1,374,566 research outputs found
Improving the Dynamic Performance of Five-Axis CNC Machine Tool by using the Software-in-the-Loop (SIL) Platform
No full textThe paper presents the development and implementation of a Software-in-the-loop (SIL) platform allowing the real-time simulation of the hybrid model of five-axis CNC machine tool which is implemented in SIMULINK. The interfacing between dSPACE software and the feed drives models in SIMULINK is explined. The values for the simulated positioning errors between the position demand and simulated position of orthogonal trimming head for the gantry axis are in the order of microns so proposed SIL model is validated. The accurate SIL platform could be used to build and optimise the machining process models including CNC machine tools under cutting conditions and improve machines’ dynamic performance
Eun-Sil Sung
No full text학위논문(박사)아주대학교 일반대학원 :분자과학기술학과,2013. 8CONTENTS …………………………………………………..………… 1
ABSTRACT OF DISSERTATION …………………………………… 6
LIST OF TABLES ................................................................... 9
LIST OF FIGURES ……………………………...………………..…... 10
CHAPTER 1. General introduction ………………………………… 13
1.1. Death receptor (DR) signaling ……………………………….… 13
1.2. Agonistic antibodies to DRs as cancer therapeutics ……………… 16
1.3. Combined Trials of TRAIL and Agonistic antibodies to DRs …… 17
CHAPTER 2. A novel agonistic antibody to human DR4 induces apoptotic cell death in various tumor cells ……………………..……. 20
2.1 Abstract ………………………………………..……..…………… 20
2.2 Introduction …..…………………………….……………………... 22
2.3 Materials and Methods …………………………..……………...… 25
2.3.1 Generation and Characterization of AY4 mAb against Human DR4 …………………...…...…………………………...………...… 25
2.3.2 Reagents …………………………………………..….………. 25
2.3.3 Cell lines and Cell viability assays …………………………… 26
2.3.4 Western blotting analysis ……………………………………... 27
2.3.5 Analysis of tumoricidal activity In vivo ……………………… 27
2.4 Results ……………………………..……………………………… 29
2.4.1 AY4 specifically binds to DR4 without competition with TRAIL …………….…...…...………………………...…………....… 29
2.4.2 AY4 alone induces cell death of various cancer cell lines without cytotoxicity in normal human hepatocytes …….……...…..….. 34
2.4.3 AY4 induces caspase-dependent apoptotic cell death .......…… 43
2.4.4 FADD and caspase-8 are essential for the AY4-mediated cell death …………..………..…………………………………..…….…. 47
2.4.5 AY4 activates both extrinsic and intrinsic apoptotic pathway …………………………..…………………………………….. 50
2.4.6 AY4 exhibits antitumor activity In vivo …………..………… 52
2.5 Discussion …………………………………..………………………. 54
CHAPTER 3. HDAC inhibitors synergistically potentiate DR4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cell ………………………………………………………………………...… 59
3.1 Abstract ………………………………………………………….. 59
3.2 Introduction ……...……………………………………………… 60
3.3 Material and Methods ……………………………………………. 64
3.3.1 Materials ……………………………..……………………… 64
3.3.2 Cell lines and cell cultures …………………..……………… 64
3.3.3 Cell viability assays ………………………..………………... 65
3.3.4 Western blotting analysis ……………………..……………... 70
3.3.5 Flow cytometry ……………………………..……………… 70
3.3.6 RNA interference ……………………………..……………… 70
3.3.7 Statistical analysis …………………………………..………. 71
3.4 Results …………………………………………………………… 72
3.4.1 Responses of various leukemia cell lines to AY4 or TRAIL alones ......................................................................................... 72
3.4.2 Screening of chemotherapeutic agents for synergistic effects on TRAIL-mediated cytotoxicity in various leukemia cell lines … 75
3.4.3 SAHA and VPA synergize both AY4- and TRAIL-induced apoptosis ………………………………………………...…… 77
3.4.4 Cell surface expression levels of DR4 and DR5 are not closely correlated with AY4 or TRAIL-induced cytotoxicity ……….. 80
3.4.5 SAHA and VPA synergize with both AY4- and TRAIL-mediated cell death by amplifying both extrinsic and intrinsic apoptotic pathways …………………………..………………………….. 83
3.4.6 Combined treatment of AY4 or TRAIL with either SAHA or VPA downregulates expression level of c-FLIP …………………... 86
3.4.7 Combined treatment of AY4 or TRAIL with either SAHA or VPA downregulates expression levels of anti-apoptotic proteins ….. 90
3.4.8 Combined treatment of AY4 with the other agents does not significantly affect expression level of anti-apoptotic proteins ……………………………………………………………….. 92
3.5 Discussion ………………………………………..……………….. 93
CHAPTER 4. The proteasome inhibitor potentiates TRAIL receptor agonist-induced apoptosis by stabilizing tBid and Bik in human head and neck squamous cell carcinoma cells …………………………….. 100
4.1 Abstract ……………………………..……………………..…….. 100 4.2 Introduction ………………………….…………………………... 102
4.3 Materials and Methods ………………………………………… 105
4.3.1 Cell lines and reagents …………………………………... 105
4.3.2 Cell treatment and viability assay ………………………. 106
4.3.3 Synergistic analysis of combined treatments ……………. 107
4.3.4 Analysis of cell surface expression of TRAIL receptors … 107
4.3.5 Western blotting and immunoprecipitation ……………… 107
4.3.6 Subcellular fractionation …………………………..……... 108
4.3.7 RNA interference …………………………………..……... 108
4.3.8 Protein stability assay ……………………………..……… 109
4.3.9 Statistical analysis ……………………………….….………. 109
4.4 Results …………………………………………………………… 110
4.4.1 MG132 sensitizes HNSCC cells to TRAIL-/AY4-mediated cell death by activating extrinsic and intrinsic apoptotic pathways .. 110
4.4.2 MG132 significantly increases DR5 surface expression and induces accumulation of proapoptotic Bik and antiapoptotic Mcl-1.. 116
4.4.3 MG132-mediated stabilization of Bik and tBid contributes to the synergistic cell death induced by combination treatment ….. 119
4.4.4 Bak, but not Bax, is constrained by Mcl-1 and Bcl-XL and released by Bik accumulated due to MG132 …………………...…….. 124
4.4.5 Knockdown of Mcl-1 and Bcl-XL significantly sensitizes HNSCC cells to TRAIL and AY4 as single agents ….………………... 127
4.4.6 Combination treatment induces translocation of tBid and Bax from the cytosol to the mitochondria …………………………...… 129
4.5 Discussion ………………………………………………….……. 133
CONCLUSION ……………………………………………………… 138
REFERENCES ……………………………………………………… 140
ABSTRACT IN KOREAN ………………………………………..... 155
PUBLICATIONS ……………………………………………………. 159MasterIdeal anti-cancer therapeutics would induce tumor cell death selectively but not normal cells. In this regard, pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5, TRAIL-R2), are attractive targets for the development of agonistic anti-cancer biologics. Recently, several agonistic monoclonal antibodies (mAb) have been developed by targeting DR4 or DR5, which induce cell death in various types of tumor in vitro and in vivo. Some of them are now under various phases of clinical trials. However, some tumor cells are resistant to TRAIL-induced apoptosis. This resistance can be overcome by its combination with anti-cancer chemotherapeutic agents [1-3].
Here I report the cell death mechanism of a mouse agonistic antibody against human DR4 and its synergistic anti-cancer mechanism in combination with anti-cancer agents in various tumor types.
In chapter 1, general introduction was described about Death Receptor (DR)-mediated signaling, developments of agonistic antibodies to DRs, and its combination therapy with anti-cancer agents.
In chapter 2, I report about characterization of a novel agonistic antibody to human death receptor 4 in various tumor cells. An agonistic mAb, AY4, raised against human DR4 in mice, which did not compete with DR4-TRAIL interaction. The anti-DR4 agonistic AY4 mAb induced caspase-dependent apoptotic cell death in various types of cancer cells without significant toxicity to normal human hepatocytes. AY4 reduced the growth of pre-established human lung tumors in a mouse xenograft model. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which were only sensitive to TRAIL and anti-DR5 agonistic mAbs but resistant to DR4-selective TRAIL mutants and other anti-DR4 agonist mAbs [4, 5].
In chapter 3, I investigated that HDACI (Histone deacetylase inhibitors) have synergistic effects on AY4-mediated cell death in the T-cell acute lymphoblastic leukemia (T-ALL) cells. In this study, I investigate the specific role of DR4 in inducing apoptosis by studying the susceptibility of various leukemia cell lines to AY4 alone or in combination with various chemotherapeutic agents, in comparison with TRAIL. I found that most leukemia cell lines are intrinsically resistant to AY4 or TRAIL alone, but two T-ALL cell lines (CEM-CM3, CCRF-CEM) become substantially sensitized by combined treatment of AY4 or TRAIL with HDACI, either SAHA or VPA, by synergistic amplification of both the extrinsic and intrinsic apoptotic pathways. All of the combined treatments synergistically downregulated the expression of several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-XL, XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. These results demonstrate that the HDACIs have synergistic effects on AY4-mdiated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.
In chapter 4, I investigated the effects of the proteasome inhibitor MG132 in combination with TRAIL or AY4 on sensitization of TRAIL- and AY4-resistant human HNSCC (head and neck squamous cell carcinoma cells) cell lines. This study provides a novel mechanism for the synergistic apoptotic cell death, which involves the generation and stabilization of tBid and the stabilization of Bik. These results indicate that tBid directly activates Bax, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-XL, suggesting a major role for the mitochondrial apoptotic pathway in the synergistic apoptotic cell death mediated by combination treatment in HNSCC cells. Moreover, restriction of Bak to the mitochondria by Mcl-1 and Bcl-XL is an important mechanism of resistance to DR5-/DR4-mediated apoptotic cell death in HNSCC cells. The potent synergy between the proteasome inhibitor and TRAIL receptor agonists suggests that the combination of the two agents may offer a new therapeutic strategy to improve HNSCC treatment.
In conclusion, I studied cell death mechanism of an agonistic anti-DR4 antibody in various tumor types. However, it has resistance to TRAIL or AY4 in many tumor cells, especially, T-ALL and HNSCC cells. To overcome the resistance, I evaluated AY4 in combination with various therapeutic agents to compare TRAIL-mediated combination effects. Here, I studied AY4-mediated cell death and molecular mechanism in treatment with the combination of various anti-cancer agents with TRAIL or AY4 in most of which are completely resistant to TRAIL and AY4 as single agents. These results suggest that the combination with anti-DR4 agonistic mAb AY4 and general anti-cancer agents can be effective in resistant tumor cells
A Strong Correlation between Serum soluble IL-2 Receptor (sIL-2R) and Atypical Lymphocytosis
No full textActivated lymphocytes morphologically change into large aberrant cells known as atypical lymphocytes (atyLy). AtyLy are seen in various non-neoplastic conditions such as viral infection of Epstein-Barr virus, cytomegalovirus and hepatitis viruses. These activated cells release various cytokines or soluble receptors such as soluble interleukin-2 receptor (sIL-2R) and Fas-receptor (Fas-R). Accordingly, we measured serum sIL-2R in 25 pediatric patients. The data and other hematological/biochemical parameters were analyzed by the statistical processing method of Principle Component Analysis (PCA). 23 out of 25 patients with atypical lymphocytosis-related conditions (atyLy/lymphocyte ratio >5%) were found to have higher serum sIL-2R levels than the cut-off-value of 400 U/mL. The correlation between sIL-2R and the atyLy/lymphocyte ratio was the best indicator for discriminating the severity of disease. The first component (contribution ratio: 0.384) of PCA showed that lymphocyte activity was mostly represented by sIL-2R, lactate dehydrogenase, white blood cell count, lymphocyte count, lymphocyte percentile and atyLy/lymphocyte ratio.
Conclusively, these findings suggest a strong correlation between serum sIL-2R level and atypical lymphocytosis
Non-native species in Ialian freshwater habitats: a macroecological assessment of invasion drivers.
No full textThe relative role of propagule pressure, abiotic and biotic variables as determinants of non-native species occurrence differs among studies, hindering the synthesis of emergent patterns in invasion ecology and preventing generalisation for conservation actions. In order to produce a broad and general assessment of the occurrence of alien species in aquatic habitats, we proposed a macroecological approach to assess the drivers of occurrence of alien species in all biota (microorganisms, plants and animals) across several natural habitats in freshwater ecosystems in Italy, and we generalised the results of the analysis to provide a risk map of the occurrence of alien species.
We determined that abiotic climatic variables were good predictors of alien species occurrence. Indeed, these variables, together with propagule pressure, expressed as the proximity to major inhabited areas, and differences in the receiving community, expressed as the native species richness, played a crucial role in determining the number of alien species. Furthermore, we found evidence of an influence of body size in determining the occurrence of the non-native species. By using the predictions of our model, we addressed the probability of the occurrence of alien species in freshwater habitats across the whole country and highlighted areas at higher risk
Identification of the ds-sIL-6R and a novel protease-derived sIL-6R from human serum via mass spectrometry (MS).
No full text(A) Schematic illustration of the procedure to precipitate total sIL-6R from human serum. A representative coomassie-stained sodium dodecyl sulfate (SDS) gel and a western blot of the precipitated protein are shown on the right. The SDS gels were run under nonreducing conditions (see Materials and Methods for details), and the region of the SDS gel excised for MS is indicated with a red box. The precipitated sIL-6R is detected with an antibody that specifically recognizes the ectodomain of the human IL-6R (4–11). (B) Proteomics workflow including disulfide bond reduction, thiol alkylation, enzymatic N-deglycosylation, proteolysis in presence of 50% H218O, LC-MS/MS analysis, and data interpretation. (C) MS/MS spectrum (higher-energy collisional dissociation [HCD]) of the C-terminal peptide of the ds-sIL-6R identified via database searching. The N-glycan site Asn-350, which is modified to an Asp-350 because of PNGase F treatment, is shown in green. (D) MS/MS spectrum (electron-transfer dissociation [ETD]) of the C-terminal peptide of the protease-derived sIL-6R identified via database searching. The N-glycan site Asn-350, which is modified to an Asp-350 because of PNGase F treatment, is shown in green. One of the identified O-glycan structures on Thr-352 is shown.</p
Quantitative Features of serum sIL-2R level in Patients with Mature B-Cell Lymphoma– Involvement of LDH
No full textSerum Lactate Dehydrogenase (LDH) activity and soluble IL-2 Receptor-alpha (sIL-2R) levels are monitored as a marker of
disease activity in patients with lymphoma. Although adult T-cell leukemia (ATL) cells are well known to release large amounts of
sIL-2R,it remains unclear to what extent B-cell lymphoma cells shed sIL-2R in sera. Subtypes of mature B-cell lymphoma, including
CD25+ hairy leukemic cells, were examined for the characteristics of sIL-2R levels in each subtype. In normal controls, the
serum sIL-2R mean value was 260u/mL.The median serum sIL-2R value for 64 B-cell lymphoma cases was 506 u/mL;by subtype
the median values were as follows: 1157 u/mL for 7 cases of chronic lymphocytic leukemia/hairy cell leukemia (CLL/HCL), 451
u/mL for 38 cases of diffuse large B-cell lymphoma (DLBCL), and 456 u/mL for 19 cases of follicular Lymphoma (FL). The median
values of serum LDH activity by the above subtypes were 175 IU/mL, 204 IU/mL, and 198 IU/mL, respectively. There was
distinct inter-subtype and inter-patient variation of serum sIL-2R.In particular, inter-case variation could be grouped into value for
three concentration ranges: less than 300 u/mL, 300-1000 u/mL, and greater than 1000 u/mL. Cases with serum sIL-2R values
of 1000 u/mL or more tended to have an especially high sIL-2R to LDH ratio, suggesting a close relationship between high sIL-2R
and CD25-expressing lymphoma cells. With respect to sIL-2R and LDH levels, CLL/HCL, DLBCL, and FL showed similar distributions.
Moreover, for sIL-2R levels exceeding 1000u/mL, sIL-2R levels were randomly high according to the LDH status. Conclusively,
the combination of serum sIL-2R level and LDH activity can provide a better understanding of characteristics of subtypes
of mature B-Cell Lymphoma and can be used as a reliable surrogate marker for evaluating numerical and biological data
SIL FieldWorks
No full textFieldWorks consists of software tools that help you manage linguistic and cultural data. FieldWorks supports tasks ranging from the initial entry of collected data through to the preparation of data for publication: * dictionary development * interlinearization of texts * cultural records, which can be categorized using the Outline of Cultural Materials * bulk editing of many fields * morphological analysis * complex non-Roman scripts using Unicode and SIL-developed Graphite * multi-user editing capability over a local area network
Dense seismic network provides new insight into the 2007 Upptyppingar dyke intrusion
No full textFactors such as network geometry, network size and phase-picking accuracy have significant
effects on the precision of seismic hypocentre locations. In turn, the precision of the hypocentral locations
dictates the degree to which morphological details within seismic swarms may be resolved. The Icelandic
national seismic network (SIL) is designed to monitor seismic activity across large expanses of Iceland in realtime
using automated earthquake detection and location software. Here we examine the performance of the
SIL network relative to a much denser, local network of seismometers deployed around the Askja volcano in
the Northern Volcanic Zone. A subset of earthquakes from the 2007–2008 dyke intrusion beneath Mt. Upptyppingar
is used to compare single- and multi-event hypocentral locations. Specifically, we highlight 288, high
signal-to-noise ratio events that occurred during an intensive sequence of earthquakes from 6–24 July 2007,
when the temporary Askja network was active. A careful refinement of phase onsets recorded by our wellconfigured,
dense network of receivers reveals hypocentres clustered tightly on a planar structure, interpreted
as a dyke dipping at 49. The root-mean-square (RMS) misfit to the plane (114 m) is only slightly greater than
the uncertainties in relative locations of the earthquakes themselves, and constitutes a three-fold reduction in
RMS misfit over SIL relative locations. The improved precision, facilitated predominantly by a more favourable
network size and configuration, permits a more detailed analysis of the intrusion
A novel method for SIL Verification based on system degradation using reliability block diagram
No full textSafety integrity level (SIL) verification plays a critical role in reliability assessment of safety related systems. However, current methods available for SIL verification are too complicated to be applied in practice. Therefore, a novel method for SIL verification, which is based on system degradation using reliability block diagram (RBD), is proposed in this paper. The key idea of the method proposed is to perform RBD analysis and calculation of average probability of dangerous failure on demand (PFDG) at each stage of system degradation, which is caused by failures of redundant channels. The method has been applied to several classical redundant architectures of safety related systems, and could make the SIL verification process simpler. Further, the formulae obtained are identical with those given in IEC 61508
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