677 research outputs found
Development of titanium-based scaffolds for spinal prosthesis
No full textThe author was selected to join Assistant Professor Cleo Choong research group together with a collaboration project with Singapore Institute of Manufacturing Technology, Singapore for his Final Year Project with the project title as “Development of Titanium-based Scaffolds for Spinal Prosthesis”. Throughout this project, the author was under the guidance of Assistant Professor Cleo Choong as School of Materials Science & Engineering supervisor, Nanyang Technological University as well as Dr. He Zeming and Dr. Saeed Maleksaeedi as direct institute supervisors.
During this 1-year of research work, the author had been exposed to various types of learning experiences as well as hands-on opportunities for example seeing how the research milestones were planned and taking charged of carrying it out, using different types of characterisation testing machines such as Instron materials characterisation machine, Stylus Profilometer (Taylor-Hobson Form Talysurf Series 2) etc., understanding various process flows required in a research in order to link up all the research data and results via program used for data analysis.
Lastly the experience gained had enabled the author to further enhance his technical knowledge, develop a better safety culture as well as make learning a more enjoyable and fun journey. Besides, the author experienced culture differences as he worked together with exchange students from University of Sydney, Australia, improving his communication skills in the process.Bachelor of Engineering (Materials Engineering
Selecting tyrosine kinase inhibitors for gastrointestinal stromal tumor with secondary KIT activation-loop domain mutations
No full text[[abstract]]Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary?secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation
MAPK phosphatase-1 represents a novel antiinflammatory target of glucocorticoids in the human endothelium
No full textGlucocorticoids are well-established anti-
inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NF-κB. In recent years, however, transcription factorindependent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase-1 (MKP-1) as a pivotal mediator of the anti-inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF-α-activated human endothelial cells and used the adhesion molecule E-selectin as inflammatory read-out parameter. Dex is known to reduce the expression of E-selectin, which is largely regulated by NF-κB. Here, we communicate that Dex at low concentrations (1–100 nM) markedly attenuates E-selectin expression without affecting NF-κB. Importantly, Dex is able to increase the expression of MKP-1, which causes an inactivation of TNF-α-induced p38 MAPK and mediates inhibition of E-selectin expression. In endothelial MKP-1ˉ/ˉ cells differentiated from MKP-1ˉ/ˉ embryonic stem cells and in MKP-1-silenced human endothelial cells, Dex did not inhibit TNF-α-evoked E-selectin expression. Thus, our findings introduce MKP-1 as a novel and crucial mediator of the anti-inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP-1 as an important and promising antiinflammatory drug target
Cooperative and Selective Lithium Complexation of 2,11,13,22-Tetraaza-5,8,16,19- tetraoxa-1,12-dioxocyclodocosanes
No full textCOPB2 gene silencing inhibits colorectal cancer cell proliferation and induces apoptosis via the JNK/c-Jun signaling pathway.
No full textObjectivesColorectal cancer (CRC) is one of the most common malignant human tumors. It is associated with high morbidity and mortality rates. In recent years, tumor gene therapy has emerged as a promising new approach for colorectal cancer therapy. Herein, we identify and analyze the role of COPB2 (coatomer protein complex, subunit beta 2) in proliferation and apoptosis of CRC cells.MethodsTo investigate the role of COPB2 in the proliferation and apoptosis of CRC cells, a shCOPB2 vector and a shCtrl vector were constructed for transfection into RKO and HCT116 cells. Cells proliferation was subsequently measured via cell counting kit-8 (CCK8) assay and Celigo cell counting assay. Apoptosis was measured via flow cytometry. The activity level of Caspase 3/7 was measured. Finally, the level of several JNK/c-Jun apoptosis pathway-related proteins were measured to characterize the mechanism of apoptosis.ResultsOur results showed that the proliferation rate was decreased and the apoptosis rate was increased in shCOPB2-treated RKO and HCT116 cells compared to those in controls. After the silencing of COPB2, JNK/c-Jun signal pathway activation was increased, the expression levels of apoptosis pathway-related proteins, such as Bad, p53 and Caspase 3, were also increased.ConclusionCOPB2 gene silencing can inhibit RKO and HCT116 cells proliferation and induce apoptosis via the JNK/c-Jun signaling pathway
Avaliação da estrutura genética da população atual de Santa Catarina com diferentes marcadores moleculares para aplicação na genética forense
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2014A identificação genética humana está centrada na utilização de um conjunto de marcadores autossômicos denominado microssatélite (Short Tanden Repeats-STRs). Todavia em algumas situações, como indivíduos com alto grau de parentesco, casos de deficiência paterna, amostras biológicas degradadas ou pouca quantidade, têm-se focado na utilização de outras classes de marcadores genéticos: inserções/deleções (InDels), do cromossomo X (X-STRs), do cromossomo Y (Y-STRs) e na região hipervariável 1 do DNA mitocondrial (mtDNA-HVR1). Visando avaliar a extensão da diversidade genética da população de Santa Catarina, indivíduos não aparentados foram genotipados para os marcadores do tipo (1) InDels, incluídos no Investigator DIPplex Kit®, (2) STRs autossômicos, incluídos no Investigator HDplexTM Kit®, (3) miniSTRs, incluídos no Investigator Hexaplex ESS Kit®, (4) STRs do cromossomo X, através do Investigator Argus X-12 Kit®. Foram determinadas as frequências alélicas e heterozigoses observada e esperada (Ho e He), e a eficácia dos parâmetros forenses: Poder de Discriminação (PD), Probabilidade de Coincidência (PC), Índice de Paternidade Típico (TPI), Poder de Exclusão de Paternidade (PE) e Conteúdo da Informação Polimórfica (PIC), sendo que não foi observado nenhum desvio (pAbstract: Human genetic identification is centered on use of microsatellite markers (Short Tanden Repeats-STRs). However, in some situations as individuals with a high degree of kinship, cases of paternal disability, degraded biological samples and in low amounts of samples have been focused on the use of other markers such as: insertions/deletions (InDels), X chromosome markers (X-STRs), Y chromosome markers (Y-STRs) and hypervariable region 1 of mitochondrial DNA (HVR1-mtDNA). Thereby, to evaluate the extent of genetic diversity of the Santa Catarina population, unrelated individuals were genotyped for: (1) InDels markers, included in the DIPplex Investigator DIPplex Kit®, (2) autosomal STRs, included in the Investigator HDplexTM Kit®, (3) miniSTRs, included in the Investigator Hexaplex ESS Kit® and (4) the X chromosome STRs, through the Investigator Argus X -12 Kit®. Allele frequencies, observed and expected heterozygosity (Ho, He) were determined, and the effectiveness of forensic parameters such as Power of Discrimination (PD), Probability of Coincidence (PC), Typical Paternity Index (TPI), Paternity Exclusion Power (PE) and Polymorphic Information Content (PIC). No deviation was observed (p <0.05) for the loci analyzed, except DXS10135 and DXS1079 Investigator Argus X-12 Kit®. All loci showed a high degree of genetic polymorphism. The highest PIC was observed in SE33 (0.948) and the lowest was observed in D6S474 (0.745). Forensic parameters were calculated based on allele frequencies for the Investigator HDplexTM and Investigator Hexaplex ESS® Kits. Combined Discrimination Power (CDP) and Combined Power of Exclusion (PEC) were 0.999999999999999999997752854927 and 0.99999999978062285, respectively. Allele frequencies for the 16 analyzed STR loci were compared with those of other populations from different geographical distributions. Genetic distance (Dsw) showed proximity between the Santa Catarina population, the European populations (France, Italy) and the sampled African population. The second group consisted of populations that do not significantly participated in the formation of Brazilian identity (Japan, Mexico, Colombia) and two isolated populations of Amerindian from Brazil. Allele frequencies and the statistical parameters of the Investigator Argus X-12 Kit® were obtained and the most polymorphic marker was DXS10135 with 23 polymorphic alleles and less polymorphic marker was DXS8378 with 5 alleles. Power of discrimination in Females (PDF) was 0.9999999999999999103669, while the Power of Discrimination in Males (PDM) was 0.999999999688867. Combined Power of Exclusionfor Trios and Duos were 0.99999999867687 and 0.999999589803, respectively. Linkage Disequilibrium tests were performed for all pairs of loci and only DXS7132-DXS10074 remained significant after Bonferroni correction (p < 0.0008). Analysis of genetic distance was used and it was verified that the population of Santa Catarina had similarity with European populations (Germany, Denmark and Portugal) followed by North African populations (Morocco and Somalia) and distant from the populations of Shanghai and Greenland because these populations not participate in the formation of the Santa Catarina identity. Another objective of this project were the genetic characterization of the variability of patrilineal population of Santa Catarina through seventeen STRs located in a non-recombinant region of the Y chromosome, included in the AmpFISTR®YfilerTM Kit. After analysis, 305 haplotypes were identified and 292 of these were unique (96%). Comparing the results obtained in this study with data from Portuguese, Spanish, Italians, Germans, Africans and other Brazilian populations was observed the important contribution of Europeans from the Iberian Peninsula in the composition of the Santa Catarina population. Variability of matrilineal population of Santa Catarina was also characterized by the sequencing of the mtDNA hypervariable region 1 (mtDNA-HVR1). This technique was the standardized and implemented in the Forensic Genetics section of the Instituto Geral de Perícias do Estado de SC (IGP-SC). During the data analysis, 221 haplotypes (n = 342) were identified, these haplotypes were classified into 85 geographic subhaplogrupos. Considering the largest haplogroups, these results showed an important contribution of the European haplogroup H (32.16%). Amerindian haplogroups A, B, C and D represent 25.15% of the population in contrast to the African haplogroup L that totalize 7.02%. Besides the genetic contribution for the understanding of the history of Santa Catarina population, the realization of this thesis resulted in the implementation of new techniques in the Forensic Genetics section of the IGP-SC, including sixty new genetic markers in their analysis and the mitochondrial DNA sequencing to solve the forensic cases of high complexity
SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma
No full textClaudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development
miR-148b-3p functions as a tumor suppressor in GISTs by directly targeting KIT
No full textAbstract Background Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. Methods miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117IHC+/KITmutation GISTs and 19 CD117IHC−/wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3. Results Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117IHC+/KITmutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3’-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients. Conclusion miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs
Rectangular Contact Lithography For Circuit Performance Improvement And Manufacture Cost Reduction
No full textAn Optical Lithography Method Is Disclosed That Uses Double Exposure Of A Reusable Template Mask And A Trim Mask To Fabricate Regularly-Placed Rectangular Contacts In Standard Cells Of Application-Specific Integrated Circuits (Asics). A First Exposure Of The Reusable Template Mask With Periodic Patterns Forms Periodic Dark Lines On A Wafer And A Second Exposure Of An Application-Specific Trim Mask Remove The Unwanted Part Of The Dark Lines And The Small Cuts Of The Dark Lines Left Form The Rectangular Regularly-Placed Contacts. All Contacts Are Placed Regularly In One Direction While Unrestrictedly In The Perpendicular Direction. The Regular Placement Of Patterns On The Template Mask Enable More Effective Use Of Resolution Enhancement Technologies, Which In Turn Allows A Decrease In Manufacturing Cost And The Minimum Contact Size And Pitch.; Since There Is No Extra Application-Specific Mask Needed Comparing With The Conventional Lithography Method For Unrestrictedly-Placed Contacts, The Extra Cost Is Kept To The Lowest. The Method Of The Invention Can Be Used In The Fabrication Of Standard Cells In Application-Specific Integrated Circuits (Asics) To Improve Circuit Performance And Decrease Circuit Area And Manufacturing Cost.published_or_final_versio
Single-cell transcriptome reveals a novel mechanism of C-Kit+-liver sinusoidal endothelial cells in NASH
No full textAbstract Aim To understand how liver sinusoidal endothelial cells (LSECs) respond to nonalcoholic steatohepatitis (NASH). Methods We profiled single-LSEC from livers of control and MCD-fed mice. The functions of C-Kit +-LSECs were determined using coculture and bone marrow transplantation (BMT) methods. Results Three special clusters of single-LSEC were differentiated. C-Kit +-LSECs of cluster 0, Msr1 +-LSECs of cluster 1 and Bmp4 + Selp +-VECs of cluster 2 were revealed, and these cells with diverse ectopic expressions of genes participated in regulation of endothelial, fibrosis and lipid metabolism in NASH. The number of C-Kit +-primary LSECs isolated from MCD mice was lower than control mice. Immunofluorescence co-staining of CD31 and C-KIT showed C-Kit +-LSECs located in hepatic sinusoid were also reduced in NASH patients and MCD mice, compared to AIH patients and control mice respectively. Interestingly, lipotoxic hepatocytes/HSCs cocultured with C-Kit +-LSECs or the livers of MCD mice receipting of C-Kit +-BMCs (bone marrow cells) showed less steatosis, inflammation and fibrosis, higher expression of prolipolytic FXR and PPAR-α, lower expression of TNF-α and α-SMA. Furthermore, coculturing or BMT of C-Kit +-endothelial derived cells could increase the levels of hepatic mitochondrial LC3B, decrease the degree of mitochondrial damage and ROS production through activating Pink1-mediated mitophagy pathway in NASH. Conclusions Hence, a novel transcriptomic view of LSECs was revealed to have heterogeneity and complexity in NASH. Importantly, a cluster of C-Kit +-LSECs was confirmed to recovery Pink1-related mitophagy and NASH progression
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