Browsing by Subject "GFP"
Now showing items 1-8 of 8
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(2017)Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV--synuclein (-syn) to target substantia nigra dopamine neurons to produce an -syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.
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(Helsingin yliopisto, 2019)This study is a cross-validation of a hierarchical theory-based model of personality trait factors that comprises hypotheses regarding which personality constructs predict specific job performance criteria. The personality measures include the Big Five dimensions together with the Need for Achievement factor. The predictor variables have been conceptually aligned with specific criterion variables that are clusters of competencies. The model consists of six one-to-one predictor-criterion relationships that are paired up into three higher-order relationships which in turn are aggregated into a single score of General Factor of Personality (GFP) on the predictor side and overall work performance on the criterion side. The original study conducted in 2015 (N=929) was based on an international sample of participants from various organisations, whereas this sample consists of employees from a single global company (N=109). The aim was to explore the similarities and differences in the results in comparison to the original data. All the participants completed the same online personality self-assessment with 31 psychometric scales and a 360-feedback tool measuring 22 competencies. At least one external reviewer nominated by the participant completed a review rating on those competencies. Principal Components were extracted to investigate how well the model fits this data and the results compared to the results from the original study. Correlations between the first-order and second-order (composite) variables were also checked. Finally, regression analyses were conducted to test nine hypotheses derived from the theoretical model. The results of this study show that there is a clear relationship between the GFP and the overall performance as the observed validity is r = .39 which is even higher than in the original study were this value was r = .23. Out of the six personality factors, Extraversion and Conscientiousness are the only significant predictors of various job performance outcome in this data and, all in all, three hypotheses out of nine are fully confirmed and a fourth one partially. The results are also discussed with view to what kind of a role a specific company culture or expected behaviours of people working in certain job roles might play on the results.
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(Helsingin yliopisto, 2022)Lactobacilli especially Limosilactobacillus reuteri’s strains inhabit the GI tract of humans with glycerol/diol dehydratase activity metabolizing glycerol and producing a broad-spectrum antimicrobial system called reuterin. It consists of 3-hydroxypropanal (3-HPA), acrolein, and its derivatives. Due to the toxic activity of ubiquitous acrolein, an analytical toolbox to determine acrolein formation by food cultures is needed. We developed assays to estimate microbial formation of acrolein using a colorimetric method based on tryptophan and a fluorescence-based approach with 2-amino-1-methyl benzimidazole (AMBI) as a probe. We compared tryptophan and AMBI-based quantification of reuterin produced by resting cells or during the growth of Lb. reuteri DSM 20016. With 600 mM glycerol, resting cells produced 329 ± 35 mM 3-HPA as quantified by HPLC-RI, and 390 ± 13 mM of 3-HPA/acrolein based on the colorimetric method with 3-HPA as standard. Acrolein (40 ± 11 mM) was detected using an AMBI probe. We also detected 3-HPA and acrolein formation during the exponential growth phase in the presence of 50 mM glycerol in different media. Also, as acrolein induces redox stress, redox potential and sensitivity to reuterin/acrolein of the engineered green fluorescence protein (roGFP2) were determined. Ultimately, the roGFP2 gene was tried to clone in E. coli (JM109) using the pTH1mp constitutive expression vector to establish as a biosensor for acrolein detection. Our results suggest that quantification of acrolein by fluorescence-based approaches and biosensors constitute novel methods to estimate any risk of acrolein formation in presence of glycerol/diol dehydratase-positive microbes and glycerol during food fermentation.
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(University of Helsinki, 2000)
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(2017)Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.
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(Helsingfors universitet, 2017)The leaves of healthy potato (Solanum tuberosum L.) plants and leaves systemically infected with Potato virus A (PVA; a monopartite positive sense single stranded RNA genome, genus: Potyvirus, family: Potyviridae) have been previously analysed and compared for the nuclear proteome to better understand the role of nucleus in plant virus infection. The analysis indicated that a small GTP-binding protein (ras-related protein; rabE1) is found in the nuclei of PVA-infected leaves but is absent from nuclei of healthy leaves. Therefore, to confirm the differential localization, this study was conducted. Subcellular localization of proteins was studied by tagging the protein with a marker protein, green fluorescent protein (GFP). The mRNA of ras-like protein, rabE1, of potato cv. Pentland Crown was cloned and expressed with GFP fused to the N- or C-terminus of rabE1 (GFP-ras and ras-GFP respectively). Plants of Nicotiana benthamiana were inoculated with PVA by two methods. Firstly, the Agrobacterium strains expressing GFP-ras or ras-GFP were mixed with the Agrobacterium strain expressing red fluorescent protein (RFP) or PVA-RFP and introduced to the healthy leaves of N. benthamiana plants and observed under epifluorescence microscope three days post inoculation. Secondly, PVA-RFP was agroinoculated to two or three basal leaves of young N. benthamiana plants and plants were grown for 12 days to allow systemic infection. Thereafter, PVA infected upper leaves and healthy leaves were infiltrated with GFP-ras or ras-GFP and the leaves were observed under epifluorescence microscope three days after infiltration. In healthy plants, GFP-ras localized to the cytoplasm and ras-GFP localized mostly to the cytoplasm and in few cells it was found both in the nucleus and cytoplasm. Whereas, in PVA infected cells, GFP-ras localized to the cytoplasm and in some cells to both nucleus and cytoplasm suggesting that PVA infection increases GFP-ras localization to the nucleus. However, no difference was observed in localization of ras-GFP in PVA infected and healthy leaves. The hypothesis of the research is thus partially true. Our study shows that rabE1 can be differentially localized in the healthy and PVA-infected plant cells.
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(2016)In the vast majority of electrophysiological studies on cognition, participants are only measured once during a single experimental session. The dearth of studies on test-retest reliability in magnetoencephalography (MEG) within and across experimental sessions is a preventing factor for longitudinal designs, imaging genetics studies, and clinical applications. From the recorded signals, it is not straightforward to draw robust and steady indices of brain activity that could directly be used in exploring behavioral effects or genetic associations. To study the variations in markers associated with cognitive functions, we extracted three event-related field (ERF) features from time-locked global field power (GFP) epochs using MEG while participants were performing a numerical N-back task in four consecutive measurements conducted during two different days separated by two weeks. We demonstrate that the latency of the MI70, a neural correlate associated with cognitive functions such as working memory, was a stable parameter and did not show significant variations over time. In addition, the M170 peak amplitude and the mean amplitude of late positive component (LPP) also expressed moderate-to-strong reliability across multiple measures over time over many sensor spaces and between participants. The M170 amplitude varied more significantly between the measurements in some conditions but showed consistency over the participants over time. In addition we demonstrated significant correlation with the M170 and LPP parameters and cognitive load. The results are in line with the literature showing less within-subject fluctuation for the latency parameters and more consistency in between-subject comparisons for amplitude based features. The within-subject consistency was apparent also with longer delays between the measurements. We suggest that with a few limitations the ERF features show sufficient reliability and stability for longitudinal research designs and clinical applications for cognitive functions in single as well as cross-subject designs. (C) 2016 Elsevier Inc. All rights reserved.
