. Designed method can thus also detect minor changes in gene expression. The designed and built qRT-PCR method can be used in the future for monitoring gene expression changes also for new akinete specific genes, and the method can be further optimized for screening natural water samples.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2009)Clostridium botulinum is an anaerobic rod shaped bacterium. It forms endospores that are commonly found in soils and aquatic sediments. C. botulinum produces one of the most potent neurotoxins that causes dangerous neuroparalytic disease called botulism. Food-borne botulism is an intoxication due to ingestion of preformed neurotoxin in foods. When favouring minimally processed food which safety depends almost only on chilled storage the risk of neurotoxin formation increases. It is essential to know the mechanisms of cold tolerance of C. botulinum when improving the safety of high-risk foods. A rapid temperature downshift causes decrease in the synthesis of most of the proteins in the cell. However the synthesis of structurally related cold shock proteins (Csp) reaches the maximum level after cold shock. It is assumed that these proteins are important for bacterial cold shock response. The exact function and meaning of cold shock proteins is not fully understood but they are believed e.g. to inhibit the formation of unwanted secondary structures of nucleic acids and regulate the synthesis of other proteins. The genome of C. botulinum ATCC 3502 strain contains three cold shock protein coding genes: cspA, cspB and cspC. The meaning of the cold shock proteins for the growth of ATCC 3502 was studied by comparing the growth of wild type strain and cspB- and cspC -mutant strains at different temperatures. The alterations in the expression of cspA-, cspB- and cspC -genes after cold shock were also studied. The results confirm that cspB and cspC genes are related to cold tolerance of the C. botulinum ATCC 3502 strain. cspB and cspC mutant strains grew at 20 °C at a reduced rate compared to C. botulinum ATCC 3502 wild type strain. There was no difference in growth rate between the strains grown at 37 °C and 44 °C. With quantitative RT-PCR, a significant twofold increase in the expression of cspA and cspB was detected for C. botulinum ATCC 3502 wild type strain while the expression of cspC stayed at normal level after cold shock. In cspB mutant strain the expression of cspA increased almost fivefold after cold shock. In cspC mutant strain after cold shock the trend of the expression of the other csp-genes was to decrease or stay at normal level. The results of quantitative RT-PCR method indicate some ability of csp genes to compensate for the functions of down regulated csp gene by other csp genes.
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(2021)Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.
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(2013)Listeria monocytogenes on elintarvikevälitteinen, opportunistinen patogeeni, joka voi aiheuttaa ihmiselle listerioosin. Kannat sietävät hyvin jääkaappilämpötiloja ja sopeutuvat erilaisiin elinympäristöihin. Elintarvikelaitoksissa listeriat ovat usein pysyviä laitoskantoja, jotka kontaminoivat tuotteita usein pitkällä aikavälillä. Tämän maisterin tutkielman laboratorio-osuus tehtiin syyslukukauden 2012 aikana Eläinlääketieteellisen tiedekunnan elintarvikehygienian ja ympäristöterveyden osastolla, mikrobiologisen elintarviketurvallisuuden huippuyksikössä. Tarkoituksena oli tutkia DEAD-Box RNA helikaasien vaikutusta L. monocytogenes EGD-e –kannan flagellan ilmenemiseen ja kiinnittymiseen. Kirjallisuuden mukaan L. monocytogenes –kantojen kiinnittymiseen vaikuttavat suuresti mahdollisesti adhesiivisina toimivien flagellojen läsnäolo sekä solujen liikkuvuus. L. monocytogenes EGD-e–kannalla on neljä DEAD-Box RNA helikaasigeeniä (lmo0866, lmo1246, lmo1450 ja lmo1722), joista osa vaikuttaa kasvuun ja liikkuvuuteen. Kaksi geeneistä vaikuttaa lisäksi positiivisesti bakteerin selviämiseen kylmän, etanolin ja emäksisen tai hapettavan kasvuympäristön aiheuttamassa stressitilanteessa. L. monocytogenes EGD-e mutanttikannoista ?lmo0866 ja ?lmo1450 on todettu liikkumattomiksi ja ?lmo1722 osittain liikkuvaksi. Nämä mutanttikannat ovat lisäksi kylmänherkkiä DEAD-Box RNA helikaasimutanttikannat ja villityyppi kuvattiin TEM-tekniikalla ja kahden flagellageenin (flhA ja motA) ilmentymistä mitattiin qRT-PCR:lla. Lopuksi kantojen kiinnittymistä tutkittiin mikrotiitterilevymenetelmällä. Kaikki tutkimukset tehtiin kahdessa lämpötilassa (+25 ja +37°C:ssa). Elektronimikroskooppikuvat ja qRT-PCR –tulokset olivat toisiaan tukevia. Mutanttikanta ?lmo0866 ei tuottanut flagelloja lainkaan, kun taas kannalla ?lmo1450 flagellojen tuotto oli heikompaa villityyppiin verrattuna +25°C:ssa. Kannat ?lmo1246 ja ?lmo1722 vastasivat villityyppiä flagellan esiintymisen suhteen. Mutanttikannoista ?lmo0866 ilmensi +25°C:ssa molempia tutkittuja flagellageenejä yli 20-kertaa vähemmän kuin villityyppi. Myös geenin lmo1450 poistaminen johti flagellageenien ilmentymisen vähenemiseen. Mutanttikannan ?lmo1722 osalta ilmentyminen oli alentunut vain toisen tutkitun flagellageenin (motA) osalta. Geenin lmo1246 deleetio ei vaikuttanut flagellageenien ilmentymiseen. Kiinnittymisessä eroja ei havaittu helikaasimutanttien ja villityypin välillä kummassakaan tutkitussa lämpötilassa, joten yksittäiset DEAD-Box RNA helikaasigeenit eivät vaikuta L. monocytogenes EGD-e –kannan kiinnittymiseen. L. monocytogenes –bakteerin kiinnittymiseen polystyreenipinnalle vaikuttavat kirjallisuuden mukaan useat eri tekijät, kuten inkubointilämpötila, -aika, ravinteet ja kanta. Flagellan ja liikkuvuuden merkitys kiinnittymiseen ei ole mahdollisesti niin suuri kuin on aiemmin oletettu.
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(2023)Heart failure is a global health issue that can result from various factors, one of which is myocardial infarction. The adult human heart has limited regenerative capacity to cover the loss of cardiomyocytes after myocardial infarction with new cardiomyocytes. The main responses to the loss of cardiomyocytes are fibrotic scar formation and the hypertrophy of remaining cardiomyocytes. Prolonged hypertrophy eventually leads to heart failure. Current treatments for heart failure only relieve the symptoms. Inducing cardiac regeneration could be one possible way to prevent and treat heart failure. Thus, to develop medical treatments that enhance the regenerative capacity, a comprehensive understanding of precise cellular mechanisms behind heart regeneration is crucial. The objective of this study was to establish a high-content analysis method for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) utilizing the Cell Painting assay to identify and categorize morphological alterations induced by various compounds in hiPSC-CMs. To evaluate the morphological impacts, dozens or even hundreds of cell features were measured at the same time. hiPSC-CMs were exposed to two hypertrophy inducers, endothelin-1 and angiotensin II, and to doxorubicin, which is known to be a cardiotoxic compound. In addition, the effects of a GATA4- targeting compound, C-2021, on hiPSC-CMs were examined. C-2021, was expected to have antihypertrophic effect on the cells. Previously used methods, proBNP staining and qPCR, were used to validate the novel method. According to proBNP staining and qPCR, endothelin-1 induced cardiomyocyte hypertrophy greater than angiotensin II. Compound C-2021 did not show statistically significant antihypertrophic properties after hypertrophic stimuli, but some tendency the alleviate the hypertrophy was noticed. Moreover, by utilizing different data processing programs a novel analysis method was developed. With this method, the different treatment groups were clustered based on the morphological alterations caused by compounds exposures. The hiPSC-CMs exposed to endothelin-1, angiotensin II or doxorubicin showed a different morphological profile compared to the control group hiPSC-CMs. Compound C-2021 was also observed to affect cell morphology. However, the data analysis still needs improvements in order to detect which cell features these compounds affect.
Now showing items 1-5 of 5