Protein engineering strategy for the stabilization of HIV-1 α-helical peptides

2019 Spring.Includes bibliographical references.Many disease-relevant protein-protein interactions (PPIs) contain an alpha helix and helical binding cleft at their interface. Disruption of these interactions with helical peptide mimics is a validated therapeutic strategy. However, short peptides typically do not fold into stable helices, which significantly lowers their in vivo stability. Researches have reported methods for helical peptide stabilization but, these approaches rely on laborious, and often expensive, chemical synthesis and purification. The research I have preformed aims to stabilize disease-relevant helices through protein engineering. In contrast to chemically constrained helical peptides, a protein can be expressed in a cellular system on a much larger scale. Recently, we reported a new strategy termed "helix-grafted display" that overcomes the traditional hurdles of helical mimics and applied it to the challenge of suppressing HIV entry. Our helix grafted proteins, potently inhibits formation of the extracellular PPI involving C-peptide helix, and HIV gp41 N-peptide trimer, as tested in HIV CD4+ cells. Further optimization of the helical sequence by yeast display yielded new proteins that suppress HIV-1 entry and express substantially better in E. coli. Furthermore, fusion proteins designed to improve the serum stability of these helix grafted proteins have been made that potently suppress HIV-1 entry. Collectively, I report a potential cocktail of evolved HIV-1 entry inhibitors that are functional against an Enfuvirtide-resistant strain and are designed for serum stabilities that rival current monoclonal antibody drugs

Using pulsed mode scanning electron microscopy for cathodoluminescence studies on hybrid perovskite films

Abstract: The use of pulsed mode scanning electron microscopy cathodoluminescence (CL) for both hyperspectral mapping and time-resolved measurements is found to be useful for the study of hybrid perovskite films, a class of ionic semiconductors that have been shown to be beam sensitive. A range of acquisition parameters is analysed, including beam current and beam mode (either continuous or pulsed operation), and their effect on the CL emission is discussed. Under optimized acquisition conditions, using a pulsed electron beam, the heterogeneity of the emission properties of hybrid perovskite films can be resolved via the acquisition of CL hyperspectral maps. These optimized parameters also enable the acquisition of time-resolved CL of polycrystalline films, showing significantly shorter lived charge carriers dynamics compared to the photoluminescence analogue, hinting at additional electron beam-specimen interactions to be further investigated. This work represents a promising step to investigate hybrid perovskite semiconductors at the nanoscale with CL

Optical emission from focused ion beam milled halide perovskite device cross‐sections

Funder: Jardine Foundation and Cambridge TrustFunder: Cambridge RoyceFunder: Dr. Christian Monachon from AttolightAbstract: Cross‐sectional transmission electron microscopy has been widely used to investigate organic–inorganic hybrid halide perovskite‐based optoelectronic devices. Electron‐transparent specimens (lamellae) used in such studies are often prepared using focused ion beam (FIB) milling. However, the gallium ions used in FIB milling may severely degrade the structure and composition of halide perovskites in the lamellae, potentially invalidating studies performed on them. In this work, the close relationship between perovskite structure and luminescence is exploited to examine the structural quality of perovskite solar cell lamellae prepared by FIB milling. Through hyperspectral cathodoluminescence (CL) mapping, the perovskite layer was found to remain optically active with a slightly blue‐shifted luminescence. This finding indicates that the perovskite structure is largely preserved upon the lamella fabrication process although some surface amorphisation occurred. Further changes in CL due to electron beam irradiation were also recorded, confirming that electron dose management is essential in electron microscopy studies of carefully prepared halide perovskite‐based device lamellae

Enabling planetary science across light-years. Ariel Definition Study Report

Ariel, the Atmospheric Remote-sensing Infrared Exoplanet Large-survey, was adopted as the fourth medium-class mission in ESA's Cosmic Vision programme to be launched in 2029. During its 4-year mission, Ariel will study what exoplanets are made of, how they formed and how they evolve, by surveying a diverse sample of about 1000 extrasolar planets, simultaneously in visible and infrared wavelengths. It is the first mission dedicated to measuring the chemical composition and thermal structures of hundreds of transiting exoplanets, enabling planetary science far beyond the boundaries of the Solar System. The payload consists of an off-axis Cassegrain telescope (primary mirror 1100 mm x 730 mm ellipse) and two separate instruments (FGS and AIRS) covering simultaneously 0.5-7.8 micron spectral range. The satellite is best placed into an L2 orbit to maximise the thermal stability and the field of regard. The payload module is passively cooled via a series of V-Groove radiators; the detectors for the AIRS are the only items that require active cooling via an active Ne JT cooler. The Ariel payload is developed by a consortium of more than 50 institutes from 16 ESA countries, which include the UK, France, Italy, Belgium, Poland, Spain, Austria, Denmark, Ireland, Portugal, Czech Republic, Hungary, the Netherlands, Sweden, Norway, Estonia, and a NASA contribution

PhD

dissertationChromosome translocations occur when DNA is transferred from one chromosome to another or is exchanged between chromosomes. The process is known to start with two nonhomologous, intact chromosomes and to end with two nonhomologous derivative chromosomes carrying DNA from the translocation partner. However, what happens in between is very poorly understood. There are a number of reasons for this. One is that chromosome translocations probably occur by a number of different mechanisms. Based on breakpoint sequences and epidemiological studies of diseases characterized by chromosome translocations, many hypotheses have been made as to how translocations actually arise. These include misrepair of double-strand breaks by homologous and nonhomologous recombinational repair pathways and errors of site-directed recombination pathways. The process of translocation is poorly understood also because there is no selection system in a model organism to detect the untargeted types of translocations present in human diseases. It has become apparent over the last 10 years that although various eukaryotes preferentially use different double-strand break repair systems, all eukaryotes are capable of repairing double-strand breaks in essentially the same ways. Therefore the development of a chromosome translocation selection system in a model organism would be of great benefit in investigating this process. This dissertation describes the development and characterization of a novel genetic selection system for chromosome translocations in the yeast, Saccharomyces cerevisiae. The system specifically selects translocations between a translocation YAC and any chromosome in the yeast genome. The translocation products are analyzed by pulsed-field gel electrophoresis, Southern blot hybridization, PCR, and breakpoint sequencing. Such analyses provide insights into how translocations are happening within yeast and provide directions to pursue in future assays. Production of a specific double-strand break in the YAC increases the translocation rate as would be expected if formation of a double-strand break is rate-limiting in translocation. The assay works in yeast strains that have been made deficient in homologous recombination by deletion of RAD52 and limitation of homology between the YAC and the yeast genome. Product analysis and rates also suggest differences between haploid and diploid strains. The current results suggest that it will be possible to use this assay in yeast to study chromosome translocation in ways relevant to the processes in mammalian systems

Using pulsed mode scanning electron microscopy for cathodoluminescence studies on hybrid perovskite films

Abstract: The use of pulsed mode scanning electron microscopy cathodoluminescence (CL) for both hyperspectral mapping and time-resolved measurements is found to be useful for the study of hybrid perovskite films, a class of ionic semiconductors that have been shown to be beam sensitive. A range of acquisition parameters is analysed, including beam current and beam mode (either continuous or pulsed operation), and their effect on the CL emission is discussed. Under optimized acquisition conditions, using a pulsed electron beam, the heterogeneity of the emission properties of hybrid perovskite films can be resolved via the acquisition of CL hyperspectral maps. These optimized parameters also enable the acquisition of time-resolved CL of polycrystalline films, showing significantly shorter lived charge carriers dynamics compared to the photoluminescence analogue, hinting at additional electron beam-specimen interactions to be further investigated. This work represents a promising step to investigate hybrid perovskite semiconductors at the nanoscale with CL

Using pulsed mode scanning electron microscopy for cathodoluminescence studies on hybrid perovskite films

Abstract: The use of pulsed mode scanning electron microscopy cathodoluminescence (CL) for both hyperspectral mapping and time-resolved measurements is found to be useful for the study of hybrid perovskite films, a class of ionic semiconductors that have been shown to be beam sensitive. A range of acquisition parameters is analysed, including beam current and beam mode (either continuous or pulsed operation), and their effect on the CL emission is discussed. Under optimized acquisition conditions, using a pulsed electron beam, the heterogeneity of the emission properties of hybrid perovskite films can be resolved via the acquisition of CL hyperspectral maps. These optimized parameters also enable the acquisition of time-resolved CL of polycrystalline films, showing significantly shorter lived charge carriers dynamics compared to the photoluminescence analogue, hinting at additional electron beam-specimen interactions to be further investigated. This work represents a promising step to investigate hybrid perovskite semiconductors at the nanoscale with CL

Research data supporting "Using pulsed mode scanning electron microscopy for cathodoluminescence studies on hybrid perovskite films"

The original data files of the CL maps are found in the data folder. Each map can be opened using Hyperspy in Python. The TRCL files are found in the data folder, inside the TRCL folder. It can be opened using Origin Pro. In that same folder, the time evolution of the CL spectrum is also found. In the si folder (inside the data folder), there are some spreadsheets for the metadata of each scan and the CASINO simulation. In the scripts folder, there is a list of Jupyter Notebooks which show all the processing of the CL maps, as well as the fitting and the plotting methods