Comparative studies on the quaternary structure of ferritin

There remain three possibilities for the general quaternary structure of apoferritin; that the molecule consists simply of 24 identical subunits, that it consists of unspecified numbers of two or three subunits of which one or two have a molecular weight substantially less than the 18,500 proposed in the unitary hypothesis, and that it consists of varying proportions of two subunits of similar but not identical mass, the total probably 24. The second of these hypotheses admittedly carries less weight since the recent retraction by Munro's laboratory of some of its earlier findings (Zahringer et at., 1977; Linder et at., 1974), but that is still a highly controversial area.The whole subject not only has inherent biological interest but has practical importance for the study of iron metabolism and also in connection with possible differences in antigenic specificity in the radioimmunoassay of serum ferritin. The work described in this thesis was undertaken in the hope of obtaining information which might help to reconcile what appear to be inconsistencies in the work discussed in the precceding pages. It was planned:1. To study apoferritin prepared from three different ferritins: horse spleen, because it is the subject of most published structural investigation; rat liver, because it is the ferritin most commonly studied in model metabolic experiments; and human spleen, because of the growing interest in serum ferritin levels in various disease conditions.2. To prepare ferritin by three different methods in order to test the hypothesis that the multiple bands produced by SDS electrophoresis in most laboratories are an artefact of one or more stages in the isolation.3. To dissociate apoferritin from each of the three sources and analyse the products by methods as far as possible identical witn those used by the original workers:(i) dissociation in 67% acetic acid, 7 mol/£ guanidine hydrochloride and SDS(ii) analysis by gel filtration on sephadex G-100 in the appropriate buffer system, and by SDS electrophoresis and acetic acid urea electrophoresis.4. To perform cross-check experiments in which the products of one type of dissociation or analysis will be subjected to a different analytical procedure. This would involve the following experiments :(i) subjecting the products obtained from dissociation in guanidine hydrochloride to acetic acid urea electrophoresis and SDS electrophoresis.(ii) subjecting the chromatographically separated acetic acid dissociated products to acetic acid urea electrophoresis(iii) subjecting the acetic acid urea electrophoretic products to SDS electrophoresisA brief word at this point may be helpful to the reader in understanding the further layout of this thesis. Chapter three will describe the tissue source, purification, and to an extent the characterisation of the material to be studied. The following two chapters will then deal with the dissociation, chromatographic separation (Chapter 4), and electrophoretic analysis (Chapter 5), while Chapter 6 will cover the cross-check experiments. The author has decided to abandon the usual pattern which involves collecting and segregating all methods in a special chapter. Instead the methods dealing with specific analyses will be described in the relevant chapters. It is felt that the layout adopted makes the thesis more readable and the proximity of the methods to the relevant results facilitates the understanding some rather complicated experiments

Improvements to the alignment process in electron-beam lithography

Electron beam lithography is capable of defining structures with sub-10 nm linewidths. To exploit this capability to produce working devices with structures defined in multiple 'lithographic steps' a process of alignment must be used. The conventional method of scanning the electron beam across simple geometrically shaped markers will be shown inherently to limit the alignment accuracy attainable. Improvements to alignment allow precise placement of elements in complex multi-level devices and may be used to realise structures which are significantly smaller than the single exposure resist limit. Correlation based alignment has been used previously as an alignment technique, providing improvements to the attainable accuracy and noise immunity of alignment. It is well known that the marker pattern used in correlation based alignment has a strong influence on the magnitude of the improvements that can be realised. There has, to date, however, been no analytical study of how the design of marker pattern affects the correlation process and hence the alignment accuracy possible. This thesis analyses the correlation process to identify the features of marker patterns that are advantageous for correlation based alignment. Several classes of patterns have been investigated, with a range of metrics used to determine the suitability and performance of each type of pattern. Penrose tilings were selected on this basis as the most appropriate pattern type for use as markers in correlation based alignment. A process for performing correlation based alignment has been implemented on a commercial electron beam lithography tool and the improvements to the alignment accuracy have been demonstrated. A method of measuring alignment accuracy at the nanometer scale, based on the Fourier analysis of inter-digitated grating has been introduced. The improvements in alignment accuracy realised have been used to facilitate the fabrication of 'nanogap' and 'nanowire' devices - structures which have application in the fields of molecular electronics and quantum conduction. Fabrication procedures for such devices are demonstrated and electrical measurements of such structures presented to show that it is a feasible method of fabrication which offers much greater flexibility than the existing methods for creating these devices

Alignment verification for electron beam lithography

Alignment between lithography layers is essential for device fabrication. A minor defect in a single marker can lead to incorrect alignment and this can be the source of wafer reworks. In this paper we show that this can be prevented by using extra alignment markers to check the alignment during patterning, rather than inspecting vernier patterns after the exposure is completed. Accurate vernier patterns can often only be read after pattern transfer has been carried out. We also show that by using a Penrose tile as a marker it is possible to locate the marker to about 1 nm without fully exposing the resist. This means that the marker can be reused with full accuracy, thus improving the layer to layer alignment accuracy. Lithography tool noise limits the process

Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

Open Access funded by British Heart Foundation Under a Creative Commons license Acknowledgments Our thanks go to Gioia Polidori Francisco for training and discussions, Kate Watt and Yvonne Turnbull for technical and laboratory managerial support, Kadri Oras and Laura Ferguson for experimental support, Po-Lin So and Bruce Conklin (Gladstone Institutes) for providing their unpublished protocols, and Yukio Nakamura for discussion. This research is supported by the British Heart Foundation (PG/12/75/29851) and the Institute of Medical Sciences. A.S.B. was supported by the British Heart Foundation (FS/12/37/29516).Peer reviewedPublisher PD

InGaN/GaN Laser Diodes with High Order Notched Gratings

We report on InGaN/GaN distributed feedback laser diodes with high order gratings emitting at a single wavelength around 428 nm. The 39th order notched gratings have the advantage of a simplified fabrication route with no need for overgrowth. The laser ridge and grating were formed by electron beam lithography followed by ICP etching. The as-cleaved lasers emitted in the pulsed regime with a peak single-mode output power of 15 mW. Optimization of the grating design should lead to higher power single wavelength operation

Suppression of Epithelial to Mesenchymal Transitioning (EMT) Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue towards Functional Insulin Producing β-Like Cells

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer

C/EBP␤ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression *

cAMP-dependent protein kinase induction of PPAR␥ coactivator-1␣ (PGC-1␣) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1␣ in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1␣ and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1␣ reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBP␤, but not C/EBP␣ and C/EBP␦, bound to the CRE on the PGC-1␣ promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBP␤ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBP␤ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBP␤ rescued cAMP-inducible PGC-1␣ and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBP␤ on the CRE in PGC-1␣ proximal promoter

Generation of Functional Beta-Like Cells from Human Exocrine Pancreas

<div><p>Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin⁺ cells. The resultant β-like cells expressed insulin protein levels at ~15–30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.</p></div

Pancreatic Transcription Factors Containing Protein Transduction Domains Drive Mouse Embryonic Stem Cells towards Endocrine Pancreas

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm. Cells were subsequently plated as a monolayer and treated with different combinations of the modified recombinant transcription factors Pdx1 and MafA. The results demonstrate that each transcription factor was efficiently taken up by the cells, where they were localized in the nuclei. RT-qPCR was used to measure the expression levels of pancreatic markers. After the addition of Pdx1 alone for a period of five days, followed by the combination of Pdx1 and TAT-MafA in a second phase, up-regulation of insulin 1, insulin 2, Pdx1, Glut2, Pax4 and Nkx6.1 was observed. As assessed by immunocytochemistry, double positive insulin and Pdx1 cells were detected in the differentiated cultures. Although the pattern of pancreatic markers expression in these cultures was comparable to that of a mouse transformed β-cell line (MIN-6) and human islets, the expression levels of insulin observed in the differentiated ES cell cultures were several orders of magnitude lower. This suggests that, although PTD-TFs may prove useful in studying the role of exogenous TFs in the differentiation of ES cells towards islets and other pancreatic lineages, the amount of insulin generated is well below that required for therapeutically useful cells

Distributed feedback InGaN/GaN laser diodes

We have realised InGaN/GaN distributed feedback laser diodes emitting at a single wavelength in the 42X nm wavelength range. Laser diodes based on Gallium Nitride (GaN) are useful devices in a wide range of applications including atomic spectroscopy, data storage and optical communications. To fully exploit some of these application areas there is a need for a GaN laser diode with high spectral purity, e.g. in atomic clocks, where a narrow line width blue laser source can be used to target the atomic cooling transition. Previously, GaN DFB lasers have been realised using buried or surface gratings. Buried gratings require complex overgrowth steps which can introduce epi-defects. Surface gratings designs, can compromise the quality of the p-type contact due to dry etch damage and are prone to increased optical losses in the grating regions. In our approach the grating is etched into the sidewall of the ridge. Advantages include a simpler fabrication route and design freedom over the grating coupling strength.Our intended application for these devices is cooling of the Sr+ ion and for this objective the laser characteristics of SMSR, linewidth, and power are critical. We investigate how these characteristics are affected by adjusting laser design parameters such as grating coupling coefficient and cavity length