1,721,328 research outputs found
Regulation of the redox homeostasis during polyglutamine misfolding in Huntington’s Disease
No full textHuntington’s Disease (HD) is one of many neurodegenerative diseases that are associated with protein misfolding, aggregation and oxidative stress. While several changes in the redox homeostasis have been shown to occur in HD animal models and HD brains, the formal relationships between intracellular protein misfolding that occurs in HD, redox dysregulation and cellular toxicity are unknown. Therefore, several cellular models of intracellular polyglutamine (polyQ) protein misfolding were established for mechanistic studies.Various in vitro transient and stable cell expression systems expressing an N-terminal fragment of huntingtin (htt) (httExon 1, httEx1) with/or without a polyQ expansion and fused to fluorescent proteins were characterized. Mutant httEx1 (mhttEx1) constructs expressed in both neuronal and non-neuronal cell lines produced early polyQ aggregates and intracellular inclusion bodies (IBs) followed by cell toxicity that increased over time in time-course experiments. Using oxidation-sensitive probes, reactive oxygen species (ROS) were measured in polyQ-expressing cells using single, live-cell imaging analysis by confocal microscopy or population assays in order to explore the relationship between polyQ aggregation, ROS production and cellular toxicity. This study highlighted an early increase in ROS due to the expression of aggregation-prone mhttEx1 in both transient and stable cellular systems that coincided with polyQ aggregation, but preceded cell death. Suppression of ROS and toxicity was achieved by two antioxidant compounds (L-NAC and Trolox). Moreover, the use of MitoQ (Coenzyme Q10 covalently attached to triphenylphosphonium cation (TPP+)) at nanomolar concentrations abrogated the increased ROS due to mhttEx1 suggesting a mitochondrial origin of ROS.Given that molecular chaperones regulate the folding/misfolding of proteins and are involved in the regulation of the cellular redox homeostasis, the role of the redoxactivatable chaperone DJ-1 in HD was investigated. Protein expression analysis in HD cell models, a rodent model of HD and human HD brain samples showed an up-regulation of DJ-1 protein expression compared to control samples. Oxidation of DJ-1 was also elevatedin the human HD cortex. To test for a functional role of DJ-1 elevation and oxidation in HD, DJ-1 was overexpressed with wild-type or mhttEx1 in cell lines and mouse primary astrocytes. Overexpression of DJ-1 accelerated mhttEx1 aggregation and toxicity both of which could be suppressed by exposure of cells to mild oxidants suggesting that DJ-1, when redox-activated to a chaperone, modulates polyQ aggregation and toxicity. This hypothesis was tested by overexpression of mhttEx1 with a DJ-1 mutant lacking a critical redox activatable cysteine (Cys106). The C106S-DJ-1 mutant lost its ability to reduce polyQ aggregation and toxicity under oxidising conditions upon co-expression with mhttEx1 suggesting that DJ-1 indeed functions as a modulator of polyQ misfolding and toxicity.Together this work suggests that ROS may be produced during polyQ aggregation and is involved in cellular toxicity. This study also shows that DJ-1 regulates both, polyQ aggregation and toxicity in cell models and given the increased DJ-1 expression in vitro and in vivo (human HD), this protein could be a potential target for HD therapy
Sirtuin 4 Isoform 1 Represses HBV Replication
No full textMasterI. INTRODUCTION 1
II. MATERIALS AND METHODS 4
1. Vector construction 4
2. Transfection and cell culture 6
3. Core particle and immunoblotting 7
4. Northern and southern blottings 7
5. SDS-PAGE and Western blotting 8
6. Co-immunoprecipitation 9
7. Statistical analysis 9
III. RESULTS 10
1. SIRT4 expression in HBV-associated hepatocellular carcinoma (HCC) tumor and adjacent non-tumor tissues 10
2. Isoforms of SIRT4 12
3. HBV replication upregulates the endogenous SIRT4 protein 14
4. Overexpression of SIRT4.1 downregulates HBc protein level, core particle formation, and HBV RI DNA synthesis 16
5. Overexpression of SIRT4.1 downregulates HBV mRNA synthesis 18
6. Overexpression effect of catalytically inactive SIRT4.1 (H161Y) mutant on HBc protein level, core particle formation, and HBV RI DNA synthesis 20
7. Effect of overexpression of catalytically inactive SIRT4.1 (H161Y) mutant on HBV mRNA synthesis 22
8. SIRT4.1 acts as upregulator of AMPK 25
9. SIRT4.1 binds to AMPK 27
IV. DISCUSSION 30
V. REFERENCES 3
Sirtuin 4 Isoform 1 Represses HBV Replication
No full text학위논문(석사)--아주대학교 일반대학원 :의생명과학과,2023. 8I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
1. Vector construction 4
2. Transfection and cell culture 6
3. Core particle and immunoblotting 7
4. Northern and southern blottings 7
5. SDS-PAGE and Western blotting 8
6. Co-immunoprecipitation 9
7. Statistical analysis 9
III. RESULTS 10
1. SIRT4 expression in HBV-associated hepatocellular carcinoma (HCC) tumor and adjacent non-tumor tissues 10
2. Isoforms of SIRT4 12
3. HBV replication upregulates the endogenous SIRT4 protein 14
4. Overexpression of SIRT4.1 downregulates HBc protein level, core particle formation, and HBV RI DNA synthesis 16
5. Overexpression of SIRT4.1 downregulates HBV mRNA synthesis 18
6. Overexpression effect of catalytically inactive SIRT4.1 (H161Y) mutant on HBc protein level, core particle formation, and HBV RI DNA synthesis 20
7. Effect of overexpression of catalytically inactive SIRT4.1 (H161Y) mutant on HBV mRNA synthesis 22
8. SIRT4.1 acts as upregulator of AMPK 25
9. SIRT4.1 binds to AMPK 27
IV. DISCUSSION 30
V. REFERENCES 33MasterSirtuins (SIRTs), NAD+-dependent, class III histone deacetylases, are important in cellular processes, including cellular apoptosis, metabolism, stress resistance, and genomic stability. Various liver defects are associated with SIRTs. Among 7 mammalian SIRTs, SIRTs 1–7, the nuclear SIRT1, the cytoplasmic SIRT2, and the mitochondrial SIRT3 are well-studied. However, the mitochondrial SIRT4 and SIRT5 and the nuclear SIRT6 and SIRT7 are relatively less studied. Recent data suggests that mitochondrial SIRTs protect against the development of age-related chronic diseases. SIRT4, an ADP-dependent NAD+ transferase, regulates insulin secretion, fatty acid oxidation, and other cellular metabolic functions, such as glutamine metabolism. Recently, SIRT4 was suggested to act as a tumor-suppressor and to involve in hepatocellular carcinoma (HCC). Nonetheless, there is no conclusion yet on whether SIRT4 has either positive or negative impact on HCC. Since hepatitis B virus (HBV) infection can cause primary HCC, I investigated whether SIRT 4 has any role(s) on HBV replication, Among 2 isoforms of SIRT4, I focused on SIRT4 isoform 1 (SIRT4.1), a main isoform of SIRT4. SIRT4 was over-expressed in HBV replicating cells. I found that SIRT4.1 WT over-expression reduced HBV mRNA transcription, HBc protein level, core particle formation, replicative intermediate HBV DNA, also known as relaxed circular, double-stranded linear, and single-stranded DNA, synthesis in HBV replicating-HepG2 and -Huh7 cells. Additionally, I discovered that the endogenous SIRT4 level was increased in HBV replicating-HepG2 cells and the endogenous SIRT4 level was shown to be increased in biopsied HBV-associated HCC tumor tissues compared to adjacent non-tumor tissues. Furthermore unlike SIRT4.1 WT, the SIRT4.1 catalytically inactive H161Y mutant did not reduce HBV replication. I found that when overexpressed SIRT4.1 down-regulates HBV replication, AMPK, a known down-regulator of HBV replication, was activated. Moreover co-immunoprecipitation experiment reveals the interaction between SIRT4.1 and AMPK, suggesting that SIRT4.1 is involved in the restriction of HBV replication through an activation of AMPK. Even though further studies are required, our results can reveal a new approach of controlling hepatitis B on the molecular basis
Renormalisation of postquantum-classical gravity
No full textOne of the obstacles to reconciling quantum theory with general relativity, is constructing a theory which is both consistent with observation, and and gives finite answers at high energy, so that the theory holds at arbitrarily short distances. Quantum field theory achieves this through the process of renormalisation, but famously, perturbative quantum gravity fails to be renormalisable, even without coupling to matter. Recently, an alternative to quantum gravity has been proposed, in which the geometry of spacetime is taken to be classical rather than quantum, while still being coupled to quantum matter fields [1, 2]. This can be done consistently, provided the dynamics is fundamentally stochastic. Here, we find that the pure gravity theory is formally renormalisable. We do so via the path integral formulation by relating the classical-quantum action to that of quadratic gravity which is renormalisable. Because the action induces stochastic dynamics of space-time, rather than deterministic evolution of a quantum field, the classical-quantum theory is free of tachyons and negative norm ghosts. The key remaining question is whether the renormalisation prescription retains completely positive (CP) dynamics. This consideration appears to single out the scale invariant and asymptotically free theory. We give further evidence that the theory is CP, by showing that the two-point function of the scalar mode is positive. To support the use of precision accelerometers in testing the quantum nature of spacetime, we also compute the power spectral density of the acceleration. The results presented here have a number of implications for inflation, CMB data, and experiments to test the quantum nature of spacetime. They may also provide a way to compute probabilities in the regime of quantum gravity where spacetime can be treated as effectively classical
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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