Are Social Networks Watermarking Us or Are We (Unawarely) Watermarking Ourself?

In the last decade, Social Networks (SNs) have deeply changed many aspects of society, and one of the most widespread behaviours is the sharing of pictures. However, malicious users often exploit shared pictures to create fake profiles, leading to the growth of cybercrime. Thus, keeping in mind this scenario, authorship attribution and verification through image watermarking techniques are becoming more and more important. In this paper, we firstly investigate how thirteen of the most popular SNs treat uploaded pictures in order to identify a possible implementation of image watermarking techniques by respective SNs. Second, we test the robustness of several image watermarking algorithms on these thirteen SNs. Finally, we verify whether a method based on the Photo-Response Non-Uniformity (PRNU) technique, which is usually used in digital forensic or image forgery detection activities, can be successfully used as a watermarking approach for authorship attribution and verification of pictures on SNs. The proposed method is sufficiently robust, in spite of the fact that pictures are often downgraded during the process of uploading to the SNs. Moreover, in comparison to conventional watermarking methods the proposed method can successfully pass through different SNs, solving related problems such as profile linking and fake profile detection. The results of our analysis on a real dataset of 8400 pictures show that the proposed method is more effective than other watermarking techniques and can help to address serious questions about privacy and security on SNs. Moreover, the proposed method paves the way for the definition of multi-factor online authentication mechanisms based on robust digital features

Machine Learning Advances for Practical Problems in Computer Vision

Convolutional neural networks (CNN) have become the de facto standard for computer vision tasks, due to their unparalleled performance and versatility. Although deep learning removes the need for extensive hand engineered features for every task, real world applications of CNNs still often require considerable engineering effort to produce usable results. In this thesis, we explore solutions to problems that arise in practical applications of CNNs. We address a rarely acknowledged weakness of CNN object detectors: the tendency to emit many excess detection boxes per object, which must be pruned by non maximum suppression (NMS). This practice relies on the assumption that highly overlapping boxes are excess, which is problematic when objects are occluding overlapping detections are actually required. Therefore we propose a novel loss function that incentivises a CNN to emit exactly one detection per object, making NMS unnecessary. Another common problem when deploying a CNN in the real world is domain shift - CNNs can be surprisingly vulnerable to sometimes quite subtle differences between the images they encounter at deployment and those they are trained on. We investigate the role that texture plays in domain shift, and propose a novel data augmentation technique using style transfer to train CNNs that are more robust against shifts in texture. We demonstrate that this technique results in better domain transfer on several datasets, without requiring any domain specific knowledge. In collaboration with AstraZeneca, we develop an embedding space for cellular images collected in a high throughput imaging screen as part of a drug discovery project. This uses a combination of techniques to embed the images in 2D space such that similar images are nearby, for the purpose of visualization and data exploration. The images are also clustered automatically, splitting the large dataset into a smaller number of clusters that display a common phenotype. This allows biologists to quickly triage the high throughput screen, selecting a small subset of promising phenotypes for further investigation. Finally, we investigate an unusual form of domain bias that manifested in a real-world visual binary classification project for counterfeit detection. We confirm that CNNs are able to ``cheat'' the task by exploiting a strong correlation between class label and the specific camera that acquired the image, and show that this reliably occurs when the correlation is present. We also investigate the question of how exactly the CNN is able to infer camera type from image pixels, given that this is impossible to the human eye. The contributions in this thesis are of practical value to deep learning practitioners working on a variety of problems in the field of computer vision

Further studies on the immunopathogenesis of canine atopic dermatitis

Various aspects of the pathogenesis of canine atopic dermatitis have been elucidated such as the role of certain allergens, antigen presenting cells, allergen-specific T lymphocytes, IgE and mast cells. However, gaps in our understanding still remain, such as the role of antigen-specific IgG and cytokine subsets. Canine atopic dermatitis is commonly treated using allergen-specific immunotherapy, but the mechanisms of its action are still incompletely understood. The studies in this thesis investigate further some aspects of the immunopathogenesis of canine atopic dermatitis and the changes that occur during allergen-specific immunotherapy.To investigate IgG responses to D.farinae antigens, a semi-quantitative, Western blot, digital image analysis system was developed and validated. Both healthy and atopic dogs mounted D. /armae-specific total IgG, IgGl and IgG4 responses to separated antigens. D. farinae-specific IgG2 and IgG3 responses were difficult to detect. The profile of IgG binding was similar in the two groups, both in terms of the number of bands recognised and their molecular weights. The most commonly recognised band in both groups was a 98 kDa antigen, most likely to be the major allergen Der f 15. These results indicate that D. farinae antigens are recognised by the canine immune system regardless of whether or not a dog is atopic. They also demonstrate that antibody class switching to IgE in atopic dogs does not appear to inhibit IgG production. The IgG antibody response does not appear to be protective against the development of clinical atopic disease, but whether or not it plays any role in the pathogenesis of the disease remains to be determined.In atopic dogs undergoing allergen-specific immunotherapy (ASIT) against D. farinae with alum-precipitated vaccines, there was no significant increase in D. farinae-specific total IgG, IgGl or IgG4, even in dogs showing apparent clinical improvements. In contrast, ASIT using aqueous vaccines resulted in significant increases in D. farinae-specific IgG responses. These results suggest that aqueous ASIT may elicit the production of IgG blocking antibodies in atopic dogs, an effect not detected using alum-precipitated vaccines.To investigate the cytokine milieu in dogs with atopic dermatitis, real-time quantitative RT-PCR was used to detect the expression of mRNA transcripts of the Thl cytokine IFN-y, the Th2 cytokine IL-4, the Treg cytokine TGF-|3 and inducible NO synthase (iNOS) as a measure of the innate immune response. The housekeeping gene for 18S ribosomal RNA (rRNA) was chosen as internal control to normalise variations in the amount of starting material between samples and between individuals. The expression of TGF-p and iNOS were lower in lesional skin compared to non-lesional skin in atopic dogs, whilst IFN-y was expressed in a significantly higher level in lesional skin compared to healthy controls. IL-4 expression did not differ between the groups. These cytokine profiles show distinct differences to those reported using non real-time, semi-quantitative RT-PCR. These differences may reflect the various methodological approaches used and illustrate the limitations inherent in such techniques for the quantification of cytokine expression

Chemokines (CCL3, CCL4, CCL5) inhibit ATP-induced release of IL-1beta by monocytic cells

ATP and chemokines are among the first inflammatory mediators that can enter the circulation via damaged blood vessels at the site of injury, leading to an activation of the host’s immune response. The main function of chemokines is leukocyte mobilization, guiding immune cells towards the injured tissue along a chemotactic concentration gradient. In monocytes, ATP typically triggers inflammasome assembly, a multiprotein complex necessary for the maturation and secretion of IL-1beta. IL-1beta is a potent inflammatory cytokine of innate immunity, essential for pathogen defense. However, excessive IL-1beta may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1beta, by engaging a cholinergic signaling pathway. LPS-primed human monocytic U937 cells were treated with chemokines in the presence or absence of nAChR antagonists or iPLA2beta inhibitors and concomitantly stimulated with the P2X7 agonist BzATP. IL-1beta concentration was determined in the cell culture supernatants. Silencing of the chemokine receptor and iPLA2b gene expression was achieved by transfecting cells with the appropriate siRNA. CCL3, CCL4, and CCL5 dose-dependently inhibited BzATP-stimulated release of IL-1beta, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. The inhibitory effect of CCL3 was blunted after silencing CCR1 or iPLA2beta gene expression by siRNA and was sensitive to antagonists of nAChRs containing subunits alpha7 and alpha9/alpha10. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1beta release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1beta from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nAChR subunits alpha7 and alpha9/alpha10. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1beta is minimized

ATP-sensitive Potassium Channels and Cardiac Arrhythmia

PhDATP-sensitive potassium channels (KATP) open in response to metabolic challenge. They form of pore subunits (Kir6.1 or Kir6.2) and modulatory subunits (SUR1, SUR2A or SUR2B) and are ubiquitously expressed. Differential subunit composition between cardiac chambers was investigated, as were atrial anti-arrhythmic effects of KATP modulation. Selective pharmacology of KATP openers and inhibitors was confirmed in a heterologous expression system through whole-cell patch clamp. Isolated HL-1 cells (a murine atrial cardiomyocyte model) and murine atrial cardiomyocytes showed identical KATP pharmacological responses representing Kir6.2/SUR1 channels. Relative quantification of murine whole atrial RNA concurred, and was distinct from the ventricles (Kir6.2/SUR2). Human whole heart RNA from normal hearts exhibited a different pattern with no obvious chamber specificity. Kir6.1-/- and Kir6.2-/- mice demonstrated that both pore types contribute to electrophysiological parameters in isolated atrial cardiomyocytes, but Kir6.2 appears more important. In atrial tissue (Langendorff hearts), Kir6.2-/- more than Kir6.1-/- mice demonstrated increased effective refractory periods and reduced conduction velocity at baseline, and during hypoxia, compared to wildtype. A trend to reduced arrhythmogenicity was observed during programmed electrical stimulation in the Kir6.2-/- mouse. In syncytia of spontaneously beating HL-1 cells, KATP activation with diazoxide was met with rotational to uniform wavefront organisation and silencing of electrical activity in a dose-dependent manner, reversed with channel blockade. In Langendorff mouse hearts KATP inhibition reversed hypoxia induced slowing of spontaneous sinus node activation, but pharmacological activation alone did not, suggesting different mechanisms with hypoxic channel activation. Thus, both pore subunits contribute to the cardiac electrophysiology of murine atria, but Kir6.2 appears more important. HL-1 cells exhibit identical KATP pharmacology to murine atrial myocytes, which have a differential subunit composition compared to the ventricle. Any human cardiac KATP differential subunit expression needs further exploration. KATP activation and inhibition have anti-arrhythmic effects and this might be explored further clinically.Medical Research Council MR/L016230/1

Pharmacological characterisation of neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are the targets for the endogenous neurotransmitter acetylcholine and represent a diverse family of ligand-gated ion channels. They are expressed in the neuromuscular junction, the peripheral nervous system and the central nervous system. In the brain, the most prevalent subtypes are the heteromeric α4β2 and homomeric α7 nAChRs. Neuronal nAChRs are implicated in numerous physiological and pathophysiological functions and are therefore important targets for therapeutic drug discovery for conditions such as Alzheimer’s disease, schizophrenia and tobacco addiction. This thesis aims to further our understanding of the pharmacological and molecular characteristics of neuronal nAChRs. Acetylcholine activates nAChRs by binding at an extracellular orthosteric site. Previous studies have described several ligands that potentiate agonist-evoked responses by binding to an allosteric site of the α7 nAChRs that is distinct from the acetylcholine binding site. These ligands, termed positive allosteric modulators (PAMs) can be described as type I, when they have little or no effect on desensitisation, or type II, when they dramatically slow down the fast desensitisation kinetics of the α7 nAChR subtype. Here, a novel series of α7-selective PAMs with a range of effects on receptor desensitisation is described, using recombinant human receptors. This series consists of PAMs with type I and type II profiles, in addition to PAMs with intermediate properties on desensitisation, therefore increasing the nAChR pharmacological toolbox. Furthermore, the effect of a number of mutations on the pharmacological properties of the receptor is investigated. Three point mutations, two in the transmembrane domain (L247T and M260L) and one in the N-terminal domain (W54A), are shown to have the ability to convert PAMs into agonists. Moreover, the M260L mutation displays this property only with PAMs that have a significant effect on receptor desensitisation. These observations can provide insights into the role of these residues on receptor gating and desensitisation. In addition to the studies on recombinant receptors, the expression and functional properties of nAChRs in neurons derived from human induced pluripotent stem cells (iPSC) is examined. The iPSC-derived neurons represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels in a native environment

The Role of Forebrain Cholinergic Signalling In Regulating Hippocampal Function And Neuropathology

Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric disorders, including Alzheimer’s Disease (AD). Cumulative use of drugs with anticholinergic activity is associated with increased risk for dementia and AD. Also, cholinergic function has been implicated in predicting the development of key neuropathological hallmarks seen in AD. However, the relationship between cholinergic dysfunction and conservation of cognitive ability as well as neuronal cell maintenance is not fully understood. Here, we tested how information processing and distinct molecular mechanisms associated with AD are regulated by cholinergic tone in genetically-modified mice in which cholinergic transmission was altered by targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine storage and release. We assessed the long-term consequences of loss of central cholinergic signalling for hippocampal vulnerability to age-induced stress. We show that deletion of forebrain-specific ACh release leads to age-related increases in neuronal vulnerability, protein aggregation, tau Thr-231 phosphorylation and misfolding, and neuroinflammation. Moreover, inhibition of forebrain cholinergic neurotransmission led to a disturbance in adult hippocampal neurogenesis, highlighted by decreased proliferation and cell survival in neural precursor cells. Additionally, we measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of Paired Associates Learning task (PAL). Acquisition in the mouse PAL task was impaired in forebrain-specific VAChT-deficient mice, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signalling. In contrast, spatial memory was relatively preserved. Moreover, we assessed the functional consequence of impaired neurogenesis by testing pattern separation using a Location Discrimination task. Mice with compromised cholinergic signalling were impaired when stimuli were presented with small separation, but not when stimuli were presented with high separation, suggesting that deficient cholinergic tone has major consequences on pattern separation. The pathological changes in the hippocampus we observed in VAChT-deficient mice have important consequences as they presented age-related deterioration in spatial navigation. Our findings provide a refined understanding of the importance of acetylcholine in modulating molecular mechanisms and key cognitive behaviours involved in AD

The mechanisms of metabolic regulation of the cloned equivalent of the vascular Katp/Kndp channel.

At the molecular level, Katp is an octameric protein complex composed of an inwardly rectifying K+ channel subunit (Kir6.*) which forms the channel pore and a regulatory sulphonylurea receptor subunit (SUR). By sensing intracellular nucleotide concentrations, Katp channels couple the membrane potassium conductance of a cell to its metabolic state. To understand the molecular basis of metabolic regulation of the Katp/Kndp channel Kir6.1/SUR2B, the cell-based Rubidium-86 (86Rb+) efflux assay was employed, using HEK293 cells as the expression system. 86Rb+ efflux was activated on the addition of lOpM levcromakilim and metabolic poisoning (induced by 20mM 2-deoxyglucose and 2.5mM sodium cyanide), and inhibited by lOpM glibenclamide. The subsequent 86Rb+ distribution between intracellular and extracellular space was determined via the measurement of Cherenkov radiation, the relative amount of 86Rb+ in the cell supernatant being a direct measure of channel activity. The functionality of selected mutants was also investigated under physiological conditions by the perforated patch- clamp method, whereby currents were evoked via voltage clamp recordings over 1000ms voltage steps between -150mV and +50mV from a holding potential of-80mV. AN-Kir6.1-AC truncations were made in an attempt to understand the role of the pore- forming subunit pharmacologically in channel gating, as has previously been shown for Kir6.2AC26. Mutants for which the RXR motif was removed were surface expressed (as shown via irnmunofluorescent staining using an anti-HA-fluorescein conjugated antibody), and showed increased basal efflux in the absence of SUR2B which was reduced by inhibitors which bind to the pore-forming subunit (lmM BaCh or lOOpM PNU-37883A). However, these mutants did not display intrinsic ATP sensitivity, as for AN-K.ir6.2-AC, and for Kir6.1AC48/AN13 this was confirmed by perforated patch clamping. When Kir6.1AC48/AN13 was expressed with SUR2B, glibenclamide was able to reverse efflux induced on metabolic inhibition, but it was suspected that under this condition the sterical conformation of the channel changed which prevented Kir6.*-specific inhibitors from binding (since they were not able to reverse metabolically induced efflux). In contrast, application of either ImM BaCb or lOOuM PNU-37883A was able to reverse metabolically induced efflux for Kir6.2AC26/SUR2B. On the introduction of mutations into NBD1 or NBD2 (K708A and K1349M respectively) of SUR2B, Kir6.1/SUR2B K708A/K1349M was shown to be non-functional, whereas Kir6.2/SUR2B K708A/K1349M displayed normal pharmacology on the addition of KCOs and metabolic inhibitors. In conclusion, Kir6.1/SUR2B is sensitive to metabolic poisoning in an analogous fashion to Kir6.2/SUR2B. but the Kir6.1 pore-forming subunit does not display intrinsic metabolic sensitivity, unlike Kir6.2. Metabolic sensitivity of Kir6.1/SUR2B is determined by both nucleotide binding domains of SUR2B, and finally the pharmacology of metabolically attenuated currents is different from those activated by a Katp channel opener