No description available.

When Y-1 mouse adrenal tumor cells were treated with sodium orthovanadate, an intermediate filament (IF) inhibitor in BHK21-F cells, there was no change in the amount of 20α-dihydroprogesterone produced. A neurofilament inhibitor, β, β'-iminodipropionitrile (IDPN), enhanced the ability of Y-1 cells to produce steroid in response to ACTH by acting on the plasma membrane. Electron microscopy of Y-1 cells extracted with Triton X-100 revealed that both vanadate and IDPN caused the aggregation of cytoskeletal and granular structures in the perinuclear area. The steroidogenic effects of IDPN suggest that the perinuclear aggrergation of cytoskeletal structures may result from the detachment of IF from the plasma membrane, while the reason for the cytoskeletal changes by vanadate is unknown.

Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25. / by Daniel C. Chauss. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243

The chondrocyte is the only cell type in articular cartilage, and its role is to maintain cartilage integrity by synthesizing and releasing macromolecules into the extracellular matrix (ECM) or breaking down its damaged constituents (Stockwell, 1991). The two major constituents of the ECM are type II collagen and aggrecans (aggregating proteoglycans). Proteoglycans have a high negative charge which attracts cations and increases the osmolarity, while also lowering the pH of the interstitial fluid. The fibrillar collagen matrix constrains ECM swelling that results from the Donnan osmotic pressure produced by proteoglycans (Wilkins et al., 2000). Activities of daily living produce fluctuating mechanical loads on the tissue which also alter the mechano-electro-chemical environment of chondrocytes embedded in the ECM. These conditions affect the physiology and function of chondrocytes directly (Wilkins et al., 2000; Guilak et al., 1995; Guilak et al., 1999). Relatively few studies of in situ chondrocyte mechanics have been reported in the biomechanics literature, in contrast to the more numerous experimental studies of the mechanobiological response of live cartilage explants to various culture and loading conditions. Analyses of chondrocyte mechanics can shed significant insights in the interpretation of experimental mechanobiological responses. Predictions from carefully formulated biomechanics models may also generate hypotheses about the mechanisms that transduce signals to chondrocytes via mechanical, electrical and chemical pathways. Therefore, computational tools that can model the response of cells, embedded within a charged hydrated ECM, to various loading conditions may serve a valuable role in mechanobiological studies. Computational modeling has become a necessary tool to study biomechanics with complex geometries and mechanisms (De et al., 2010). Usually, theoretical and computational models of cell physiology and biophysics are formulated in 1D, deriving solutions by solving ordinary differential equations, such as cell volume regulation (Tosteson and Hoffman, 1960), pH regulation (Boron and De Weer, 1976), and Ca2+ regulation (Schuster et al., 2002). Cell modeling software, such as The Virtual Cell (vcell.org Moraru et al. (2008)), analyze stationary cell shapes and isolated cells. To model the cell-ECM system while accounting for ECM deformation, the fibrillar nature of the ECM, interstitial fluid flow, solute transport, and electrical potential arising from Donnan or streaming effects, we adopt the multiphasic theory framework (Ateshian, 2007). This framework serves as the foundation of multiphasic analyses in the open source finite element software FEBio (Maas et al., 2012; Ateshian et al., 2013), which was developed specifically for biomechanics and biophysics, and offers a suitable environment to solve complex models of cell-ECM interactions in 3D. In the studies proposed here, we will extend the functionality of FEBio to further investigate the cell-ECM system. These extensions and studies are summarized in the following chapters: Chapter 1: This introductory chapter provides the general background and specific aims of this dissertation. Chapter 2: Cell-ECM interactions depend significantly on the ECM response to external loading conditions. For fibrillar soft tissues such as articular cartilage, it has been shown that modeling the ECM using a continuous fiber distribution produces much better agreement with experimental measurements of its response to loading. However, evaluating the stress and elasticity tensors for such distributions is computationally very expensive in a finite element analysis. In this aim we develop a new numerical integration scheme to calculate these tensors more efficiently than standard techniques, only accounting for fibers that are in tension. Chapter 3: Cell-ECM interactions also depend significantly on accurate modeling of selective transport across the cell membrane. However, the thickness of this membrane is typically three orders of magnitude smaller than the cell size, which poses significant numerical challenges when modeling the membrane using the finite element method, such as element locking. To date, no existing finite element software offers a multiphasic membrane element. In this aim, we formulate and implement a new membrane element in FEBio, which can accommodate fluid and solute transport within the biphasic and multiphasic framework, to model passive and selective transport across the cell membrane. Chapter 4: This aim extends Aim 2 to incorporate reactions across multiphasic membrane elements in FEBio, to model the conformational reactions of cell membrane transporters, such as carrier-mediated transporters and membrane pumps. This implementation is verified against standard models for the regulation of cell volume, pH, and Ca2+. Chapter 5: This final chapter provides a summary of the advances contributed in this dissertation, along with suggestions for future aims related to the topics covered here. With the completion of these aims, we have extended the modeling capabilities for cell physiology and mechanobiology to more complex multicellular systems embedded within their ECM, while subjected to a range of varying mechanical, electrical or chemical loading conditions.

It is generally believed that ribosome-inactivating proteins (RIPs) are transported to their intracellular targets to express their toxicity. However, studies on the uptake, intracellular transport and apoptotic mechanism of type I RIPS and the cell-binding B chain of type II RIPS are rare. This study is to investigate some problems in these aspects of RIP toxicity. / RCA caused a cell loss at the minimal dose of 50 nM at 24 hr. The main type of cell death was apoptosis, which peaked at 12 hr. The apoptosis proceeded through an extrinsic pathway that involved the activation of caspase-8, but not caspase-9. / RTA caused cell loss at the minimal dose of 50 nM at 24 hr. The main type of cell death was apoptosis, which peaked at 12 hr. RTA was internalised via a clathrin-dependent RME. Like the TCS transport, RTA was not found in the Golgi apparatus. The apoptosis proceeded via the extrinsic pathway that involved the activation of caspase-8 and caspase-3. However, on live rabbits, RTA caused necrotic skin damage. / RTB caused cell loss at the minimal dose of 100 nM at 24 hr. The main type of cell death was initially necrosis, but later became apoptosis, which peaked at 12 hr. / TCS caused a decrease in cell number at the minimum effective dose of 800 nM at 24 hr post administration. The main type of cell death was apoptosis, which peaked at 12 hr. / These results show that (1) the cell-binding B chain is not a precondition for RIP toxicity, because TCS and RTA are also toxic to cells; (2) RTB itself is toxic; (3) without the binding of the B chain to cell surface, the entry and intracellular transport of type I RIPS differ from those of the type II; and (4) both RIPs and single B chain can induce apoptosis. Additionally, the results from live rabbits and cultured cells show that in vivo and in vitro toxicity may involve different cell death mechanisms. RTB-treated NIH 3T3 cells may serve as a model for the switch of cell death from necrosis to apoptosis. (Abstract shortened by UMI.) / We studied trichosanthin (TCS) and ricin A chain (RTA), which are type I RIPS, ricinus communis agglutinin (RCA), which is a type II RIP, and ricin B chain (RTB), which is the cell-binding chain of ricin and RCA. / Sha Ou. / "August 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3547. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 185-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Mak, Keng Hou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 77-91). / Abstracts in English and Chinese. / Thesis Committee --- p.2 / Declaration --- p.3 / Table of Contents --- p.4 / List of Abbreviations --- p.6 / List of Figures --- p.8 / Abstract --- p.10 / Chapter Chapter 1 --- Introduction --- p.12 / Chapter 1.1 --- Background --- p.12 / Chapter 1.1.1 --- Overview of apoptosis --- p.12 / Chapter 1.1.2 --- Synopsis of the apoptotic pathway --- p.13 / Chapter 1.1.3 --- Defining apoptosis --- p.14 / Chapter 1.1.4 --- Interaction between pro- and anti-apoptotic factors determines cell fate --- p.14 / Chapter 1.1.5 --- DNA fragmentation during the execution phase --- p.15 / Chapter 1.1.6 --- Current understanding of the point of commitment in apoptosis --- p.16 / Chapter 1.1.7 --- Previous studies and hypotheses related to the reversibility of late-state apoptosis --- p.16 / Chapter 1.1.8 --- Unanswered questions --- p.19 / Chapter 1.2 --- "Hypothesis and objectives, Study models and Significance" --- p.19 / Chapter 1.2.1 --- Hypothesis and objectives --- p.19 / Chapter 1.2.2 --- Study models --- p.20 / Chapter 1.2.3 --- Significance --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter Chapter 3 --- Results --- p.30 / Chapter 3.1 --- Dying cells reversed execution stage of apoptosis after removal of apoptotic stimuli --- p.30 / Chapter 3.2 --- Dying cells reversed apoptosis after DNA damage --- p.37 / Chapter 3.3 --- Genetic alterations and transformation occurred after reversal of apoptosis --- p.43 / Chapter 3.4 --- Investigating molecular mechanism driving reversal of apoptosis --- p.50 / Chapter 3.4.1 --- Preparation and characterization of samples for microarray --- p.50 / Chapter 3.4.2 --- Gene ontology enrichment analysis of the expression profile during reversal of apoptosis --- p.52 / Chapter 3.4.3 --- Interfering stress response or anti-apoptotic factors during the reversal of apoptosis drove cells to terminal death --- p.56 / Chapter Chapter 4 --- Discussion --- p.62 / Chapter 4.1 --- "Reversal of apoptosis in ""normal cells"" was observed" --- p.62 / Chapter 4.2 --- Cells surviving apoptosis had their genomes damaged and altered --- p.63 / Chapter 4.3 --- Transformation occurred after reversal of apoptosis --- p.65 / Chapter 4.4 --- Investigating molecular mechanism driving reversal of apoptosis --- p.65 / Chapter 4.5 --- Summary --- p.68 / Chapter Chapter 5 --- Perspectives --- p.70 / Chapter 5.1 --- Could reversal of apoptosis be evolutionarily advantageous? --- p.70 / Chapter 5.2 --- "Reversal of apoptosis as an ""individualistic"" behavior against organismal integrity" --- p.71 / Chapter 5.3 --- Proposed studies --- p.72 / Chapter 5.3.1 --- Other apoptotic targets that may leave persistent effects --- p.72 / Chapter 5.3.2 --- Post- caspase activation regulation of apoptosis --- p.74 / Chapter 5.3.3 --- Identifying correlation between reversal of apoptosis and cancer --- p.74 / Chapter 5.3.4 --- Single cell methods and cell tracking system for further studies --- p.75 / Chapter 5.3.5 --- Notes on studying reversal of apoptosis in relation to phagocytosis --- p.76 / References --- p.77

Yau, Pak Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-167). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / List of abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Table of content --- p.xii / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1. --- Introduction / Chapter 1.1.1. --- Tumor hypoxia --- p.2 / Chapter 1.1.2. --- Non-coding RNA --- p.6 / Chapter 1.1.3. --- Long non-coding RNAs: regulation and related diseases --- p.7 / Chapter 1.1.3.1. --- aHIF and cancer --- p.11 / Chapter 1.1.4. --- Objective --- p.11 / Chapter Chapter Two: --- Regulation of HIF-lα by aHIF --- p.13 / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- aHIF: a natural antisense long non-coding RNAs --- p.14 / Chapter 2.1.2. --- The relationship between aHIF and HIF-lα --- p.15 / Chapter 2.1.3. --- HIF-lα regulation --- p.19 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Cell culture --- p.22 / Chapter 2.2.2. --- Western blot analysis --- p.22 / Chapter 2.2.3. --- RNA isolation and reverse transcription --- p.23 / Chapter 2.2.4. --- Quantitative Real-time PCR --- p.23 / Chapter 2.2.5. --- Plasmids construction --- p.24 / Chapter 2.2.5.1. --- aHIF over-expression clone --- p.24 / Chapter 2.2.5.2. --- Luciferase reporter with HIF-lα 3'UTR --- p.25 / Chapter 2.2.5.3. --- HIF-lα and PTB over-expression vector --- p.25 / Chapter 2.2.5.4. --- PTB knock-down vector --- p.30 / Chapter 2.2.6. --- Stable Clone --- p.30 / Chapter 2.2.7. --- Transfection --- p.31 / Chapter 2.2.8. --- Luciferase reporter assay --- p.31 / Chapter 2.2.9. --- Statistical analysis --- p.32 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Effect of aHIF (FL) on HIF-lα expression --- p.33 / Chapter 2.3.2. --- Effect of aHIF (FL) on HIF-lα 3，UTR --- p.33 / Chapter 2.3.3. --- Effects of aHIF (OL) and aHIF (NOL) on HIF-lα level --- p.37 / Chapter 2.3.4. --- Effects of aHIF (NOL) and aHIF (OL) on HIF-lα 3，UTR --- p.39 / Chapter 2.3.5. --- Effect of aHIF (FL) on HIF-lα 3' UTR in PTBi cells --- p.41 / Chapter 2.3.6. --- Effect of aHIF (NOL) on HIF-lα 3，UTR in PTBi cells --- p.43 / Chapter 2.3.7. --- Effect of aHIF (OL) on HIF-lα 3' UTR in PTBi cells --- p.45 / Chapter 2.4 --- Discussion / Chapter 2.4.1. --- aHIF regulates HIF-la through HIF-la 3' UTR (FL) --- p.47 / Chapter 2.4.2. --- Factors involved in aHIF- HIF-lα interaction --- p.53 / Chapter Chapter Three: --- aHIF regulates drug sensitivity through BNIP3 --- p.58 / Chapter 3.1 --- Introduction / Chapter 3.1.1. --- aHIF and drug sensitivity --- p.59 / Chapter 3.1.2. --- BNIP3: its regulation and functions --- p.61 / Chapter 3.1.3. --- Taxol and its action mechanism --- p.67 / Chapter 3.1.4. --- Objective --- p.69 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Cell culture --- p.70 / Chapter 3.2.2. --- Cell viability assay --- p.70 / Chapter 3.2.3. --- Western blot analysis --- p.70 / Chapter 3.2.4. --- Plasmid construction --- p.71 / Chapter 3.2.5. --- Transfection --- p.71 / Chapter 3.2.6. --- Stable clone formation --- p.71 / Chapter 3.2.7. --- Quantitative real-time PCR --- p.71 / Chapter 3.2.8. --- Annexin V binding assay --- p.72 / Chapter 3.2.9. --- DNA fragmentation assay --- p.72 / Chapter 3.2.10. --- Detection of mitochondrial membrane potential by flow cytometry --- p.73 / Chapter 3.2.11. --- Cytochrome c and AIF translocation assay --- p.73 / Chapter 3.2.12. --- Statistical analysis --- p.74 / Chapter 3.3 --- Results / Chapter 3.3.1. --- Effect of aHIF on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.2. --- Effect of HIF-lαi on Taxol and vincristine sensitivity in HepG2 cells --- p.75 / Chapter 3.3.3. --- Effect of aHIF on Taxol-induced apoptosis --- p.78 / Chapter 3.3.4. --- HIF-1α regulation of BNIP3 expression --- p.78 / Chapter 3.3.5. --- Effect of aHIF on BNIP3 expression --- p.81 / Chapter 3.3.6. --- BNIP3 expression in BNIP3i stable transfectant --- p.81 / Chapter 3.3.7. --- The response of BNIP3i cells towards Taxol and vincrisinte --- p.84 / Chapter 3.3.8. --- Effect of BNIP3 on Taxol and vincristine sensitivity in BNIP3i cells --- p.84 / Chapter 3.3.9. --- Taxol- or vincristine- induced apoptosis in BNIP3i cells --- p.87 / Chapter 3.3.10. --- "Effects of aHIF, HIF-lα and BNIP3 on Taxol-induced apoptosis in HepG2 cells" --- p.89 / Chapter 3.3.11. --- Caspases activation in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.12. --- Mitochondrial membrane depolarization in Taxol - or vincristine - induced apoptosis in BNIP3i cells --- p.91 / Chapter 3.3.13. --- AIF and cytochrome c expressions in BNIP3i cells --- p.92 / Chapter 3.3.14. --- Effect of aHIF on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.15. --- Effect of HIF-lα on other chemo- and radio-therapeutics in HepG2 cells --- p.96 / Chapter 3.3.16. --- BNIP3i cells became more sensitivity to a number of drugs --- p.99 / Chapter 3.3.17. --- BNIP3i became more resistance to some drugs --- p.99 / Chapter 3.4 --- Discussion / Chapter 3.4.1. --- aHIF affected Taxol sensitivity through BNIP3 --- p.102 / Chapter 3.4.2. --- Mechanism of BNIP3 regulated Taxol or vincristine induced apoptosis --- p.106 / Chapter 3.4.3. --- Possible roles of BNIP3 in response to other therapeutics --- p.110 / Chapter Chapter Four: --- aHIF regulation of tumorigenesis --- p.116 / Chapter 4.1 --- Introduction / Chapter 4.1.1. --- aHIF in cancer biology --- p.117 / Chapter 4.1.2. --- Ras proteins --- p.118 / Chapter 4.1.3. --- K-Ras and cancers --- p.121 / Chapter 4.1.4. --- Regulation of Ras --- p.122 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1. --- Cell culture --- p.124 / Chapter 4.2.2. --- Western blot analysis --- p.124 / Chapter 4.2.3. --- Plasmids construction --- p.124 / Chapter 4.2.4. --- Transfection --- p.124 / Chapter 4.2.5. --- Cell growth assay --- p.124 / Chapter 4.2.6. --- Soft agar assay --- p.125 / Chapter 4.2.6. --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1. --- Effect of aHIF and HIF-lα on cell proliferation --- p.127 / Chapter 4.3.2. --- Effect of aHIF and HIF-lα on anchorage-independent growth --- p.127 / Chapter 4.3.3. --- Effect of aHIF and HIF -lα on K-Ras expression --- p.130 / Chapter 4.3.4. --- Effect of FTS on cell transfected with aHIF or HIF-lα --- p.130 / Chapter 4.4 --- Discussion / Chapter 4.4.1. --- Role of aHIF in tumorigenesis --- p.133 / Chapter 4.4.2. --- Proposed pathways of aHIF-regulated tumorigenesis --- p.136 / Chapter Chapter Five: --- General discussion and conclusion --- p.140 / Chapter 5.1 --- General discussion --- p.141 / Chapter 5.2 --- Conclusion --- p.146 / Chapter 5.3 --- Future perspectives --- p.147 / Chapter 5.3.1 --- Role ofPTB in aHIF-HIF-lα interaction --- p.147 / Chapter 5.3.2 --- Effect of aHIF (OL) on HIF-lα mRNA 3' UTR --- p.147 / Chapter 5.3.3 --- Effect ofaHIF on AIF --- p.148 / Chapter 5.3.4 --- Confirmation of the involvement of K-Ras --- p.148 / Chapter Chapter Six --- References --- p.150 / Chapter 6.1 --- References --- p.151

No description available.

Endothelial cells (EC) exposed to oscillatory shear stress (OS) experience oxidative stress as a signature of atherosclerosis. Conversely, unidirectional laminar shear stress (LS) reduces reactive oxygen species (ROS) levels and inflammatory responses. Peroxiredoxins (PRX) are antioxidant enzymes that reduce hydrogen peroxide, but have yet to be investigated in response to shear stress. We hypothesized that LS, compared to OS, promotes increased expression of PRX, which in turn influences the balance of ROS in EC. In this study, we identified all six PRX family members in bovine aortic endothelial cells (BAEC). Furthermore, we revealed that PRX are regulated by shear stress in EC. When compared to OS and static culture (ST), exposure to chronic LS upregulated PRX1 levels intracellularly. LS also upregulated PRX5 relative to ST, but not OS. In addition, PRX exhibited broad subcellular localization in BAEC, but these patterns did not change in response to shear stress. To establish the functional importance of PRX1 in shear stress-dependent redox balance, we next examined the role of PRX1 in LS-mediated hydrogen peroxide regulation. Here, Amplex Red assay was used to measure ROS levels in BAEC. Depletion of PRX1 using siRNA resulted in significantly higher ROS levels following LS, OS, and ST, while PRX5 depletion did not. These findings indicated that chronic exposure to LS upregulates PRX1 expression to keep ROS levels low in EC. To identify the pathway by which atheroprotective LS stimulates PRX1 protein production, we also undertook gene expression studies. We discovered that LS upregulates Prdx1 gene in a time-dependent manner compared to OS or ST. However, this increase in expression was not due to stabilization of Prdx1 mRNA. In addition, Prdx1 promoter analysis revealed a Nrf2 transcription factor binding site 160bp upstream of the gene. Nrf2 overexpression promoted basal PRX1 protein production, while Nrf2 depletion reduced Prdx1 mRNA following exposure to LS. Collectively, our work illustrated that LS affects PRX1 by inducing the Prdx1 gene, in part via the transcription factor Nrf2. Moreover, this discovery of PRX1 as a mechanosensitive antioxidant may contribute important insights into endothelial cell biology and provide a novel therapeutic target for vascular diseases.