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Characterisation of immortalised human foetal retinal progenitor cell linesHasan, Shazeen Mumtaz January 2008 (has links)
Inherited retinal dystrophies and age-related macular degeneration (AMD) are leading causes of blindness. Current treatments only slow disease progression in a minority of patients, therefore it is important to develop new treatments that can regenerate lost cells, restore retinal function and halt disease progression. Retinal progenitor cells have the potential to replace degenerating photoreceptors in retinal diseases. This thesis explores the viability of using immortalised human foetal retinal progenitor cells for this purpose, and aims to elucidate the trophic factors required, in vitro, to drive these cells towards retinal cell lineages. We also examine their potential to survive, integrate and differentiate in the diseased and developing rat retina. Two human foetal retinal progenitor cell lines were established by infecting retinal foetal tissue with the temperature-sensitive tsT-SV40 antigen. The immortalised progenitor cells were compared to primary human foetal retinal progenitor cultures, and both were shown to express similar markers of neural progenitor cells and early neuronal cell types. Several trophic factors were investigated, including serum, retinoic acid and conditioned medium, with respect to their ability to influence gene expression. Serum appeared to induce expression of ganglion cell markers, and conditioned medium stimulated cell proliferation. Cells were also grafted into neonatal hooded Lister rats and RCS dystrophic rats, and neither the primary nor the immortalised progenitor cells demonstrated integration into the retina. In the RCS rat, cells were engulfed by host macrophage/microglia and showed no signs of integration or expression of neuronal markers. Immortalised cells, in vivo, stained positively for Ki67 and low levels of nestin, but exhibited limited ability to differentiate or integrate. These data indicate that immortalised progenitor cells maintain many characterisics of unimmortalised progenitor cells, and suggest that immortalisation of retinal progenitors may have long term therapeutic possibilities.
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A study of hemolysis in pulsatile blood pumpsAlami, Mohamed Ali January 1975 (has links)
No description available.
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Structural and functional studies of Ly49B, a key immune receptor in myeloid cellsMickiewicz, Katarzyna Maria January 2013 (has links)
Murine Ly49 receptors are predominantly expressed on natural killer (NK) cells and play a key role in regulating immunological activity. The Ly49 family contains both inhibitory and activating receptors, which bind class I major histocompatibility complex (MHC I) or MHC-like molecules. Regulation of NK cell activity is the result of finely balanced signalling, which is delivered by the two receptor types upon ligand binding. Once activated, NK cells play an essential role in the elimination of cancerous and virus infected cells. Ly49B differs from other members of the Ly49 family in that it is expressed on the surface of myeloid, rather than NK cells. It shares only 50 % sequence identity with other Ly49s and contains an additional 20 amino acids at its carboxyl-terminus. Furthermore, there are significant variations between Ly49Bs from different mouse strains. These inter- and intra-molecular variations suggest that different allelic forms could possess distinct structural and functional properties when compared with one another and with other members of the Ly49 family. This thesis seeks to provide an in-depth characterisation of the biochemical properties of Ly49B in comparison to other Ly49 receptors. In this study a series of Ly49B mutants, created by site-directed mutagenesis, was used to identify residues critical for monoclonal anti-Ly49B antibody and ligand binding. Flow cytometric analysis of the mutants revealed that C57 Ly49B-specific residues, L166 and K167, are important for the integrity of the monoclonal 1A1 anti-Ly49B antibody epitope. The equivalent BALB/c Ly49B residues, W166 and N167, together with residue C251 were shown to play an essential role in MHC class I ligand binding. Immunoprecipitation and Western blotting were used to demonstrate that Ly49B exists in multiple molecular forms in transfected and native cells, each of which most likely represents the receptor at different stages of glycosylation. Similar results have never before been presented for any Ly49 receptor. Finally, a method for refolding and purification of the extracellular portion of Ly49B was developed, following expression in Escherichia coli.
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The role of HLA class I antibody in endothelial cell activation and allograft rejectionNaemi, Fatmah Mohammed A. January 2014 (has links)
Background: Antibody-mediated rejection is one of the major causes of acute and chronic rejection. This is mediated by endothelium microvascular inflammation and leukocyte migration to the graft. However, the role of donor specific HLA class I antibodies in inducing allograft rejection in the absence of complement is not fully understood. In this project, the mechanisms by which HLA class I antibodies induce endothelial cell-leukocyte interactions were examined. Methods: Human microvascular endothelial cells (HMEC-1) were stimulated with HLA class I antibody either mouse monoclonal (W6/32) or from sensitized kidney patients. The activation of cell signaling pathways was examined using Western blotting. The expression of adhesion molecules and chemokines were determined using flow cytometry and q-PCR, respectively. Monocyte adhesion and migration was examined using chemotaxis and flow based adhesion assays. Results: HMEC-1 cells stimulated with W6/32 antibody showed phosphorylation of a transcription factor CREB by a mechanism dependent on the protein kinase A pathway. W6/32 also induced significant expression of the adhesion molecules VCAM-1 and ICAM-1 (P<0.001) in a mechanism dependent on the PI3K/Akt pathway. Additionally, stimulated cells showed significant up-regulation in IL-6, CXCL8, CXCL1, CCL5 and CXCL10. The expression of CXCL8 was significantly reduced by knocking down CREB (p<0.001). Conditioned media from W6/32-treated cells was able to induce significant THP-1 monocyte migration compared to control (p<0.001). Furthermore, monocytes flowing at 0.5 dyne/cm2 significantly adhered to HMEC-1 cells-treated with F(ab)2-fragments of W6/32 (p<0.001). HLA class I alloantibodies from patients induced phosphorylation of CREB and a significant upregulation of VCAM-1, ICAM-1 and CXCL8. Monoclonal human HLA-B58 antibody was also able to induce CREB phosphorylation. Conclusion: Exposure of microvascular endothelial cells to HLA class I antibodies induce an activation of endothelial cell signaling that are responsible for upregulation of adhesion molecules and chemokines. These mediators enhance the interaction between donor endothelium and recipient leukocytes during cellular rejection. Strategies that block endothelium-leukocyte interaction might reduce the incidence of allograft rejection and improve allograft survival.
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The role of disease-linked genetic variation in the regulation of gene transcriptionSoderquest, Katrina January 2013 (has links)
Genome-Wide Association Studies have found genetic variation from across the genome to be associated with various diseases and other traits. In many cases, the exact mechanism by which a genetic variant increases or decreases the risk of a particular condition is poorly understood. Many efforts are now underway to combine data on disease-associated genetic variation with other genome-wide data to understand the way in which genetic variation can alter genomic regulation and affect disease risk. In this project we examine whether disease-associated genetic variation, in the form of single nucleotide polymorphisms, can be found in binding sites for the transcription factors T-bet in Th1 cells and GATA3 in Th1 or Th2 cells. We hypothesise that, in some cases, variation within binding sites for these transcription factors could alter transcription factor binding affinity and subsequent gene regulation. As ’master regulators’ of T helper cell lineage commitment, T-bet and GATA3 play a central role in the fate of a naïve T helper cell and the development of an immune response. We find several single nucleotide polymorphisms in our transcription factor binding sites, some of which are near other genomic features such as potential enhancer elements. Furthermore, we find an enrichment of immune related SNPs in T-bet and GATA3 binding sites compared to the total catalogue of Genome-Wide Association Study hits. We then develop a medium throughput assay which combines oligonucleotide pulldown and flow cytometry to test whether such SNPs alter transcription factor binding in vitro. We find three SNPs, rs1465321, rs11135484 and rs1006353 which alter binding of T-bet in vitro. Of these, rs1465321 is associated with Crohn’s disease, coeliac disease and ulcerative colitis and is in an intron for IL18R1. Therefore, we examine the role of T-bet in IL18R1 expression, IL-18 signalling and two mouse models of disease.
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Heritability and missing heritability : can twin studies be trusted?Trzaskowski, Maclej January 2013 (has links)
'Missing heritability' is the discrepancy between the amount of variance explained by specific single nucleotide polymorphisms (SNPs) identified in genome-wide association (GWA) and twin-estimated heritability. Four categories of explanations have been proposed for missing heritability: (1) additive effect sizes of common single nucleotide polymorphisms (SNPs) used in GWAS are too small to detect with current sample sizes; (2) rare variants are not captured by commercial arrays; (3) nonadditive effects (allelic, gene-gene or gene-environment interactions); (4) twin estimates of heritability are inflated. A recently developed quantitative method that uses GWA data- Genome-wide Complex Trait Analysis (GCTA)- has made it possible to explore these issues as it allows to compare quantitative twin-based estimates with quantitative DNA-based estimates. I use data from an on-going longitudinal study of 14,000 twins (7000 pairs) born in the UK between 1994 and 1996 called the Twins Early Development Study (TEDS) to investigate the following: the proportion of twin heritability that can be explained by additive effects of common SNPs (Chapters 2, 3 and 4); increasing heritability across development in the presence of strong genetic stability (Chapters 5 and 6); and genetic pleiotropy (Chapter 7). In Chapters 2, 3 and 4, Iapply univariate twin, GWA and GCTA methods to demonstrate that although we are still far from closing the gap between heritability and the actual genetic variants, there still is scope for discovery of common additive genetic effects. In Chapters 5, 6 and 7, I employ bivariate GCTA, polygenic predictor scores (PGS) and twin estimates from the same sample to confirm that twin estimates and DNA estimates of genetic pleiotropy and stability concur. In conclusion, in this thesis I provide evidence that much of the so-called 'missing heritability' can be explained by common additive genetic effects and that phenomena from twin research can be replicated using DNA alone.
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The preparation and characterisation of cells and their scaffolds suitable for tissue repair applicationsPittarello, Ledo January 2008 (has links)
In this research, a tissue engineering approach was investigated for the fabrication of homogeneous polymer/cells scaffolds suitable for applications in the clinic, in particular for the treatment of coronary disease and atherosclerosis. Sodium alginate chemically modified with an RGD-containing peptide promoting cell attachment was chosen as the natural polymer for the preparation of matrix/cells constructs. Several conditions were investigated to optimise the derivatisation of the biomaterial, and no major dissimilarities were identified among alginates with different guluronic/mannuronic acid compositions and viscosities during the derivatisation process. Successful incorporation of the GRGDY-pentapeptide onto the alginate was achieved. Amino acid analysis, which allowed a reliable quantification of the degree of peptide attached, demonstrated that the complete pentapeptidic sequence was present in the alginate after completion of the derivatisation procedure. However, a significant loss of the peptide's final tyrosine residue was consistently observed during derivatisation. Studies were also performed using multipotent fibroblast-like plastic-adherent cells recovered from frozen bone marrow samples of adult patients, here defined as human "mesenchymal stem cells" (hMSCs). Prior to the cell immobilisation in the alginate matrix, the optimal conditions for cell expansion in monolayer culture were identified. Cell samples were assessed according to their percent viability at each expansion passage, doubling time, cell growth rate and specific glucose consumption rate. Overall, the highest cell growth kinetics, highest cell viabilities and lowest doubling times were observed for the combined use of serum-/bFGF-supplemented low-glucose DMEM medium and non-coated culture vessels in a 21% dissolved oxygen tension atmosphere. hMSCs expanded for four subsequent passages under these monolayer conditions proved to retain their undifferentiated state and their ability to differentiate toward multiple phenotypes. Finally, alginate/cells beads were prepared and maintained in the optimal culture conditions identified in the monolayer studies. Constructs prepared with either hMSCs or human foreskin fibroblasts in either unmodified alginate or alginate coupled with the GRGDY-pentapeptide were analysed. After immobilisation, both cell types failed to adhere to the matrix and to acquire their characteristic elongated, spindle-shaped morphology. Lack of cell proliferation and.
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An investigation into the regulation of gene expression in response to ER stressZhao, Xuechan January 2014 (has links)
β-cell dysfunction is a major feature of the development of type 2 diabetes (T2D). Endoplasmic reticulum (ER) stress has been shown to play an important role in β-cell survival and death, and has been shown to be an important factior in the development of diabetes and many other diseases. The unfolded protein response (UPR) is a unique adaptive pathway which can improve cell survival during ER stress through the activation of three ER stress transducers: PERK, IRE1 and ATF6. However, if ER stress remains unresolved, UPR signalling triggers the apoptotic pathway through, for example, expression of the pro-apoptotic protein CHOP, which is a downstream target of ATF4. In addition, eukaryotic initiation factor 5 (eIF5) has been reported to play a role in the regulation of ATF4. Therefore, this thesis aimed at investigating the expression of eIF5, ATF4 (activating transcriptional factor 4) and CHOP (C/EBP homolog protein) in response to ER stress. The studies outlined in this thesis demonstrate that the URP is induced in response to thapsigargin-induced ER stress. In MIN6 cells, the up-regulation of ATF4 and CHOP in response to ER stress is independent of the PERK-eIF2α pathway. However, IRE1 activation is required for the up-regulation of ATF4 and CHOP in response to ER stress in a variety of cell lines. Surprisingly, IRE1-mediated ATF4 and CHOP expression is independent of translation and transcription. In addition, mRNA polyadenylation appears to be required for the up-regulation of ATF4 and CHOP. Preliminary evidence also suggests that inhibition of the IRE1 pathway results in inhibition of miR214 expression. This could lower the miRNA214-mediated up-regulation of ATF4.
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A role for giantin in primary ciliogenesis through control of the localisation of the cytoplasmic dynein-2 intermediate chainsAsante, David January 2014 (has links)
Properly assembled primary cilia play fundamental roles in the development and function of nearly all cells. The cilium is assembled by extension of a microtubule bundle, the axoneme from the mother centriole ensheathed by a specialised membrane, the ciliary membrane which is continuous with the plasma membrane. Intraflagellar transport (IFT) traffics ciliary components along the length of the nascent cilium and is required for the assembly, maintenance and function of the cilium. The microtubule motor, cytoplasmic dynein-2 mediates retrograde IFT from the axoneme tip to base and has been shown to be necessary for cilia assembly. Vital cilia machinery constituents localise to the centrosome. The Golgi is positioned adjacent to the centrosome in most cells and both organelles polarise towards the apical plasma membrane during ciliogenesis. Furthermore, some Golgi-localised proteins have been shown to execute cilia-related functions. Here, the transmembrane Golgi protein giantin (GOLG81) is shown to be required for cilia formation. Giantin is not required for the RABll-RABIN8-RAB8 pathway that is implicated in the early stages of ciliary membrane formation. Instead, giantin depletion caused mislocalisation of the intermediate chains of dynein-2, WDR34 and WDR60. Highly effective suppression of giantin, WDR34 or WDR60 in cells completely inhibited primary cilia formation; while, partial depletion of giantin, WDR34 or WDR60 permitted the assembly of cilia, albeit of abnormally increased lengths. These data implicate giantin in ciliogenesis through control of dynein-2 localisation. Unlike the cytoplasmic dynein-l complex, existing knowledge of cytoplasmic dynein-2 composition is limited. This work details the subunit composition of the human cytoplasmic dynein-2 motor complex. WDR34 and WDR60 are genuine dynein-2 intermediate chains and associate with the known heavy (DYNC2Hl) and light intermediate (LlC3) chains of dynein-2. Dynein-2 possesses all the' known dynein-l light chains in addition to exclusive possession of the light chain, TCTEX-2. The only dynein-l accessory factor found to be shared by dynein-2 was NUDCD3, whose depletion resulted in mislocalisation of WDR34, failure of cilia assembly and assembly of abnormally long cilia in partial knockdowns. No interactions were found between dynein-2 and dynactin, LlSl, NUDE/NUDEL or BICD2. NUDCD3, thus, presents a common avenue for regulating dynein-l and dynein-2 functions, indicating a functional interplay between the two motors. This novel biochemical characterisation of the human cytoplasmic dynein-2 complex could potentially advance research into 1FT and dynein-2 function in ciliopathies
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The role of epicardial adipose tissue adipokines in the genesis of postoperative atrial fibrillationViviano, Alessandro January 2015 (has links)
Objectives: We aim to explore the Epicardial Adipose Tissue (EAT) proteome to identify its role in the genesis of postoperative atrial fibrillation (AF). Methods: EAT samples were collected in 76 patients with no history of AF undergoing CABG. 50 were incubated in culture medium. Conditioned media representing EAT secretome were harvested and proteins extracted from the remaining tissues. Samples were analysed by two-dimensional difference in-gel electrophoresis (2D-DIGE) and high-performance liquid chromatographytandem mass spectrometry (HPLC-MSIMS). Gene expression analysis was performed on 24 samples (8 AF vs. 16 controls). Findings were validated by Western blotting. Results: 20 secretome samples (10 vs. 10) and 12 EAT extracts (6 vs. 6), divided according to development of postoperative AF, were analysed by proteomics. The proteomics analysis returned 35 differentially expressed protein spots in the secretome and 16 differentially expressed protein spots in the EAT extracts (p<O.05, fold change> 1.2). Gelsolin (GSN) was significantly reduced in AF (p=O.03). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) was upregulated in AF (p=O.04). SBP1 was increased in AF for the secretome and reduced in the EAT extracts. Catalase (CATA) was downregulated in AF. GSN was consistently found to be downregulated in the AF group by three independent techniques. Conclusions. Systemic inflammatory response to cardiac surgery and oxidative stress are known triggers for AF. GSN exerts antinflammatory properties by severing actin filaments. DDAH2 degrades nitric oxide synthase inhibitor ADMA (asymmetric dimethylarginine), known to increase oxidative stress.
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