Human-Centric Machine Vision

Recently, the algorithms for the processing of the visual information have greatly evolved, providing efficient and effective solutions to cope with the variability and the complexity of real-world environments. These achievements yield to the development of Machine Vision systems that overcome the typical industrial applications, where the environments are controlled and the tasks are very specific, towards the use of innovative solutions to face with everyday needs of people. The Human-Centric Machine Vision can help to solve the problems raised by the needs of our society, e.g. security and safety, health care, medical imaging, and human machine interface. In such applications it is necessary to handle changing, unpredictable and complex situations, and to take care of the presence of humans

The development and validation of a murine model for studying the role of histamine receptors in acute and chronic itch

Itch (pruritus) is an unpleasant sensation of the skin, which evokes the desire to scratch. The condition commonly presents in clinical practice, as a symptom of systemic disease and various skin disorders, such as atopic dermatitis. Itch has tended to be closely associated with pain, and older evidence suggests that the sensations involve the same sensory nerves, firing at different frequencies. Partly because of this, and the subjective cognitive nature of the condition, itch has been relatively ignored as a research area and there is a general lack of effective animal models for studying itch. This has restricted detailed studies into putative mediators of itch and their mechanism(s) of action.The present studies were undertaken to develop and validate acute and chronic models of itch in mice, based on the combined use of behavioural tests in awake mice and in vivo electrophysiological recordings from itch afferents in anaesthetized animals. The hypothesis was that scratching behaviour can be evoked in mice using intradem1al injections of pruritogenic drugs and that this can be measured automatically and objectively to provide a reliable indicator of itch. A further hypothesis was that electrophysiological recordings made in vivo from murine cutaneous sensory nerves can be used to distinguish between pruritogens and algogens.The model of itch that was developed is based on injection of histamine into the back of the mouse neck to evoke scratching of the area by the hind paws. Histamine is a pruritogen in both humans and mice, although the lack of effectiveness of traditional H 1-receptor antihistamines in treating all clinical itch disorders suggests other mediators are also responsible for pruritus. The studies demonstrated that scratching in mice can be induced using histamine and other pruritogens (e.g. trypsin and 5-HT) in a reproducible dose dependent manner. Scratching was established as a response to itch-provoking agents, but not to painful stimuli. A novel mechanism of histamine evoked scratching involving H4 receptors was discovered. Chronic itching 111 response to topical application of dinitrochlorobenzene was also established. A robust automated method for the detection and measurement of scratching in mice was developed, which considerably enhances accuracy and reduces the time taken, in comparison with manual observation of scratching.In vivo electrophysiological recordings showed that pruritogens evoke a pattern of response in cutaneous nerves distinct in nature from that evoked by algogenic stimuli. However, nerves responded to both stimuli, suggesting that in mice, there are probably no independent 'pruritoceptors', unlike the situation in man. In summary, scratching in mice can be recorded automatically and used as a reproducible quantitative measure of itch. This model can be used for further studies on putative mediators of itch to establish their mechanism of action. Knowledge from such studies should provide understanding of the sensation of itch in man, and should facilitate the development of novel therapies specific for pruritus

Topical administration of EGF suppresses immune response and protects skin barrier in DNCB-induced atopic dermatitis in NC/Nga mice

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response

Etude expérimentale des dynamiques temporelles du comportement normal et pathologique chez le rat et la souris

155 p.Modern neuroscience highlights the need for designing sophisticated behavioral readout of internal cognitive states. From a thorough analysis of classical behavioral test, my results supports the hypothesis that sensory ypersensitivity might be the cause of other behavioural deficits, and confirm the potassium channel BKCa as a potentially relevant molecular target for the development of drug medication against Fragile X Syndrome/Autism Spectrum Disorders. I have also used an innovative device, based on pressure sensors that can non-invasively detect the slightest animal movement with unprecedented sensitivity and time resolution, during spontaneous behaviour. Analysing this signal with sophisticated computational tools, I could demonstrate the outstanding potential of this methodology for behavioural phenotyping in general, and more specifically for the investigation of pain, fear or locomotion in normal mice and models of neurodevelopmental and neurodegenerative disorders

Contributions of MyD88-dependent receptors and CD11c-positive cells to corneal epithelial barrier function against Pseudomonas aeruginosa.

Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (-/-) or TLR4 (-/-) corneas, but not TLR2 (-/-), TLR5 (-/-), TLR7 (-/-), or TLR9 (-/-), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (-/-) or TLR5 (-/-) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1β, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (-/-) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions

Molecular Mediators of Acute and Chronic Itch in Mouse and Human Sensory Neurons

Itch is a distinct sensation that arises from the activation of small-diameter pruriceptive nerve fibers innervating the skin. Recent strides in the field have identified several histamine-dependent and -independent pruriceptive pathways and receptors that contribute to acute and chronic itch. The work presented in this thesis further investigates the molecular mechanisms involved in the signaling, development, and sensitization of itch in mouse and human. Most pruritogen receptors are Gq-Protein Coupled Receptors (GqPCR), which canonically activate protein kinase C (PKC); however, little is known about whether specific PKC isoforms regulate itch. The first study in this thesis demonstrates that the isoform PKCδ contributes to histamine-induced scratching, but not histamine-independent itch. Our studies show that PKCδ is expressed in dorsal root ganglia (DRG), where it mediates sensory neuron responses to histamine. To investigate the mechanisms behind a common form of chronic pruritus, the second study in this thesis applied a mouse model of dry skin itch to test changes in sensory neuron structure and function. We found that dry skin was marked by a significant increase in epidermal nerve fiber innervation independent of scratching. Furthermore, dry skin was associated with a selective increase in non-peptidergic, Ret-positive fibers and a functional expansion of the proportion of chloroquine-sensitive neurons. Epidermal hyperinnervation and increased growth factor levels in the skin of patients with pruritic skin diseases suggest a potential role for neurotrophic factors (NTFs) in itch. In our third study, we tested the hypothesis that NTF signaling modulates pruritogen-evoked itch. Pretreatment with nerve growth factor (NGF) selectively potentiated histamine-induced scratching and increased the proportion of histamine-responsive sensory neurons. Artemin pretreatment, on the other hand, potentiated scratching induced by chloroquine, and increased the proportion of chloroquine-responsive neurons. Interestingly, aberrations in endogenous TrkA-NGF signaling significantly impacted normal pain sensation, but did not play a role in histamine- and chloroquine-induced itch. In the final study of this thesis, we developed a protocol to surgically extract human DRG from organ donors and culture dissociated human primary sensory neurons. Using this approach, we performed functional studies to characterize the pruritogen- and algogen- responsive sensory neuron subpopulations in humans. We found that NGF and artemin pretreatment did not change histamine and chloroquine responses in vitro, indicating a potential functional difference between mouse and human sensory neurons

Interleukin 31 mediates MAP kinase and STAT1/3 activation in intestinal epithelial cells and its expression is upregulated in inflammatory bowel disease

Background/aim: Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31R\textgreeka) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation.Methods: Expression studies were performed by RT-PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays.Results: Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF-\textgreeka, IL1\textgreekb, IFN-\textgreekg, and sodium butyrate stimulation increased IL31, IL31R\textgreeka, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK-1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNF\textgreekDARE mouse model of ileitis but in inflamed colonic lesions compared to non-inflamed tissue in patients with Crohn's disease (CD; average 2.4-fold increase) and in patients with ulcerative colitis (UC; average 2.6-fold increase) and correlated with the IL-8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88).Conclusion: IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation

The role of dietary arachidonic acid and docosahexaenoic acid in preventing the phenotype observed with highly unsaturated fatty acid deficiency

The physiological roles of highly unsaturated fatty acids (HUFA), mainly arachidonic acid (AA, 20:4ω6) and docosahexaenoic acid (DHA, 22:6ω3), are not completely understood. In order to study specific functions for AA and DHA, a delta-6 desaturase knockout (D6D-/-) mouse was created. D6D is a key enzyme in synthesizing HUFA from the precursor dietary essential fatty acids, linoleic acid (LA, 18:2ω6) or α-linolenic acid (ALA, 18:3ω3). By disrupting D6D expression, LA and ALA provided in the diet will not be metabolized to HUFA. Phenotype of the D6D-/- mouse is therefore specific to lack of AA and/or DHA and consists of ulcerative dermatitis, male infertility, gastrointestinal ulcers, and hepatic lipidosis. New insight on specific AA and DHA roles was established through dietary prevention of HUFA deficiency phenotype. The absence of a D6D isozyme had to be assessed before further characterizing HUFA roles with the D6D-/- mouse model. The presence of a D6D isozyme would interfere with the creation of HUFA deficiency. The primary D6D isozyme candidate was Fads3 gene due to its increased gene expression in D6D-/- liver and homology to the Fads2 gene that encodes for D6D. Cloning and transfection of Fads3 into cultured HEK293 cells confirmed lack of D6D activity (Chapter 3). The order of appearance of D6D-/- phenotype due to HUFA deficiency had yet to be determined. A D6D-/- time course study (Chapter 4) characterized the mouse at different ages in order to follow sequence of HUFA deficiency pathology. The amount of HUFA in D6D-/- at weaning was comparable to control mouse indicating the presence of HUFA stores that most likely result from HUFA passed on from the mother. Subsequent HUFA depletion with age correlated with severity of D6D-/- phenotype. Male infertility, gastrointestinal erosions, and hepatic lipidosis are the first observed HUFA deficiency phenotype to appear at 6 weeks of age, followed by impaired antibody response at 9 weeks, and ulcerative dermatitis by 21 weeks of age. HUFA supplementation studies helped determine specific roles for AA and DHA in preventing HUFA deficiency phenotype. Hepatic lipidosis was prevented by either AA or DHA (Chapter 5). AA essentiality was specific to skin and gastrointestinal function since DHA supplementation was unsuccessful in preventing ulcerative dermatitis or gastrointestinal ulcers (Chapter 6). DHA essentiality was specific to male reproduction as indicated by full restoration of spermatogenesis, sperm counts, and sperm motility (Chapter 7). The role of DHA in spermatogenesis is related to acrosome biogenesis, a process which relies on vesicle fusion (Chapter 8). The immune system (Chapter 9) was further characterized following up on splenomegaly and thymic atrophy observations of the first characterization of the D6D-/-. HUFA deficiency results in decreased antibody response indicating essentiality for HUFA in immune function. In summary, these studies showed for the first time a specific requirement for AA in skin, and of DHA in male reproduction. The mechanism behind DHA requirement in male fertility has been linked to acrosome biogenesis. Future research done with the D6D-/- mouse model will help develop hypothesis on other potential mechanisms behind the essentiality of AA and DHA. Understanding how HUFA maintain tissue homeostasis will help in the development of treatments for diseases that result from an altered essential fatty acid metabolism