Some Considerations on a Calculus with Weak References

Weak references are references that do not prevent the object they point to from being garbage collected. Most realistic languages, including Java, SML/NJ, and OCaml to name a few, have some facility for programming with weak references. Weak references are used in implementing idioms like memoizing functions and hash-consing in order to avoid potential memory leaks. However, the semantics of weak references in many languages are not clearly specified. Without a formal semantics for weak references it becomes impossible to prove the correctness of implementations making use of this feature. Previous work by Hallett and Kfoury extends λgc, a language for modeling garbage collection, to λweak, a similar language with weak references. Using this previously formalized semantics for weak references, we consider two issues related to well-behavedness of programs. Firstly, we provide a new, simpler proof of the well-behavedness of the syntactically restricted fragment of λweak defined previously. Secondly, we give a natural semantic criterion for well-behavedness much broader than the syntactic restriction, which is useful as principle for programming with weak references. Furthermore we extend the result, proved in previously of λgc, which allows one to use type-inference to collect some reachable objects that are never used. We prove that this result holds of our language, and we extend this result to allow the collection of weakly-referenced reachable garbage without incurring the computational overhead sometimes associated with collecting weak bindings (e.g. the need to recompute a memoized function). Lastly we use extend the semantic framework to model the key/value weak references found in Haskell and we prove the Haskell is semantics equivalent to a simpler semantics due to the lack of side-effects in our language.

Plasmid biology of natural Lactococcus lactis strains and molecular mechanisms of bacteriophage-host interaction

Lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P are bacteriocins offering promising perspectives in terms of preservation and shelf-life extension of food products and should find commercial application in the near future. The studies detailing their characterization and bio-preservative applications are reviewed. Transcriptomic analyses showed a cell wall-targeted response of Lactococcus lactis IL1403 during the early stages of infection with the lytic bacteriophage c2, which is probably orchestrated by a number of membrane stress proteins and involves D-alanylation of membrane lipoteichoic acids, restoration of the physiological proton motive force disrupted following bacteriophage infection, and energy conservation. Sequencing of the eight plasmids of L. lactis subsp. cremoris DPC3758 from raw milk cheese revealed three anti-phage restriction/modification (R/M) systems, immunity/resistance to nisin, lacticin 481, cadmium and copper, and six conjugative/mobilization regions. A food-grade derivative strain with enhanced bacteriophage resistance was generated via stacking of R/M plasmids. Sequencing and functional analysis of the four plasmids of L. lactis subsp. lactis biovar. diacetylactis DPC3901 from raw milk cheese revealed genes novel to Lactococcus and typical of bacteria associated with plants, in addition to genes associated with plant-derived lactococcal strains. The functionality of a novel high-affinity regulated system for cobalt uptake was demonstrated. The bacteriophage resistant and bacteriocin-producing plasmid pMRC01 places a metabolic burden on lactococcal hosts resulting in lowered growth rates and increased cell permeability and autolysis. The magnitude of these effects is strain dependent but not related to bacteriocin production. Starters’ acidification capacity is not significantly affected. Transcriptomic analyses showed that pMRC01 abortive infection (Abi) system is probably subjected to a complex regulatory control by Rgg-like ORF51 and CopG-like ORF58 proteins. These regulators are suggested to modulate the activity of the putative Abi effectors ORF50 and ORF49 exhibiting topology and functional similarities to the Rex system aborting bacteriophage λ lytic growth.

A benchmark comparison between reconfigurable, intelligent and autonomous wireless inertial measurement and photonic technologies in rehabilitation

Advanced sensory systems address a number of major obstacles towards the provision for cost effective and proactive rehabilitation. Many of these systems employ technologies such as high-speed video or motion capture to generate quantitative measurements. However these solutions are accompanied by some major limitations including extensive set-up and calibration, restriction to indoor use, high cost and time consuming data analysis. Additionally many do not quantify improvement in a rigorous manner for example gait analysis for 5 minutes as opposed to 24 hour ambulatory monitoring. This work addresses these limitations using low cost, wearable wireless inertial measurement as a mobile and minimal infrastructure alternative. In cooperation with healthcare professionals the goal is to design and implement a reconfigurable and intelligent movement capture system. A key component of this work is an extensive benchmark comparison with the 'gold standard' VICON motion capture system.

Axial symmetry and transverse trace-free tensors in numerical relativity

Transverse trace-free (TT) tensors play an important role in the initial conditions of numerical relativity, containing two of the component freedoms. Expressing a TT tensor entirely, by the choice of two scalar potentials, is not a trivial task however. Assuming the added condition of axial symmetry, expressions are given in both spherical and cylindrical coordinates, for TT tensors in flat space. A coordinate relation is then calculated between the scalar potentials of each coordinate system. This is extended to a non-flat space, though only one potential is found. The remaining equations are reduced to form a second order partial differential equation in two of the tensor components. With the axially symmetric flat space tensors, the choice of potentials giving Bowen-York conformal curvatures, are derived. A restriction is found for the potentials which ensure an axially symmetric TT tensor, which is regular at the origin, and conditions on the potentials, which give an axially symmetric TT tensor with a spherically symmetric scalar product, are also derived. A comparison is made of the extrinsic curvatures of the exact Kerr solution and numerical Bowen-York solution for axially symmetric black hole space-times. The Brill wave, believed to act as the difference between the Kerr and Bowen-York space-times, is also studied, with an approximate numerical solution found for a mass-factor, under different amplitudes of the metric.

Financial modelling with 2-EPT probability density functions

The class of all Exponential-Polynomial-Trigonometric (EPT) functions is classical and equal to the Euler-d’Alembert class of solutions of linear differential equations with constant coefficients. The class of non-negative EPT functions defined on [0;1) was discussed in Hanzon and Holland (2010) of which EPT probability density functions are an important subclass. EPT functions can be represented as ceAxb, where A is a square matrix, b a column vector and c a row vector where the triple (A; b; c) is the minimal realization of the EPT function. The minimal triple is only unique up to a basis transformation. Here the class of 2-EPT probability density functions on R is defined and shown to be closed under a variety of operations. The class is also generalised to include mixtures with the pointmass at zero. This class coincides with the class of probability density functions with rational characteristic functions. It is illustrated that the Variance Gamma density is a 2-EPT density under a parameter restriction. A discrete 2-EPT process is a process which has stochastically independent 2-EPT random variables as increments. It is shown that the distribution of the minimum and maximum of such a process is an EPT density mixed with a pointmass at zero. The Laplace Transform of these distributions correspond to the discrete time Wiener-Hopf factors of the discrete time 2-EPT process. A distribution of daily log-returns, observed over the period 1931-2011 from a prominent US index, is approximated with a 2-EPT density function. Without the non-negativity condition, it is illustrated how this problem is transformed into a discrete time rational approximation problem. The rational approximation software RARL2 is used to carry out this approximation. The non-negativity constraint is then imposed via a convex optimisation procedure after the unconstrained approximation. Sufficient and necessary conditions are derived to characterise infinitely divisible EPT and 2-EPT functions. Infinitely divisible 2-EPT density functions generate 2-EPT Lévy processes. An assets log returns can be modelled as a 2-EPT Lévy process. Closed form pricing formulae are then derived for European Options with specific times to maturity. Formulae for discretely monitored Lookback Options and 2-Period Bermudan Options are also provided. Certain Greeks, including Delta and Gamma, of these options are also computed analytically. MATLAB scripts are provided for calculations involving 2-EPT functions. Numerical option pricing examples illustrate the effectiveness of the 2-EPT approach to financial modelling.

Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Human intestinal microbiota and metabolites they produce in relation to host health

The aim of this thesis was to identify selected potential probiotic characteristics of Bifidobacterium longum strains isolated from human sources, and to examine these characteristics in detail using genomic and phenotypic techniques. One strain in particular Bifidobacterium longum DPC 6315 was the main focus of the thesis and this strain was used in both the manufacture of yoghurt and an animal study. In total, 38 B. longum strains, obtained from infants and adults, were assessed in vitro for the selected probiotic traits using a combined phenotypic and molecular approach. Differentiation of the 38 strains using amplified ribosomal DNA restriction analysis (ARDRA) into subspecies indicated that of the 38 bifidobacterial strains tested, 34 were designated B. longum subsp. longum and four B. longum subsp. infantis.

Mesenchymal stem cell therapy for the treatment of myocardial infarction

Mesenchymal stem cells (MSCs) are currently under investigation as repair agents in the preservation of cardiac function following myocardial infarction (MI). However concerns have emerged regarding the safety of acute intracoronary (IC) MSC delivery specifically related to mortality, micro-infarction and microvascular flow restriction post cell therapy in animal models. This thesis aimed to firstly identify an optimal dose of MSC that could be tolerated when delivered via the coronary artery in a porcine model of acute MI (AMI). Initial dosing studies identified 25x106 MSC to be a safe MSC cell dose, however, angiographic observations from these studies recognised that on delivery of MSC there was a significant adverse decrease in distal blood flow within the artery. This observation along with additional supportive data in the literature (published during the course of this thesis) suggested MSC may be contributing to such adverse events through the propagation of thrombosis. Therefore further studies aimed to investigate the innate prothrombotic activity of MSC. Expression of the initiator of the coagulation cascade initiator tissue factor (TF) on MSC was detected in high levels on the surface of these cells. MSC-derived TF antigen was catalytically active, capable of supporting thrombin generation in vitro and enhancing platelet-driven thrombus deposition on collagen under flow. Infusion of MSC via IC route was associated with a decreased coronary flow reserve when delivered but not when coadministered with an antithrombin agent heparin. Heparin also reduced MSC-associated in situ thrombosis incorporating platelets and VWF in the microvasculature. Heparin-assisted MSC delivery reduced acute apoptosis and significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and scar formation at 6 weeks post AMI. In addition, this thesis investigated the paracrine factors secreted by MSC, in particular focusing on the effect on cardiac repair of a novel MSC-paracrine factor SPARCL1. In summary this work provides new insight into the mechanism by which MSC may be deleterious when delivered by an IC route and a means of abrogating this effect. Moreover we present new data on the MSC secretome with elucidation of the challenges encountered using a single paracrine factor cardiac repair strategy.

Molecular basis of complete C4 deficiency. A study of three patients.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes, C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes and is also remarkable by the high frequency of the null alleles, C4A*Q0 and C4B*Q0. Complete C4 deficiency is exceptional because this condition appears only in homozygotes for the very rare double-null haplotype C4AQ0,BQ0. This condition in most cases gives rise to systemic lupus erythematosus and an increased susceptibility to infections. The molecular basis for complete C4 deficiency has not yet been established. Therefore we studied the DNA of three previously described C4 deficient patients belonging to unrelated families by restriction fragment length polymorphism analysis using C4 and 21-OH probes. These studies revealed a deletion of the C4B and 21-OHA genes in two patients and no deletion at all in the third patient. Therefore, complete C4 deficiency as a result of homozygosity for the C4AQ0, BQ0 haplotype is not a consequence of a deletion of the C4 genes. The molecular basis of this genetic abnormality is certainly very complex and may vary also from one case to another.

Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

The cytolytic molecules Fas ligand and TRAIL are required for murine thymic graft-versus-host disease.

Thymic graft-versus-host disease (tGVHD) can contribute to profound T cell deficiency and repertoire restriction after allogeneic BM transplantation (allo-BMT). However, the cellular mechanisms of tGVHD and interactions between donor alloreactive T cells and thymic tissues remain poorly defined. Using clinically relevant murine allo-BMT models, we show here that even minimal numbers of donor alloreactive T cells, which caused mild nonlethal systemic graft-versus-host disease, were sufficient to damage the thymus, delay T lineage reconstitution, and compromise donor peripheral T cell function. Furthermore, to mediate tGVHD, donor alloreactive T cells required trafficking molecules, including CCR9, L selectin, P selectin glycoprotein ligand-1, the integrin subunits alphaE and beta7, CCR2, and CXCR3, and costimulatory/inhibitory molecules, including Ox40 and carcinoembryonic antigen-associated cell adhesion molecule 1. We found that radiation in BMT conditioning regimens upregulated expression of the death receptors Fas and death receptor 5 (DR5) on thymic stromal cells (especially epithelium), while decreasing expression of the antiapoptotic regulator cellular caspase-8-like inhibitory protein. Donor alloreactive T cells used the cognate proteins FasL and TNF-related apoptosis-inducing ligand (TRAIL) (but not TNF or perforin) to mediate tGVHD, thereby damaging thymic stromal cells, cytoarchitecture, and function. Strategies that interfere with Fas/FasL and TRAIL/DR5 interactions may therefore represent a means to attenuate tGVHD and improve T cell reconstitution in allo-BMT recipients.

Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.

The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.