Transfer printing of AlGaInAs/InP etched facet lasers to Si substrates

InP-etched facet ridge lasers emitting in the optical C-band are heterogeneously integrated on Si substrates by microtransfer printing for the first time. 500 μm × 60 μm laser coupons are fabricated with a highly dense pitch on the native InP substrate. The laser epitaxial structure contains a 1-μm-thick InGaAs sacrificial layer. A resist anchoring system is used to restrain the devices while they are released by selectively etching the InGaAs layer with FeCl3:H2O (1:2) at 8 °C. Efficient thermal sinking is achieved by evaporating Ti-Au on the Si target substrate and annealing the printed devices at 300 °C. This integration strategy is particularly relevant for lasers being butt coupled to polymer or silicon-on-insulator (SOI) waveguides

Separatism in Brittany

The introduction to the thesis attempts to place the separatist movement in Brittany into perspective as one of the various separatist movements with in France, It contains speculation on some possible reasons for the growth of separatist feeling, and defines terms that are frequently used in the thesis. Chapter One gives an account of Breton history, tracing Brittany's evolution as an independent state, its absorption by France, the disappearance of its remaining traces of independence, and the last spasms of action to regain this independence after having become merely part of a centralised state. Chapter Two examines the beginnings and development of a renewed effort to regain some measure of independence, and covers in some detail the period from the early nineteenth century to the end of the Second World War, known in Brittany as that of the first and second emsavs. To clarify a complicated period of development, a lexicon, a list of parties, groupings and devices of the Breton movement, and two flow charts summarising the movement's development from 1914 to I939 are given at the beginning of this chapter. Chapter Three deals with the period from I945 to the present day, known in Brittany as that of the third emsav, and examines in some detail the present state of the Breton movement. Chapter Four examines the work of various Breton writers who have played some part in expressing or shaping the Bretons' awareness of their separate identity, and shows to what extent their writings reflect the historical and political development of Brittany, Chapter Five contains the writer's conclusions and one detailed examination of Breton attitudes to the Breton movement, which helps to put it into its overall Breton perspective. The most important of the appendices to the thesis is the latest available detailed breakdown of the movement

Comparison of InGaAs and InAlAs sacrificial layers for release of InP-based devices

Heterogeneous integration of InP devices to Si substrates by adhesive-less micro transfer printing requires flat surfaces for optimum attachment and thermal sinking. InGaAs and InAlAs sacrificial layers are compared for the selective undercut of InP coupons by FeCl3:H2O (1:2). InAlAs offers isotropic etches and superior selectivity (> 4,000) to InP when compared with InGaAs. A 500 nm thick InAlAs sacrificial layer allows the release of wide coupons with a surface roughness < 2 nm and a flatness < 20 nm. The InAlAs release technology is applied to the transfer printing of a pre-fabricated InP laser to a Si substrate

NF-κB regulates mica gene transcription in endothelial cell through a genetically inhibitable control site

Endothelial cells form a barrier between blood and the underlying vessel wall, which characteristically demonstrates inflammatory damage in atherosclerotic disease. MICA is a highly polymorphic ligand for the activating immune receptor NKG2D and can be expressed on endothelial cells. We hypothesized that damaged vessel walls, such as those involved in atherosclerosis, might express MICA, which could contribute to the vascular immunopathology. Immune activation resulting from MICA expression could play a significant role in the development of vascular damage. We have demonstrated that TNFα up-regulates MICA on human endothelial cells. The up-regulation is mediated by NF-κB, and we have defined the regulatory control site responsible for this at −130 bp upstream of the MICA transcription start site. This site overlaps with a heat shock response element and integrates input from the two pathways. We have shown that in atherosclerotic lesions there is expression of MICA on endothelial cells. Using lentivirus-mediated gene delivery in primary human endothelial cells, we were able to inhibit the MICA response to TNFα with a truncated HSF1 that lacked a transactivation domain. This highlights the potential for transcription-based therapeutic approaches in atherosclerotic vascular disease to reduce immune-mediated endothelial and vessel wall damage

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus‐associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signalling on NK cells, CD8+ T cells and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D‐dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1