Cellular factors that interact with the negative regulatory element of the 5'-long terminal repeat of human immunodeficiency virus type 1

Transcriptional regulation of HlV-1 gene expression has been shown to be regulated by a combination of viral and cellular proteins which bind to regulatory elements in the viral 5' long terminal repeat (5'LTR) Functionally the LTR can be divided into three regulatory regions: the TAR region, extending from nucleotides 1 to 60 relative to the start site of transcription, contains sequences with which the viral trans-activator Tat interacts. The adjacent region from nucleotides -1 to -78 contains the core promoter with elements crucial for both basal and Tat-induced expression. The third region, extending from -79 to -454, contains numerous elements with which a variety of cellular factors may interact, resulting in either positive or negative modulation of LTR-driven transcription. The work contained within this thesis describes the discovery and delineation of two new transcription factor binding sites, designated as site A and site B, within the 5'-LTR of HIV-1. The majority of the work focused on site B itself, involving the characterisation of cellular proteins that specifically interacted with the nucleotide sequences in this site. Site B was found to contain a palindromic sequence TGACC involved in protein-DNA contact separated by a 9 base-pair spacer sequence that was not important for protein binding. This palindrome resembles the consensus binding site for members of the nuclear hormone receptor super-family of transcription factors. Although several members of this super-family of transcription factors were shown to interact in vitro with site B, the predominant protein present in T-lymphocyte nuclear extracts did not correspond to any of those previously characterised. The T-cell protein was shown to have a relative molecular size of 100-110 kD for the monomeric polypeptide and bound to site B as a dimer. Maximal binding to site B required both halves of the palindrome. Functionally site B was shown to act as a repressor element of both basal transcription and of transcription activated by phorbol ester in T- lymphocytes. Site B was also shown to function as a retinoic acid response element (RARE) in a heterologous promoter. The ability to function as either a positive or negative regulatory element is a recognised characteristic of nuclear hormone response elements and in part is a function of the relative abundance of factors able to interact with the site or to form complexes with one another. The overall effect of site B upon LTR-directed transcription may similarly depend upon the complex interaction of multiple factors which themselves depend on the cell type, cell activation state and degree of differentiation

Plausibility of Image Reconstruction Using a Proposed Flexible and Portable CT Scanner

The very hot and power-hungry x-ray filaments in today's computed tomography (CT) scanners constrain their design to be big and stationary. What if we built a CT scanner that could be deployed at the scene of a car accident to acquire tomographic images before moving the victim? Recent developments in nanotechnology have shown that carbon nanotubes can produce x-rays at room temperature, and with relatively low power needs. We propose a design for a portable and flexible CT scanner made up of an addressable array of tiny x-ray emitters and detectors. In this paper, we outline a basic design, propose a strategy for reconstruction, and demonstrate the feasibility of reconstruction using experiments on a software simulation of the flexible scanner. These simulations show that reconstruction quality is stable over a wide range of scanner geometries, while progressively larger errors in the scanner geometry induce progressively larger errors. We also raise a number of issues that still need to be overcome to build such a scanner.This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation, and the Ontario Innovation Trust

Identification of Genes Whose Expression Overlaps Age Boundaries and Correlates with Risk Groups in Paediatric and Adult Acute Myeloid Leukaemia

Few studies have compared gene expression in paediatric and adult acute myeloid leukaemia (AML). In this study, we have analysed mRNA-sequencing data from two publicly accessible databases: (1) National Cancer Institute's Therapeutically Applicable Research to Generate Effective Treatments (NCI-TARGET), examining paediatric patients, and (2) The Cancer Genome Atlas (TCGA), examining adult patients with AML. With a particular focus on 144 known tumour antigens, we identified STEAP1, SAGE1, MORC4, SLC34A2 and CEACAM3 as significantly different in their expression between standard and low risk paediatric AML patient subgroups, as well as between poor and good, and intermediate and good risk adult AML patient subgroups. We found significant differences in event-free survival (EFS) in paediatric AML patients, when comparing standard and low risk subgroups, and quartile expression levels of BIRC5, MAGEF1, MELTF, STEAP1 and VGLL4. We found significant differences in EFS in adult AML patients when comparing intermediate and good, and poor and good risk adult AML patient subgroups and quartile expression levels of MORC4 and SAGE1, respectively. When examining Kyoto Encyclopedia of Genes and Genomes (KEGG) (2016) pathway data, we found that genes altered in AML were involved in key processes such as the evasion of apoptosis (BIRC5, WNT1) or the control of cell proliferation (SSX2IP, AML1-ETO). For the first time we have compared gene expression in paediatric AML patients with that of adult AML patients. This study provides unique insights into the differences and similarities in the gene expression that underlies AML, the genes that are significantly differently expressed between risk subgroups, and provides new insights into the molecular pathways involved in AML pathogenesis

Serum profiling identifies ibrutinib as a treatment option for young adults with B‐cell acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is a haematological malignancy that is characterized by the uncontrolled proliferation of immature lymphocytes. 80% of cases occur in children where ALL is well understood and treated. However it has a devastating affects on adults, where multi-agent chemotherapy is the standard of care with allogeneic stem cell transplantation for those who are eligible. New treatments are required to extend remission and prevent relapse to improve patient survival rates. We used serum profiling to compare samples from presentation adult B-ALL patients with age- and sex-matched healthy volunteer (HV) sera and identified 69 differentially recognised antigens (P ≤ 0·02). BMX, DCTPP1 and VGLL4 showed no differences in transcription between patients and healthy donors but were each found to be present at higher levels in B-ALL patient samples than HVs by ICC. BMX plays a crucial role in the Bruton's Tyrosine Kinase (BTK) pathway which is bound by the BTK inhibitor, ibrutinib, suggesting adult B-ALL would also be a worthy target patient group for future clinical trials. We have shown the utility of proto-array analysis of B-ALL patient sera, predominantly from young adults, to help characterise the B-ALL immunome and identified a new target patient population for existing small molecule therapy

Event-based Asynchronous Sparse Convolutional Networks

Event cameras are bio-inspired sensors that respond to per-pixel brightness changes in the form of asynchronous and sparse "events". Recently, pattern recognition algorithms, such as learning-based methods, have made significant progress with event cameras by converting events into synchronous dense, image-like representations and applying traditional machine learning methods developed for standard cameras. However, these approaches discard the spatial and temporal sparsity inherent in event data at the cost of higher computational complexity and latency. In this work, we present a general framework for converting models trained on synchronous image-like event representations into asynchronous models with identical output, thus directly leveraging the intrinsic asynchronous and sparse nature of the event data. We show both theoretically and experimentally that this drastically reduces the computational complexity and latency of high-capacity, synchronous neural networks without sacrificing accuracy. In addition, our framework has several desirable characteristics: (i) it exploits spatio-temporal sparsity of events explicitly, (ii) it is agnostic to the event representation, network architecture, and task, and (iii) it does not require any train-time change, since it is compatible with the standard neural networks' training process. We thoroughly validate the proposed framework on two computer vision tasks: object detection and object recognition. In these tasks, we reduce the computational complexity up to 20 times with respect to high-latency neural networks. At the same time, we outperform state-of-the-art asynchronous approaches up to 24% in prediction accuracy

Antigenic targets for the immunotherapy of acute myeloid leukaemia

One of the most promising approaches to preventing relapse is the stimulation of the body’s own immune system to kill residual cancer cells after conventional therapy has destroyed the bulk of the tumour. In acute myeloid leukaemia (AML), the high frequency with which patients achieve first remission, and the diffuse nature of the disease throughout the periphery, makes immunotherapy particularly appealing following induction and consolidation therapy, using chemotherapy, and where possible stem cell transplantation. Immunotherapy could be used to remove residual disease, including leukaemic stem cells from the farthest recesses of the body, reducing, if not eliminating, the prospect of relapse. The identification of novel antigens that exist at disease presentation and can act as targets for immunotherapy have also proved useful in helping us to gain a better understand of the biology that belies AML. It appears that there is an additional function of leukaemia associated antigens as biomarkers of disease state and survival. Here, we discuss these findings

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca2+-handling. We investigated the effects of ascending aortic banding (AoB) on Ca2+-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca2+-release at the cell edge whereas Ca2+ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na+/Ca2+ exchanger, SR Ca2+ ATPase or phospholamban. Mathematical modeling indicated that [Ca2+]i transients at the cell center were accounted for by simple centripetal diffusion of Ca2+ released at the cell edge. In contrast, caffeine (10 mM) induced Ca2+ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca2+-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca2+ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca2+-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca2+-release

Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients

Ion induced solid flow

Amorphous solids can flow over very long periods of time. Solid flow can also be artificially enhanced by creating defects, as by Ion Beam Sputtering (IBS) in which collimated ions with energies in the 0.1 to 10 keV range impact a solid target, eroding its surface and inducing formation of nanometric structures. Recent experiments have challenged knowledge accumulated during the last two decades so that a basic understanding of self-organized nano-pattern formation under IBS is still lacking. We show that considering the irradiated solid to flow like a highly viscous liquids can account for the complex IBS morphological phase diagram, relegating erosion to a subsidiary role and demonstrating a controllable instance of solid flow at the nanoscale. This new perspective can allow for a full harnessing of this bottom-up route to nanostructuring.Comment: 17 pages, 5 figure