Characterising gene regulation during epileptogenesis in different models of epilepsy

As epilepsy develops an enormous range of changes occurs in neurons. This process, epileptogenesis, involves complex spatiotemporal alterations of neuronal homeostasis and neural networks. The molecular mechanism of epileptogenesis remains obscure and gene regulation during the epileptogenic process dynamically controls various signalling and functional pathways which play an important role in defining the mechanisms of epilepsy. This thesis explores gene regulation in different in vitro models of seizure like activity, and further focuses on the temporal profiles of molecular changes during an in vivo model of epilepsy. We seek to identify important regulators of epileptogenesis which may be the targets for further study of the mechanism of epilepsy in human. The High-K+, Low-Mg2+, Kainic acid, and Pentylenetetrazole models were used to elicit seizure like activity in cortical neuronal cultures. The tetanus toxin (TeNT) model of focal neocortical epilepsy in rats was utilised to characterise gene regulation in different time points: acute, subacute and chronic stages (48-72 hours, 2 weeks, and 30 days after first spontaneous seizure, respectively). A set of candidate genes relevant to epilepsy was selected to analyse changes in mRNA expression during these in vitro and in vivo models. The mRNA expression of the different candidate genes reveals diverse regulatory behaviours in different models, as well as at different time points during the process of epileptogenesis. The cell culture model treated with Low-Mg2+ for 72 hours displayed the most similar mRNA expression profile to the in vivo model of TeNT neocortical epilepsy during subacute to chronic stages. Furthermore, in the in vivo model, GFAP, mTOR, REST, and SNAP-25 are all temporarily apparently up-regulated during epileptogenesis, while CCL2 is strongly up-regulated later when epilepsy is established. Transient down-regulation of BDNF in the acute stage, and the distinctly lower expression of GABRA5 in late stage suggest that this GABAergic signalling pathway may be down-regulated in the late phase of epileptogenesis. Our work highlights how different candidate genes are differentially regulated during epileptogenesis, and how the regulation of individual genes changes as epileptogenesis progresses

The perceptual timescape: Perceptual history on the sub-second scale

There is a high-capacity store of brief time span (∼1000 ms) which information enters from perceptual processing, often called iconic memory or sensory memory. It is proposed that a main function of this store is to hold recent perceptual information in a temporally segregated representation, named the perceptual timescape. The perceptual timescape is a continually active representation of change and continuity over time that endows the perceived present with a perceived history. This is accomplished primarily by two kinds of time marking information: time distance information, which marks all items of information in the perceptual timescape according to how far in the past they occurred, and ordinal temporal information, which organises items of information in terms of their temporal order. Added to that is information about connectivity of perceptual objects over time. These kinds of information connect individual items over a brief span of time so as to represent change, persistence, and continuity over time. It is argued that there is a one-way street of information flow from perceptual processing either to the perceived present or directly into the perceptual timescape, and thence to working memory. Consistent with that, the information structure of the perceptual timescape supports postdictive reinterpretations of recent perceptual information. Temporal integration on a time scale of hundreds of milliseconds takes place in perceptual processing and does not draw on information in the perceptual timescape, which is concerned with temporal segregation, not integration

Microengineered Biomaterials and Biosystems for Systems Immunology, Cancer Biology, and Stem Cell-based Regenerative Medicine.

Many exciting topics exist at the interface between biology and micro/nanotechnology. This dissertation will discuss interdisciplinary researches that leveraging the engineering advances in biomaterials, microfluidics and advanced manufacturing for new and better solutions for emerging problems in cancer biology, systems immunology, and stem cell-based regenerative medicine. First, this dissertation will discuss the potential of integrated microfluidic immunophenotyping assay device to perform rapid, accurate, and sensitive functional cellular immunophenotyping assays for target subpopulations of immune cells isolated directly from patient blood. This dissertation will also explore the possible technique using nanotopographic substrates for efficient capture of circulating tumor cells regardless of surface protein expression and cancer type, critical for early cancer diagnosis and for fundamental understanding of cancer metastasis. This dissertation will also provide a comprehensively profiling of the biophysical characteristics of inflammatory breast cancer stem cells at the single-cell level using multiple microengineered tools to delineate the live cell phenotypic characteristics of the model of the most metastatic breast cancer subtype. Last, this dissertation will further explore synthetic micro/nanoscale ex vivo cellular microenvironment for study and regulating human embryonic stem cell behaviors that are desirable for functional tissue engineering and regenerative medicine. These novel micro/nanoengineered functional biomaterials and biosystems will not only permit advances in engineering but also greatly contribute to improving human health.PhDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108939/1/wqchen_1.pd

Proceedings of the International Cancer Imaging Society (ICIS) 16th Annual Teaching Course

Table of contents O1 Tumour heterogeneity: what does it mean? Dow-Mu Koh O2 Skeletal sequelae in adult survivors of childhood cancer Sue Creviston Kaste O3 Locoregional effects of breast cancer treatment Sarah J Vinnicombe O4 Imaging of cancer therapy-induced CNS toxicity Giovanni Morana, Andrea Rossi O5 Screening for lung cancer Christian J. Herold O6Risk stratification of lung nodules Theresa C. McLoud O7 PET imaging of pulmonary nodules Kirk A Frey O8 Transarterial tumour therapy Bernhard Gebauer O9 Interventional radiology in paediatric oncology Derek Roebuck O10 Image guided prostate interventions Jurgen J. Fütterer O11 Imaging cancer predisposition syndromes Alexander J. Towbin O12Chest and chest wall masses Thierry AG Huisman O13 Abdominal masses: good or bad? Anne MJB Smets O14 Hepatobiliary MR contrast: enhanced liver MRI for HCC diagnosis and management Giovanni Morana O15 Role of US elastography and multimodality fusion for managing patients with chronic liver disease and HCC Jeong Min Lee O16 Opportunities and challenges in imaging metastatic disease Hersh Chandarana O17 Diagnosis, treatment monitoring, and follow-up of lymphoma Marius E. Mayerhoefer, Markus Raderer, Alexander Haug O18 Managing high-risk and advanced prostate cancer Matthias Eiber O19 Immunotherapy: imaging challenges Bernhard Gebauer O20 RECIST and RECIST 1.1 Andrea Rockall O21 Challenges of RECIST in oncology imaging basics for the trainee and novice Aslam Sohaib O22 Lymphoma: PET for interim and end of treatment response assessment: a users’ guide to the Deauville Score Victoria S Warbey O23 Available resources Hebert Alberto Vargas O24 ICIS e-portal and the online learning community Dow-Mu Koh O25 Benign lesions that mimic pancreatic cancer Jay P Heiken O26 Staging and reporting pancreatic malignancies Isaac R Francis, Mahmoud, M Al-Hawary, Ravi K Kaza O27 Intraductal papillary mucinous neoplasm Giovanni Morana O28 Cystic pancreatic tumours Mirko D’Onofrio O29 Diffusion-weighted imaging of head and neck tumours Harriet C. Thoeny O30 Radiation injury in the head and neck Ann D King O31 PET/MR of paediatric brain tumours Giovanni Morana, Arnoldo Piccardo, Maria Luisa Garrè, Andrea Rossi O32 Structured reporting and beyond Hebert Alberto Vargas O33 Massachusetts General Hospital experience with structured reporting Theresa C. McLoud O34 The oncologist’s perspective: what the oncologist needs to know Nick Reed O35 Towards the cure of all children with cancer: global initiatives in pediatric oncology Carlos Rodriguez-Galindo O36 Multiparametric imaging of renal cancers Hersh Chandarana O37 Linking imaging features of renal disease and their impact on management strategies Hebert Alberto Vargas O38 Adrenals, retroperitoneum and peritoneum Isaac R Francis, Ashish P Wasnik O39 Lung and pleura Stefan Diederich O40 Advances in MRI Jurgen J. Fütterer O41 Advances in molecular imaging Wim J.G. Oyen O42 Incorporating advanced imaging, impact on treatment selection and patient outcome Cheng Lee Chaw, Nicholas van As S1 Combining ADC-histogram features improves performance of MR diffusion-weighted imaging for Lymph node characterisation in cervical cancer Igor Vieira, Frederik De Keyzer, Elleke Dresen, Sileny Han, Ignace Vergote, Philippe Moerman, Frederic Amant, Michel Koole, Vincent Vandecaveye S2 Whole-body diffusion-weighted MRI for surgical planning in patients with colorectal cancer and peritoneal metastases R Dresen, S De Vuysere, F De Keyzer, E Van Cutsem, A D’Hoore, A Wolthuis, V Vandecaveye S3 Role of apparent diffusion coefficient (ADC) diffusion-weighted MRI for predicting extra capsular extension of prostate cancer. P. Pricolo ([email protected]), S. Alessi, P. Summers, E. Tagliabue, G. Petralia S4 Generating evidence for clinical benefit of PET/CT – are management studies sufficient as surrogate for patient outcome? C. Pfannenberg, B. Gückel, SC Schüle, AC Müller, S. Kaufmann, N. Schwenzer, M. Reimold,C. la Fougere, K. Nikolaou, P. Martus S5 Heterogeneity of treatment response in skeletal metastases from breast cancer with 18F-fluoride and 18F-FDG PET GJ Cook, GK Azad, BP Taylor, M Siddique, J John, J Mansi, M Harries, V Goh S6 Accuracy of suspicious breast imaging—can we tell the patient? S Seth, R Burgul, A Seth S7 Measurement method of tumour volume changes during neoadjuvant chemotherapy affects ability to predict pathological response S Waugh, N Muhammad Gowdh, C Purdie, A Evans, E Crowe, A Thompson, S Vinnicombe S8 Diagnostic yield of CT IVU in haematuria screening F. Arfeen, T. Campion, E. Goldstraw S9 Percutaneous radiofrequency ablation of unresectable locally advanced pancreatic cancer: preliminary results D’Onofrio M, Ciaravino V, Crosara S, De Robertis R, Pozzi Mucelli R S10 Iodine maps from dual energy CT improve detection of metastases in staging examinations of melanoma patients M. Uhrig, D. Simons, H. Schlemmer S11Can contrast enhanced CT predict pelvic nodal status in malignant melanoma of the lower limb? Kate Downey S12 Current practice in the investigation for suspected Paraneoplastic Neurological Syndromes (PNS) and positive malignancy yield. S Murdoch, AS Al-adhami, S Viswanathan P1 Technical success and efficacy of Pulmonary Radiofrequency ablation: an analysis of 207 ablations S Smith, P Jennings, D Bowers, R Soomal P2 Lesion control and patient outcome: prospective analysis of radiofrequency abaltion in pulmonary colorectal cancer metastatic disease S Smith, P Jennings, D Bowers, R Soomal P3 Hepatocellular carcinoma in a post-TB patient: case of tropical infections and oncologic imaging challenges TM Mutala, AO Odhiambo, N Harish P4 Role of apparent diffusion coefficient (ADC) diffusion-weighted MRI for predicting extracapsular extension of prostate cancer P. Pricolo, S. Alessi, P. Summers, E. Tagliabue, G. Petralia P5 What a difference a decade makes; comparison of lung biopsies in Glasgow 2005 and 2015 M. Hall, M. Sproule, S. Sheridan P6 Solid pseudopapillary tumour of pancreas: imaging features of a rare neoplasm KY Thein, CH Tan, YL Thian, CM Ho P7 MDCT - pathological correlation in colon adenocarcinoma staging: preliminary experience S De Luca, C Carrera, V Blanchet, L Alarcón, E Eyheremnedy P8 Image guided biopsy of thoracic masses and reduction of pneumothorax risk: 25 years experience B K Choudhury, K Bujarbarua, G Barman P9 Tumour heterogeneity analysis of 18F-FDG-PET for characterisation of malignant peripheral nerve sheath tumours in neurofibromatosis-1 GJ Cook, E Lovat, M Siddique, V Goh, R Ferner, VS Warbey P10 Impact of introduction of vacuum assisted excision (VAE) on screen detected high risk breast lesions L Potti, B Kaye, A Beattie, K Dutton P11 Can we reduce prevalent recall rate in breast screening? AA Seth, F Constantinidis, H Dobson P12 How to reduce prevalent recall rate? Identifying mammographic lesions with low Positive Predictive Value (PPV) AA Seth ([email protected]), F Constantinidis, H Dobson P13 Behaviour of untreated pulmonary thrombus in oncology patients diagnosed with incidental pulmonary embolism on CT R. Bradley, G. Bozas, G. Avery, A. Stephens, A. Maraveyas P14 A one-stop lymphoma biopsy service – is it possible? S Bhuva, CA Johnson, M Subesinghe, N Taylor P15 Changes in the new TNM classification for lung cancer (8th edition, effective January 2017) LE Quint, RM Reddy, GP Kalemkerian P16 Cancer immunotherapy: a review of adequate imaging assessment G González Zapico, E Gainza Jauregui, R Álvarez Francisco, S Ibáñez Alonso, I Tavera Bahillo, L Múgica Álvarez P17 Succinate dehydrogenase mutations and their associated tumours O Francies, R Wheeler, L Childs, A Adams, A Sahdev P18 Initial experience in the usefulness of dual energy technique in the abdomen SE De Luca, ME Casalini Vañek, MD Pascuzzi, T Gillanders, PM Ramos, EP Eyheremendy P19 Recognising the serious complication of Richter’s transformation in CLL patients C Stove, M Digby P20 Body diffusion-weighted MRI in oncologic practice: truths, tricks and tips M. Nazar, M. Wirtz, MD. Pascuzzi, F. Troncoso, F. Saguier, EP. Eyheremendy P21 Methotrexate-induced leukoencephalopathy in paediatric ALL Patients D.J. Quint, L. Dang, M. Carlson, S. Leber, F. Silverstein P22 Pitfalls in oncology CT reporting. A pictorial review R Rueben, S Viswanathan P23 Imaging of perineural extension in head and neck tumours B Nazir, TH Teo, JB Khoo P24 MRI findings of molecular subtypes of breast cancer: a pictorial primer K Sharma, N Gupta, B Mathew, T Jeyakumar, K Harkins P25 When cancer can’t wait! A pictorial review of oncological emergencies K Sharma, B Mathew, N Gupta, T Jeyakumar, S Joshua P26 MRI of pancreatic neuroendocrine tumours: an approach to interpretation D Christodoulou, S Gourtsoyianni, A Jacques, N Griffin, V Goh P27 Gynaecological cancers in pregnancy: a review of imaging CA Johnson, J Lee P28 Suspected paraneoplastic neurological syndromes - review of published recommendations to date, with proposed guideline/flowchart JA Goodfellow, AS Al-adhami, S Viswanathan P29 Multi-parametric MRI of the pelvis for suspected local recurrence of prostate cancer after radical prostatectomy R Bradley P30 Utilisation of PI-RADS version 2 in multi-parametric MRI of the prostate; 12-months experience R Bradley P31 Radiological assessment of the post-chemotherapy liver A Yong, S Jenkins, G Joseph P32 Skeletal staging with MRI in breast cancer – what the radiologist needs to know S Bhuva, K Partington P33 Perineural spread of lympoma: an educational review of an unusual distribution of disease CA Johnson, S Bhuva, M Subesinghe, N Taylor P34 Visually isoattenuating pancreatic adenocarcinoma. Diagnostic imaging tools. C Carrera, A Zanfardini, S De Luca, L Alarcón, V Blanchet, EP Eyheremendy P35 Imaging of larynx cancer: when is CT, MRI or FDG PET/CT the best test? K Cavanagh, E Lauhttp://deepblue.lib.umich.edu/bitstream/2027.42/134651/1/40644_2016_Article_79.pd

The Effects of Nerve Growth Factor on Adult and Aged Dorsal Root Ganglion Neurons Maintained in Primary Culture

Nerve growth factor (NGF) is, a prototype neurotrophic factor, well documented for its biochemical character, distribution and functions in embryonic and neonatal animals. However, only very tentative evidence exists for its effects on mature neurons, and results are controversial. Studying NGF by using mature neurons should be a significant and attractive field, not only for its clinical value in nerve regeneration by adults, but also for understanding the mechanisms of aging. Studies in vitro present a comparatively simple environment for analysis of the effective factors. Dorsal root ganglia (DRGs), containing an enormous heterogeneity of sensory neuronal types, are a useful model in understanding the nervous system. Hence, in the present study, mouse DRG neurons were maintained in vitro supplemented with NGF V5 cultures without NGF or with anti-NGF present, to examine any NGF effects on survival, morphological phenotype, neuropeptide expressions and neurite outgrowth (equivalent to regeneration in vitro). Comparison of adult (6 months) and aged (2 years) animals was made throughout the present study. In the survival study, a range of NGF (25-200ng/ml) was added to adult neurons in co-cultures, and neuronal survival monitored for 14 days in vitro (div). 100ng/ml NGF effectively maintained survival, compared with cultures without exogenous NGF. Aged as well as adult neurons were cultured [with non-neuronal cells (NNCs) present] without exogenous NGF or with 100ng/ml NGF added for up to 29div. NGF enhanced the survival of both adult and aged neurons (P<0.005 by ANOVA). To exclude possible endogenous NGF from NNCs, or any mediation of an NGF effect in co-culture by NNCs , a neuron- enriched culture system was also used. In these neuronal loss was avoided during cell preparation and well dispersed neurons were obtained. 100ng/ml NGF and/or 1:100 anti-NGF were re-examined in the modified neuron-enriched cultures for 9div and an enhanced survival of both adult and aged neuron persisted, although this effect was lower than in co-cultures. In general, about 20% of DRG neurons were NGF-dependent for their survival in adult and aged cultures. By size analysis of over 6500 neurons, intermediate-sized neurons (24-33mum in diameter) were predominant for both adult and aged in co-culture in the presence of NGF. The immunocytochemistiy (ICC) study using the avidin-biotin- peroxidase complex (ABC) method revealed that higher proportions of SP- immunoreactive (ir), CGRP-ir and NPY-ir aged, as well as adult, neurons were present in enriched cultures supplemented with 10 or 100ng/ml NGF than in cultures without NGF added (P<0.05-0.01 by ANOVA and t-tests), whereas SOM-ir adult and aged neurons showed little difference in cultures without or with NGF added. By counting at the peak day (9div) of SP expression, enhancement of the SP-ir subset in the presence of vs absence of NGF was similar for adult or aged neurons; the proportion of the SP-ir subset was not reduced following aging. Hence the SP-ir subset, together with the subsets of CGRP-ir and NPY-ir, were considered as NGF-dependent survival neurons in adult, and even aged mouse DRGs. Enhancement of the SP-ir proportion was more rapid in aged neuronal cultures supplemented with NGF than in cultures without exogenous NGF. Also, the staining intensities for CGRP and NPY were greater in both adult and aged cultures with NGF added than those in cultures without NGF. In addition to maintaining survival, NGF affected the mature neuronal phenotypes. Scanning election microscopy demonstrated that in the presence of NGF, abundant microvilli projections were distributed on the neuronal surface; in contrast, neurons were deformed and lost their smooth appearance, if 1:100 anti-NGF was present in enriched cultures. In the study of neurite geometry in enriched culture, major neurite length (maximal extension of a neurite), entire neurite length (lengths of major neurite plus all its branches), total neurite length (lengths of all neurite outgrowth from each neuron), soma size, neurite number (for each neuron) and branch number (per neurite) were selected as parameters. Tracing was carried out blindly at 1, 3, 6 and 9div by computer image analysis and a digitizing tablet. Neurites and neurons from at least 5 mice for each adult or aged cultures were measured directly from culture or from photomicrograph montages and data were statistically tested by ANOVA. The results demonstrated that NGF enhanced neurite outgrowth (P<0.0005) at 6 and 9div for both adult and aged neurons Total neurite lengths of adult and aged neurons were distinct in the presence of NGF; neurite length was predominantly enhanced by adult neurons, whereas increased branch lengths were evident for aged neurons. Neurites predominantly extended from intermediate- and large-sized neurons. In summary, aged neurons showed comparatively lower abilities for survival and functional peptide expression compared to adult neurons in vitro, but continued to be responsive to exogenous NGF. In addition, reconstruction of neurites by aged and adult neurons in vitro was demonstrated in the present study

REDOX PROTEOMICS IDENTIFICATION OF OXIDATIVELY MODIFIED PROTEINS AND THEIR PHARMACOLOGICAL MODULATION: INSIGHT INTO OXIDATIVE STRESS IN BRAIN AGING, AGE-RELATED COGNITIVE IMPAIRMENT

The studies presented in this work were completed with the goal ofgaining greater insight into the roles of protein oxidation in brain aging and age-relatedcognitive impairment. Aging is associated with the impairment of physiological systemssuch as the central nervous system (CNS), homeostatic system, immune system, etc.Functional impairments of the CNS is associated with increased susceptibility to developmany neurodegenerative diseases such as Alzheimer\u27s diseases (AD), Parkinson\u27s disease(PD), and amyotrophic lateral sclerosis (ALS). One of the most noticeable functionalimpairments of the CNS is manifested by cognitive decline. In the past three decades, thefree radical theory of aging has gained relatively strong support in this area. Excessiveproduction reactive oxygen species (ROS) was demonstrated as a contributing factor inage-related memory and synaptic plasticity dysfunction. This dissertation use proteomicsto identify the proteins that are oxidatively modified and post-translationally altered inaged brain with cognitive impairment and normal aging brain.Ongoing research is being pursued for development of regime to preventoxidative damage by age-related oxidative stress. Among which are those that scavengefree radicals by antioxidants, i.e. ??-lipoic acid (LA), and protecting the brains byreducing production of neurotoxic substance, i.e. reducing production of amyloid ??(A??).Therefore, proteomics were also used to identify the alteration of specific proteins in agedbrain treated with LA and antisense oligonucleotides again amyloid protein precursor.This dissertation provides evidences that certain proteins are less oxidatively modifiedand post-translationally altered in cognitively impaired aged brain treated with LA andantisense oligonucleotides against the A?? region of amyloid precursor protein (APP)(AO).Together, the studies in this dissertation demonstrated that increased oxidativestress in brain play a significant role in age-related cognitive impairment. Moreover, suchincreased oxidative stress leads to specific protein oxidation in the brain of cognitiveimpaired subject, thereby leading to cognitive function impairment. Moreover, thefunctional alterations of the proteins identified by proteomics in this dissertation mayleads to impaired metabolism, decline antioxidant system, and damaged synapticcommunication. Ultimately, impairment of these processes lead to neuronal damages andcognitive decline. This dissertation also show that several of the up-regulated andoxidized proteins in the brains of normal aging mice identified are known to be oxidizedin neurodegenerative diseases as well, suggesting that the expression levels of certainproteins may increase as a compensatory response to oxidative stress. This compensationwould allow for the maintenance of proper molecular functions in normal aging brainsand protection against neurodegeneration

The roles of STRA6, EFNB1/B2 and ARMC5 in T cell function and autoimmune diseases

Les récepteurs tyrosine kinases sont un groupe de molécules clés de signalisation, qui ont 2 fonctions: la détection des stimuli de l'environnement extérieur des cellules et la transmission de ces signaux à l’intérieur des cellules. Dans les 20 dernières années, notre laboratoire a choisi d'étudier la fonction d’Ephb6 kinase, un récepteur tyrosine kinase fortement exprimé dans les lymphocytes T.Comme Efnb1 et Efnb2 sont tous des ligands pour Ephb6, nous avons ensuite procédé à étudier leur rôle dans la fonction des cellules T in vitro et in vivo. Des cellules T spécifiques mutants (KO) dans les gènes Efnb1 ou Efnb2 ainsi que les doubles mutants Efnb1/b2 (double KO) ont été générés, mais il n’y avait que les souris double KO qui ont démontré de la déficience dans le développement des thymocytes, fonction de Th1 et Th17, la signalisation du récepteur d’IL-6, et les réponses antivirales. Des preuves solides indiquent que la reconnaissance d’auto-antigène par les cellules T est un événement précoce dans la pathogenèse de la PR. Donc, nous avons postulé que les cellules T spécifique Efnb1 / b2 double KO chez la souris peuvent protéger les souris de l’arthrite induite par collagène (CIA), un modèle de souris de la PR humaine. Nous avons trouvé que Efnb1et Efnb2 dans les cellules T étaient essentielles pour la production d'anticorps pathogéniques et de la migration des lymphocytes T vers les pattes enflammées chez les souris CIA. Notre étude clinique suggère que l'expression de EFNB1 dans les cellules T pourrait être un paramètre utile pour surveiller l'activité de la maladie de RA et la réponse de traitement. Pour élucider les événements dans le programme d'activation des lymphocytes T, nous avons exploré par l'analyse des micropuces d'ADN pour identifier des molécules qui ont été exprimées de manière différente dans le WT par rapport aux cellules T Ephb6 KO dans le stade précoce de l’activation des cellules T. Environ 30 molécules étaient sur ou sous exprimées plus de 3 fois dans les cellules T WT par rapport aux cellules T KO pendant les 16 premières heures après stimulation par l'anti-CD3. Stra6 (stimulée par le gène de l'acide rétinoïque 6) et Armc5 (Armadillo répéter contenant 5) ont été parmi ceux qui ont été validées pour leur expression altérée. STRA6 est un récepteur de haute affinité pour le plasma rétinol-binding protéine (RBP) et un médiateur pour absorption cellulaire de vitamine A. Cellules T KO et WT étaient similaires en termes de prolifération et les réponses immunitaires anti-virales de virus de la chorioméningite lymphocytaire (LCMV). Ainsi, la sur-régulation de Stra6 est soit un événement parallèle qui ne soit pas essentiel pour le programme d'activation des lymphocytes T, ou il est très essentiel que la redondance existe, et sa suppression ne montre aucun effet apparent sur l'activation des cellules T. ARMC5 est une protéine intracellulaire contenant sept répétitions en tandem d’armadillo et un domaine BTB. Les fonctions du ARMC5 dans le système immunitaire ne sont pas encore connues. Nos résultats d'hybridation in situ ont montré une expression élevée de Armc5 dans le thymus, et une expression modérée dans les ganglions lymphatiques et la rate. Nous avons généré des souris KO Armc5. Fait interessant, les cellules T Armc5 KO présentaient de la prolifération diminuée et de la différenciation compromise vers Th1 et Th17 in vitro. Les souris KO étaient résistantes à l'induction expérimentale d’encéphalite auto-immune, et ont été compromises dans les réponses immunitaires anti-LCMV. En utilisant de la levure 2-hybride test, nous avons identifié 8 protéines ARMC5-associantes, qui sont connues pour les rôles dans l'activation de la cellule, le cycle cellulaire et l'apoptose. Une étude mécanique est en cours. Nos résultats montrent que Armc5 est essentiel dans la programme d'activation/de prolifération/de différenciation des lymphocytes T. Nos études ont augmenté nos connaissances sur EFNB1, EFNB2, STRA6 et ARMC5 en biologie des lymphocytes T et leur pertinence à des troubles immunitaires dans des modèles animaux ainsi que chez l'être humaine.Receptor tyrosine kinases are a group of key signaling molecules, which have dual functions: sensing the environmental stimuli outside the cells and transmitting them into the cells. 20 years ago, our laboratory started to study the function of Ephb6 kinase, a receptor tyrosine kinase highly expressed in T lymphocytes. As both Efnb1 and Efnb2 are the ligands for Ephb6, we then proceeded to study their roles in T cell function in vitro and in vivo. T cellspecific Efnb1, Efnb2 single gene knockout (KO), as well as Efnb1/b2 double KO mice were generated, but only the double KO mice showed compromised thymocyte development, Th1 and Th17 function, IL-6 receptor signaling, and anti-virus responses. Strong evidence indicates that T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Thus, we postulated that T cell-specific Efnb1/b2 double KO in mice may protect mice from collagen-induced arthritis (CIA), a mouse model for human RA. We found that Efnb1 and Efnb2 in T cells were essential for pathogenic antibody production and T cell migration to the inflamed paws in mice with CIA. Our clinical study suggests that the expression of EFNB1 in T cells might be a useful parameter for monitoring RA disease activity and treatment responses. Naïve T cells have the ability to expansion and differentiation into effector cells once they encounter foreign antigens, during which a large number of molecules are modulated. Some of these molecules play essential regulatory roles, while others exert house keeping functions and/or act as supporters to cope with increased or changed metabolic demands. To fully elucidate events in the T cell activation program, we undertook unbiased exploration with DNA microarray analysis to identify molecules that were differentially expressed in WT versus Ephb6 KO T cells in the early T-cell activation stage. About 30 molecules were up- or down-regulated more than three folds in WT T cells compared with KO T cells. Stra6 (stimulated by retinoic acid gene 6) and Armc5 (Armadillo repeat-containing 5) were among those that had been validated for their altered expression. We generated mice with these two genes deleted to study their roles in T cell function in vitro and in vivo. STRA6 is a high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. Stra6 KO mice manifest normal spleen and thymus in size, cellularity and lymphocyte subpopulations. KO and WT T cells were similar regarding proliferation, differentiation and anti-viral immune responses to lymphocytic choriomeningitis virus (LCMV). Thus, the up-regulation of Stra6 is either a parallel event which is not essential for the T cell activation program or it is so critical that heavy redundancy exists. ARMC5 is an intracellular protein containing seven tandem armadillo repeats and one BTB domain. Functions of ARMC5 in the immune system are not known previously. Our in situ hybridization results showed high expression of Armc5 in the thymus and moderate expression in the spleen and lymph nodes. A transient increase of Armc5 expression in T cells after TCR activation was found. To investigate its roles in T cell function, Armc5 KO mice were generated. The KO mice weighed 40% less than their WT counterparts. Lymphoid organs (the thymus, spleen and lymph nodes) of the KO mice appeared to be of normal size, weight, cellularity, and lymphocyte subpopulations. Intriguingly, Armc5 KO T cells presented decreased proliferation and compromised differentiation towards Th1 and Th17 in vitro. The KO mice were resistant to experimental autoimmune encephalitis induction and were compromised in anti-LCMV immune responses. Using yeast 2-hybrid assay, we have identified 8 ARMC5-assciating proteins, which have known functions in cell cycling and apoptosis. Further mechanistic study is underway. Our results reveal that Armc5 is vital in the T cell activation/proliferation /differentiation program. Our studies have augmented our knowledge about EFNB1, EFNB2, STRA6 and ARMC5 in T cell biology and their relevance to immune disorders in animal models as well as in humans

Regulation of adipogenesis and inflammation: role(s) of adipose microRNAs

Background: Obesity is associated with elevated risk of premature death and a range of co- morbidities. It is multifactorial and heterogeneous in origin, and, stratification of this disease, depending on the range of associated pathogenicities could help identify mediators and in the design of targeted therapies. Recent research has focused on microRNA (miRs) as potential biomarkers of cardiovascular risk, as well as their role as causative agents in the obesity associated pathologies. / Aims of the project were to: / 1. Stratify obese subjects depending on systemic biomarkers of insulin resistance and inflammation. / 2. Identify and validate specific miRs associated with these phenotypes. / 3. Confirm and validate, in the whole transcriptome, targets for the specific miRs to assign functionality. / Methods: Non-diabetic, morbidly obese subjects of Arab origin were studied. Blood and adipose tissue samples were obtained before and after weight loss, along with anthropometric data. Glucose and lipids levels were determined by conventional methods. Insulin and adipokine concentrations were assayed by commercially available 2-site ELISA (R & D Systems, Oxon, UK). The population was dichotomised according to their serum insulin levels: Metabolically Healthy Obese (MHO) insulin 7.0 miU/ml. Total RNA, including miR, was extracted from peripheral blood cells, whole adipose tissue, the stromal vascular fraction and adipocytes of the abdominal subcutaneous and omental adipose tissues. miR expression was assessed using an inflammatory pathway specific array (Qiagen), and by small RNA sequencing (Ion Torrent). mRNA expression was assessed by whole transcriptome analysis (Ion Proton) and validated by PCR based microarrays (SurePrint G3 Human Gene Expression). / Results: PO, matched for age and BMI, had significantly higher serum insulin levels and HOMA index of IR, compared to MHO. They also had higher leptin, a marker of fat mass and adipocyte hypertrophy, and lower adiponectin, an endogenous insulin sensitizer. However, blood pressure, lipids and inflammatory markers, such as IL-6, MCP-1 and CRP were not significantly different between the groups. Three miRs were significantly down-regulated in the PO; miR-29, miR-144 and miR- 374, and associated with inflammation, along with miR-122, miR-302, miR-200, which were associated with hyperinsulinaemia and insulin resistance. Many of their targets, especially those of miR-29, showed elevated expression in the PO. Following surgical weight loss there was a significant reduction in insulin which correlated with an increase in the levels of expression of nine miRs; miR-9, miR-200c, miR-141, miR-124, miR- 376c, miR-302, while one was downregulated, miR-26b. Whole transcriptome analysis of mRNA in blood and adipose tissue revealed modulation of several genes in the cardiometabolic pathways in the PO compared to MHO, along with genes leading to increased fibrosis. / Conclusions: Significant differences in the expression of specific miR species occured along with insulin resistance and inflammation in PO compared to MHO. The target genes of these miRs, especially miR-29, miR-144 and miR-122, suggested fibrosis, in the presence of IR and inflammation, as a major lesion in these patients. Functional studies to explore the role of these miRs in fibrosis may offer new insights on putative therapeutic targets for this group of patients