Browsing by Subject "expression"

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  • Taha, Lamia (Helsingin yliopisto, 2021)
    The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.
  • Yoo, Bo Ram (Helsingfors universitet, 2017)
    Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.
  • Salumäe, Astrid (Helsingin yliopisto, 2020)
    In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA.
  • Salmela, Viljami R.; Ölander, Kaisu; Muukkonen, Ilkka; Bays, Paul M. (2019)
    Many studies of visual working memory have tested humans' ability to reproduce primary visual features of simple objects, such as the orientation of a grating or the hue of a color patch, following a delay. A consistent finding of such studies is that precision of responses declines as the number of items in memory increases. Here we compared visual working memory for primary features and high-level objects. We presented participants with memory arrays consisting of oriented gratings, facial expressions, or a mixture of both. Precision of reproduction for all facial expressions declined steadily as the memory load was increased from one to five faces. For primary features, this decline and the specific distributions of error observed, have been parsimoniously explained in terms of neural population codes. We adapted the population coding model for circular variables to the non-circular and bounded parameter space used for expression estimation. Total population activity was held constant according to the principle of normalization and the intensity of expression was decoded by drawing samples from the Bayesian posterior distribution. The model fit the data well, showing that principles of population coding can be applied to model memory representations at multiple levels of the visual hierarchy. When both gratings and faces had to be remembered, an asymmetry was observed. Increasing the number of faces decreased precision of orientation recall, but increasing the number of gratings did not affect recall of expression, suggesting that memorizing faces involves the automatic encoding of low-level features, in addition to higher-level expression information.
  • Lintula, Susanna; Mirtti, Tuomas; Rannikko, Antti; Bützow, Anna; Lempiäinen, Anna; Stenman, Jakob; Stenman, Ulf-Hakan; Hotakainen, Kristina (2019)
    The beta subunit of human chorionic gonadotropin (hCG beta) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p <.0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p <.0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.
  • Fawcett, Katherine A.; Obeidat, Ma'en; Melbourne, Carl; Shrine, Nick; Guyatt, Anna L.; John, Catherine; Luan, Jian'an; Richmond, Anne; Moksnes, Marta R.; Granell, Raquel; Weiss, Stefan; Imboden, Medea; May-Wilson, Sebastian; Hysi, Pirro; Boutin, Thibaud S.; Portas, Laura; Flexeder, Claudia; Harris, Sarah E.; Wang, Carol A.; Lyytikäinen, Leo Pekka; Palviainen, Teemu; Foong, Rachel E.; Keidel, Dirk; Minelli, Cosetta; Langenberg, Claudia; Bossé, Yohan; Berge, Maarten Van den; Sin, Don D.; Hao, Ke; Campbell, Archie; Porteous, David; Padmanabhan, Sandosh; Smith, Blair H.; Evans, David M.; Ring, Sue; Langhammer, Arnulf; Hveem, Kristian; Willer, Cristen; Ewert, Ralf; Stubbe, Beate; Pirastu, Nicola; Klaric, Lucija; Joshi, Peter K.; Patasova, Karina; Massimo, Mangino; Polasek, Ozren; Starr, John M.; Karrasch, Stefan; Strauch, Konstantin; Meitinger, Thomas; Rudan, Igor; Rantanen, Taina; Pietiläinen, Kirsi; Kähönen, Mika; Raitakari, Olli T.; Hall, Graham L.; Sly, Peter D.; Pennell, Craig E.; Kaprio, Jaakko; Lehtimäki, Terho; Vitart, Veronique; Deary, Ian J.; Jarvis, Debbie; Wilson, James F.; Spector, Tim; Probst-Hensch, Nicole; Wareham, Nicholas J.; Völzke, Henry; Henderson, John; Strachan, David P.; Brumpton, Ben M.; Hayward, Caroline; Hall, Ian P.; Tobin, Martin D.; Wain, Louise V. (2021)
    Background: Lung function is highly heritable and differs between the sexes throughout life. However, little is known about sexdifferential genetic effects on lung function. We aimed to conduct the first genome-wide genotype-by-sex interaction study on lung function to identify genetic effects that differ between males and females. Methods: We tested for interactions between 7,745,864 variants and sex on spirometry-based measures of lung function in UK Biobank (N=303,612), and sought replication in 75,696 independent individuals from the SpiroMeta consortium. Results: Five independent single-nucleotide polymorphisms (SNPs) showed genome-wide significant (P<5x10-8) interactions with sex on lung function, and 21 showed suggestive interactions (P<1x10-6). The strongest signal, from rs7697189 (chr4:145436894) on forced expiratory volume in 1 second (FEV1) (P=3.15x10-15), was replicated (P=0.016) in SpiroMeta. The C allele increased FEV1 more in males (untransformed FEV1 β=0.028 [SE 0.0022] litres) than females (β=0.009 [SE 0.0014] litres), and this effect was not accounted for by differential effects on height, smoking or pubertal age. rs7697189 resides upstream of the hedgehog-interacting protein (HHIP) gene and was previously associated with lung function and HHIP lung expression. We found HHIP expression was significantly different between the sexes (P=6.90x10-6), but we could not detect sex differential effects of rs7697189 on expression. Conclusions: We identified a novel genotype-by-sex interaction at a putative enhancer region upstream of the HHIP gene. Establishing the mechanism by which HHIP SNPs have different effects on lung function in males and females will be important for our understanding of lung health and diseases in both sexes.
  • Kokko, Ida-Maria (Helsingin yliopisto, 2017)
    Fans converse and unite with likeminded people who share the same objects of fandom. Modern technology has enabled the creation of online fan communities where fans express their identities and fandom. Along with fandom, fans are increasingly expressing their opinions online which has led to a more reciprocal, yet complex relationship between fans and producers. The aim of this master’s thesis is to study expressions of fandom in two online fan communities created for the video game franchise Tomb Raider: a commercial Facebook page administered by producers, and a non-commercial Facebook page administered by fans. This study also attempts to distinguish and explain potential similarities and differences in fans’ expressions of fandom between the two pages. This study relies on academic fandom studies and content analysis. Content analysis is applied to data collected from the two Facebook fan pages during the months of January, February, and March 2017. Obtained with Netvizz, an application developed by Rieder (2013) to extract specified Facebook content, the data comprises of anonymised posts and comments. It is classified into categories pertaining to Fiske’s (1992) conceptualisation of enunciative productivity and textual productivity as categories of fan productivity, which serves as a general theoretical framework for the study. According to the results, fans express their fandom on the two Facebook pages through enunciative productivity, such as fan talk and reminiscing and opinions and comparisons, and textual productivity, such as fan art, fan-made images and cosplay. Expressions of fandom are predominantly enunciative with lower amounts of textual productivity. The observed similarities between the pages concern the general themes of the pages, hegemonic topics of discussion among the fans, and forms of content and fan productivity. The results show differences in the structure, amount, and posting frequency of content, page objective, social dynamics between the administrators and the fans, and the general atmosphere of the page. The similarities and differences between expressions of fandom on the two Facebook pages can be explained by their distinct characteristics, size, and objectives as well as fans’ uses for the page. The commercial Facebook page is more official, larger in size, and it aims to motivate consumption whereas the non-commercial Facebook page is a smaller, safer place for fans to express their fandom. Fans use the commercial Facebook page for influential reasons, and the non-commercial Facebook page for recreational reasons.