NICaN Regional Supportive & Palliative Care Network

NICaN Regional Supportive & Palliative Care Network Friday 30th May 2008 Lecture Theatre, Fern House Antrim 2.00 pm - 5.00 pm Welcome, Introductions Stuart MacDonnell, Chair of the Supportive and Palliative Care network welcomed everyone to the meeting. This meeting had been rescheduled to accommodate the validation workshop for the regional palliative care model, which took place on Friday,18th April. Acknowledging the full agenda, several items were pulled forward to accommodate speakers SPC_0809_03 Modernisation and Reform of Supportive and Palliative care Mr MacDonnell welcomed Dr Sonja McIlfatrick and Dr Donna Fitzimons, members of the Phase 1 Project Team for the Modernisation and Reform of palliative care. Their presentation highlighted the journey taken by the Project Team since January 2008 - May 2008. Seeking to deliver the network vision, for any person with palliative care need, cancer or non - cancer, the project team incorporated several methodologies. The literature review identified best practice. An assessment of need including epidemiological data and review of service provision. Consultation reflected the engagement with patients, carers and professional forums, primary care and non-malignant focus groups. The breadth of consultation confirmed the evidence for the identified components of the model. These were validated at the April workshop. External review of the work was provided by Dr Phil Larkin (Galway Uni) Prof David Clark (End of Life Care Observatory, Lancaster University) and Mr Bob Neillans (Chair of the Mid Trent Palliative care network, which has been involved in the Delivering choice programme within Lincolnshire). The Guiding Principles of the model reinforced Patient and family centred care, enhanced community provision and supported by specialists. The components of the model are · Identification of patient with Palliative careened · Holistic Assessment · Integration of services · Coordination of care · End of Life Care and Bereavement Care The consultation process also highlighted the need for Increased Public and Professional Awareness. This was recognised as an encompassing component. Underpinning the model is the need for robust Education and common core values e.g. dignity, choice, advocacy, empowerment, partnership working. Stuart MacDonnell, who also chaired the steering group during the project, congratulated the Project Team for delivering the comprehensive document on schedule. The Report has been submitted to the NICaN Board and the DHSSPSNI. In addition, an outline for Phase 2 of this work has been submitted. Mr MacDonnell recognised that there is real opportunity for palliative care to benefit from the DHSSPSNI commitment to concrete developments. Phase 2 will progress the current high-level components of the model into quality services developments at a local level, demonstrating integration throughout. The methods propose continued engagement with the Delivering Choice Programme enabled through a Central and also Local Teams. The report and the Appendices care available on the NICaN website www.nican@n-i.nhs.uk SPC_0809_01 Chairman's Business · Update on the Cancer Service Framework, the document has been submitted and presented to the Departmental Programme Board. Next stages will include the review of costs and development of a implementation guidance It is hoped that the completed document should be available for public consultation in Autumn 2008. with a launch of the framework document and accompanying implementation guide in Spring 2009. Some funding has already been identified to advance key areas of work including, Advanced communication skills training, peer review and an appointment of a post to develop the cancerni.net, focusing on children and e-learning tools. · Children's and Adolescent Cancer network group , Liz Henderson is to convene a group to consider how this is to be taken forward. · NICaN appointments Recognition was given to the significant contribution made by Dr Gerard Daly during his position as NICaN Lead Clinician, particularly throughout the early establishment of the NICaN. Dr Dermott Hughes (Western Trust) has been appointed as the NICaN Medical Director. The Primary Care Director post has been advertised and it is hoped that the Director of Network will be advertised later in Summer. Endorsement of End of Life care paper. The Paper was presented and endorsed at the March 2008 NICaN Board meeting. Mr David Galloway (Director of Secondary Care) emphasised the need for this important work to be recognised within the regional model to ensure that it is reflected in future models of service delivery Congratulations were again echoed to the Chair of the End of Life Group for this work, Dr Glynis Henry, and the working group Other recognition Mr MacDonnell congratulated the significant achievements across the network. These include: · Dr Francis Robinson (Consultant Palliative Medicine, Western Trust) Awarded - Consultant of the year at the NI Health Care awards. · Mrs Evelyn Whittaker Hospice Nurse Specialist, NI Hospice, Joint Second Prize in the Development award within the International Journal of Palliative Nursing Awards, for her work in development of palliative care education in nursing homes. · Mr Ray Elder is the newly appointed Team Leader of Community Palliative care, SE Trust. · Mrs Bridget Denvir, who managed the establishment of one of the first community multiprofessional palliative care teams is moving to work with establishing integrated teams within the Belfast Trust. Bridget has been an active core member of the network and here contribution has been much appreciated. Mrs Sharon Barr will attend in future. SPC_0809_02 Minutes & matters Arising from Meeting, 13th December 2007 No amendments were made to the draft minutes from the December meeting. These will be posted on the NICaN website for future reference. Palliative Care Research Following consultation, the response to the business case for the All Ireland Institute was forwarded on 22 February 2008 to Prof David Clark. Prof Judith Hill informed the group that terms of tender are now being developed. Awareness raising across academic institutions continues to engage interest in potential partnerships. Atlantic Philantrophies have offered financial support to the venture and match funding is being sought from across jurisdictions. Previous discussions at Network meetings have endorsed the need to establish a work strand for research and development within palliative and end of life care. To identify the body of interested parties and explore the strengths and weaknesses of a collaborative model for research, a workshop, - Building collaboration for Palliative and End of life Care Research -will take place on 4 June 10am - 2pm.in the Comfort Hotel.Antrim, The workshop will be chaired by Prof David Clark, Director of the International Observatory on End of Life Care. Prof Shelia Payne, Help the Hospices Chair in Hospice Studies and co director of the Cancer Experiences Collaborative will present the Experiences and Results from Research Collaborative. Feedback from this event will be brought back to the next meeting in September. SPC_0809_04 Patient Information pathways - a pathway for advanced disease Ms Danny Sinclair, NICaN Regional Coordinator for Patient Information informed the network of how patient information pathways have been developed in line with the Cancer Services Collaborative. Emerging themes, with regard to information needs of patients with advanced disease, are being identified from the work undertaken across the tumour groups. It is important to identify all information needs to develop a generic pathway of information resources for advanced disease to be endorsed by the Supportive and Palliative care network. This could be used across the all tumour specific information pathways and across organisational boundaries. The resulting pathway could potentially be used for non- cancer condition. A group is to be established to take this work forward. The group will: · Develop a list of advanced disease information themes · .Identify when they become relevant for the patient or their carer · .Identify existing resources · .Develop resources where needed · .Participate or nominate when review is required Dr Sheila Kelly nominated Helen Hume (SETrust) Paula Kealey will also contribute to this work; a nomination from the Patient and Public Information Forum has also been identified. A date will be circulated across the network to engage further interest and establish group SPC_0809_08 Development of a Regional Syringe Driver Prescription Chart Ms Kathy Stephenson reported that the second consultation of the draft regional syringe driver prescription chart and the focus group discussions, Pilots of the chart are to be undertaken within Trust, Hospices and General Practices. SPC_0809_05 A framework for Generalist and Specialist Palliative and End of Life Care Competency Dr Kathleen Dunne, lead of the Education works strand, reported on the findings following consultation of the Education framework. The report was widely appreciated across the network and valued as a significant and timely document for the commissioning of generalist and specialist adult palliative care education. Mr MacDonnell congratulated Dr Dunne and the members of the education workstrand for developing the framework aligning its significance to the underpinning needs of the regional model Amendments will be made to the document and then forwarded to the NICaN Board for endorsement. A process of implementation will be explored and reported to the network group at the September meeting. Key target areas for generalist palliative care education were highlighted within care of the elderly and general medicine. . SPC_0809_06 Pallcareni.net-a website for people with palliative care needs Ms Danny Sinclair, reminded the group of the pending amalgamation of the CAPriCORN and NICaN website. The resulting new web address will be www. cancerni.net. Recurrent funding has been secured to ensure the development of the supportive and palliative care website.www.Pallcareni.net The new website will host good information for people with palliative care needs, regardless of diagnosis. It will be accessible via the cancerni.net portal or independently as the pallcareni portal. It will signpost people with palliative care needs to condition- specific websites. The website will also enable the communication needs of the NI Regional Supportive & Palliative Care Network. This is a very significant method of seeking to enable greater understanding of palliative care for public and professionals, as highlighted within the regional model. Currently the material from the CAPriCORN website is being migrated onto cancerni and /or pallcareni.net as appropriate. To enable the further development of this opportunity a steering group of interested individuals is to be established. Their role will be to: · Drive the development of the website so it meets the needs of public and professionals through the sourcing and development of additional content · Identify any support that is needed, e.g. technical support · Review the website as a whole as it grows (coordinating condition-specific developments) · Review the functions of the website to aid communication throughout the Supportive and Palliative care network The steering group representation should reflect the constituencies within the Supportive and Palliative Care network. Current expressions of interest have come from Heather Reid and Valerie Peacock. A date will be circulated across the network to engage further interest and establish group SPC_0809_07 Update of Guidelines workstrand Dr Pauline Wilkinson presented the current work within the guidelines workstrand. 1. Brief Holistic Assessment & Referral Criteria to Specialist Palliative Care The development of an Holistic assessment Tool will help to identify holistic need at generalist and specialist level. Recognition of complex need prompts appropriate referral to specialist palliative care. The regional referral form is compatible with the Minimum Data set. The final drafts of this work are to be circulated widely, inclusive of service framework groups, primary care, secondary care and the supportive and palliative care network. Consultation will take place during June and July. Piloting of the forms will also be undertaken. 2. Control of Pain in Cancer Patients The original guidelines where developed 2003 and are now ready for review. The Mapping exercise, undertaken in May 2007, highlighted that the Guidelines were poorly adopted. The group have reviewed the pending SIGN 2 guidelines for pain with regard to practice in Northern Ireland. These are highly evidence based and are due to be launched this Summer. Whilst an excellent resource their comprehensiveness limits their readability, this may result in poor compliance. The Guidelines group feel it is important to have accessible and user-friendly guidelines particularly for Generalists and Out of hours. There are examples of good work that has taken place across the province, but there is a need for regional consistency. Dr Wilkinson has contacted Dr Carolyn Harper (Deputy CMO) and GAIN with regard to enabling funding to progress this work. The Guidelines group hope to approach the NICaN Primary Care Group to work in collaboratively on this piece, based on the templates already available. The works should be available in both electronic and paper versions. 3. Care of the dying & Breaking bad news Dr Gail Johnston has now completed an Audit of the Care of the Dying Pathways within the EHSSB. Gail is also seeking to examine to what extent the Regional Guidelines for Breaking Bad News are being implemented in the EHSSB with a view to identifying the need for further training or organisational structures that would facilitate future uptake. 4. Advances in new Technology Syringe Drivers Dr Wilkinson reported on a presentation made to the guidelines group by Mr Jim Elliot, Principle Engineer, Cardiology & Ann McLean, and Macmillan Palliative Care Nurse RVH. There is increasing concern with regard to how devices meet the recommended safety standards and how to reduce error. New devices have 3 point checking, automatic detection of syringe, automatic flow rates, full range of alarms, battery status and data download to provide an event log. There are now 2 companies in UK who have devices that meet these safety criteria. The current Graseby syringe drivers, which have been on the market and used predominately within Northern Ireland over the past 27 years Most new devices are not compatible with the regionally available monoject syringe, however contractual changes will lead to the withdrawal of the monoject syringes in October 2008. The Guidelines group supports a regional approach to this matter. This was echoed in the Supportive and Palliative care network. An option appraisal, identifying costs, and training issues should be developed through the engagement with Trusts and DHSSPSNI. The issue of Patient safety should be raised with the DHSSPSNI. SPC_0809_09 Evaluation of Supportive and Palliative Care network Deferred to next meeting. . SPC_0809_10 Emerging Issues Mrs Anne Coyle, Bereavement Coordinator, Southern Trust, announced that the Regional Bereavement Strategy is soon to be released. Anne supported the close alignment between the content of the strategy and the work of the regional model and other workstrands within the Supportive and Palliative care network. Ms Eleanor Donaghy, Transplant Coordinator, briefly highlighted the issue of tissue donation. Each year Northern Ireland has a dearth of corneal donations. There is no upper age limit for donation and retrieval is not limited by a cancer diagnosis. Recipients do not require immunosuppressive and the transplant is lifelong. The National Blood Service provided coordination of this donation they may be contacted via 07659180773. It is hoped that Mrs Coyle and Ms Donaghy could provide more comprehensive presentations at a future meeting. Events · Irish Psycho- Oncology Group Seminar, Cork 6 June, Exploring the Struggle for meaning in Cancer · Integrated Care: Putting Research into Practice, 13June, Trinity College, Dublin · Macmillan online conference Friday 13 June 2008, 9am - 5pm · Delivering effective end of life care: developing partnership working 15 Oct 2008, 9.30 -4.15 pm London Network Meeting was closed at 5.00pm SPC_0607_ Dates of Future Meetings (please note the change of venue) 10th September 2008, 1.30 - 5pm venue to be decided15th January 2009, 1.30 - 5pm venue to be decided12th May 2009, 1.30 - 5pm venue to be decided Attendances Apologies Stuart MacDonnellLorna NevinSonja McIlfatrick Donna FitzsimonsKathleen DunnePauline WilkinsonKathy StephensonSheila KellyMarie Nugent,Anne CoyleFiona GilmourJudith HillLorna DicksonMargaret CarlinLoretta GribbenYvonne Duff Lesley NelsonLiz HendersonSue FosterCathy PayneGraeme PaynePatricia MageeGeraldine WeatherupPaula KealyCaroline McAfeeLinda WrayValerie PeacockAnn McCleanRay Elder Martin BradleyHelen HumeGillian RankinHeather MonteverdeJulie DoyleAlison PorterYvonne SmythLiz Atkinson,Glynis HenryMaeve HullyCaroline HughesAnn FinnBob BrownSharon BarrJulie DoyleJanis McCulla .

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH - 95% (81.8-99.1), EMB - 94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.

Software de gestión de reclamaciones y denuncias on-line

Alta y gestión de reclamaciones de consumo a través de la web.

Healthy breaks: tasty tips for the under fives

Today the Public Health Agency is launching a new resource pack designed to assist nursery schools and playgroups deliver a healthy breaks scheme.All nursery schools and playgroups in Northern Ireland will receive the pack - 'Healthy breaks for pre-school children' - which includes a poster and information leaflets for parents explaining why a healthy break is so important for pre-school children and some tips and ideas for healthy nutritious breaks.Judith Hanvey, Regional Food in Schools Co-ordinator, appointed jointly by the PHA and the Department of Education, said: "Break-time snacking schemes currently operate in many pre-school settings across Northern Ireland, however they are implemented in different ways. The PHA has developed this new guidance so that any existing or future schemes have the information that they need to deliver a healthy break programme."Judith explained why a healthy break is so important for pre-school pupils."Young children have small stomachs but high requirements for energy (calories) and nutrients. This means that healthy snacks between meals are very important to make sure that they get all the nourishment they need."Childhood is also an important time to establish good eating and drinking habits for later life."Healthy eating schemes give children the knowledge and opportunity to make healthier choices."The Department of Education and Department of Health, Social Services and Public Safety recently launched 'Healthy Food for Healthy Outcomes: Food in Schools Policy', which builds on the nutritional standards which were introduced in 2007 by the Department of Education.The policy encourages a 'whole school approach' to all food and drink provided and consumed in schools. This ensures that children have the opportunity to benefit from a healthy balanced diet, which can aid learning, leading to improved educational outcomes. It also requires that every school should have their own whole-school food policy in place by September 2014. Nursery schools that receive funding from the Department of Education must follow this policy.This is also in line with the position taken by the Department of Health, Social Services and Public Safety's 10 year obesity prevention strategy, A Fitter Future for All, which aims to take a consistent approach to healthy food within early years settings.

DIP-STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures.

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP-STR that relies on pairing deletion-insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP-STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP-STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.

24 Hours in the Life of HIV-1 in a T Cell Line.

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

Estudio de la usabilidad en la administración pública municipal on-line

S'ha realitzat l'anàlisi del site www.tortosa.cat, realitzant un estudi d'usabilitat centrada en l'usuari sobre webs orientades al comerç electrònic i als serveis.

NEDD4-2 and salt-sensitive hypertension.

PURPOSE OF REVIEW: NEDD4-2 is an ubiquitin-protein ligase that was originally identified as an interactor of the epithelial Na+ channel (ENaC); this interaction is defective in Liddle's syndrome, causing elevated ENaC activity and salt-sensitive hypertension. In this review we aim to highlight progress achieved in recent years demonstrating that NEDD4-2 is involved in the control of Na+ transporters that are different from ENaC, but which also play a role in salt-sensitive hypertension. RECENT FINDINGS: It has been shown that NEDD4-2 interacts with ubiquitylates and negatively regulates the thiazide-sensitive NCC (Na+,Cl- -cotransporter), both in vitro and in vivo in inducible, nephron-specific Nedd4-2 knockout mice. Moreover, evidence has been provided that NEDD4-2 is also involved in the regulation of human NHE3 (Na+,H+-exchanger 3) and NKCC2 (Na+,K+,2Cl- -cotransporter 2). SUMMARY: The emerging role of NEDD4-2 in the regulation of different Na+ transporters along the nephron and the identification of human polymorphisms in the NEDD4-2 gene (Nedd4L) related to salt-sensitive hypertension makes this ubiquitin-protein ligase an interesting target for the development of antihypertensive drugs.

The role of CTCFL/BORIS in Meiosis

The complexity of mammalian genome organization demands a complex interplay of DNA and proteins to orchestrate proper gene regulation. CTCF, a highly conserved, ubiquitously expressed protein has been postulated as a primary organizer of genome architecture because of its roles in transcriptional activation/repression, insulation and imprinting. Diverse regulatory functions are exerted through genome wide binding via a central eleven zinc finger DNA binding domain and an array of diverse protein-protein interactions through N- and C- terminal domains. CTCFL has been identified as a paralog of CTCF expressed only in spermatogenic cells of the testis. CTCF and CTCFL have a highly homologous DNA-binding domain, while the flanking amino acid sequences exhibit no significant similarity. Genome- wide mapping of CTCF binding sites has been carried out in many cell types, but no data exist for CTCFL apart from a few identified loci. The lack of high quality antibodies prompted us to generate an endogenously flag-tagged CTCFL mouse model using BAC recombination. IHC staining using anti-flag antibodies confirmed CTCFL localization to type Β spermatogonia and preleptotene spermatocytes and a mutually exclusive pattern of expression with CTCF. ChIP followed by high-throughput sequencing identified 10,382 binding sites showing 70% overlap but representing only 20% of CTCF sites. Consensus sequence analysis identified a significantly longer binding motif with prominently less ambiguity of base calling at every position. The significant difference between CTCF and CTCFL genomic binding patterns proposes that their binding to DNA is differentially regulated. Analysis of CTCFL binding to methylated regions on a genome wide scale identified approximately 1,000 loci. Methylation-independent binding of CTCFL might be at least one of the mechanisms that ensures distinct binding patterns of CTCF and CTCFL since CTCF binding is methylation- sensitive. Co-localization of CTCF with cohesin has been well established and analysis of CTCFL and SMC3 overlap identified around 3,300 binding sites from which two related but distinct consensus sequence motifs were derived. Because virtually all data for cohesin binding originate from mitotically proliferating cells, the anticipated overlap is expected to be considerably higher in meiotic cells. Meiosis-specific cohesin subunit Rec8 is specific for spermatocytes and 6 out of the 12 identified binding sites are also bound by CTCFL. In conclusion, this was the first genome-wide mapping of CTCFL binding sites in spermatocytes, the only cell type where CTCF is not expressed. CTCFL has a unique binding site repertoire distinct from CTCF, binds to methylated sequences and shows a significant overlap with cohesin binding sites. Future efforts will be oriented towards deciphering the role CTCFL plays in conversion of chromatin structure and function from mitotic to meiotic chromosomes. - La complexité de l'organisation du génome des mammifères exige une interaction particulière entre ADN et protéines pour orchestrer une régulation appropriée de l'expression des gènes. CTCFL, une protéine ubiquitaire très conservée, serait le principal organisateur de l'architecture du génome de par son rôle dans l'activation / la répression de la transcription, la protection et la localisation des gènes. Diverses régulations sont opérées, d'une part au travers d'interactions à différents endroits du génome par le biais d'un domaine protéique central de liaison à l'ADN à onze doigts de zinc, et d'autre part par des interactions protéine-protéine variées au niveau de leur domaine N- et C-terminal. CTCFL a été identifié comme un paralogue de CTCF exprimé uniquement dans les cellules spermatiques du testicule. CTCFL et CTCF ont un domaine de liaison à l'ADN très homologue, tandis que les séquences d'acides aminés situées de part et d'autre de ce domaine ne présentent aucune similitude. Une cartographie générale des sites de liaison au CTCF a été réalisée pour de nombreux types cellulaires, mais il n'existe aucune donnée pour CTCFL à l'exception de l'identification de quelques loci. L'absence d'anticorps de bonne qualité nous a conduit à générer un modèle murin portant un CTCFL endogène taggué grâce à un procédé de recombinaison BAC. Une coloration IHC à l'aide d'anticorps anti-FLAG a confirmé la présence de CTCFL au niveau des spermatogonies de type Β et des spermatocytes au stade préleptotène, et une distribution mutuellement exclusive avec CTCF. Une méthode de Chromatine Immunoprecipitation (ChIP) suivie d'un séquençage à haut débit a permis d'identifier 10.382 sites de liaison montrant 70% d'homologie mais ne représentant que 20% des sites CTCF. L'analyse de la séquence consensus révèle un motif de fixation à l'ADN nettement plus long et qui comporte bien moins de bases aléatoires à chaque position nucléotidique. La différence significative entre les séquences génomiques des sites de liaison au CTCF et CTCFL suggère que leur fixation à l'ADN est régulée différemment. Appliquée à l'échelle du génome, l'étude de l'interaction de CTCFL avec des régions méthylées de l'ADN a permis d'identifier environ 1.000 loci. Contrairement à CTCFL, la liaison de CTCF dépend de l'état de méthylation de l'ADN ; cette modification épigénétique constitue donc au moins un des mécanismes de régulation expliquant une localisation de CTCF et CTCFL à des sites distincts du génome. La co- localisation de CTCF avec la cohésine étant établie, l'analyse de la superposition des séquences de CTCFL avec la sous-unité SMC3 identifie environ 3.300 sites de liaison parmi lesquels deux mêmes motifs consensus distincts par leur séquence sont mis en évidence. La presque quasi-totalité des données sur la cohésine ayant été établie à partir de cellules en prolifération mitotique, il est probable que la similitude au sein des séquences consensus soit encore plus grande dans le cas des cellules en méiose. La sous-unité Rec8 de la cohésine propre à l'état de méiose est spécifiquement exprimée dans les spermatocytes. Or 6 des 12 sites de liaison identifiés sont également utilisés par CTCFL. Pour conclure, ce travail constitue la première cartographie à l'échelle du génome des sites de liaison de CTCFL dans les spermatocytes, seul type cellulaire où CTCFL n'est pas exprimé. CTCFL possède un répertoire unique de sites de fixation à l'ADN distinct de CTCF, se lie à des séquences méthylées et présente un nombre important de sites de liaison communs avec la cohésine. Les perspectives futures sont d'élucider le rôle de CTCFL dans le remodelage de la structure de la chromatine et de définir sa fonction dans le processus de méiose.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

PFC J2EE : botiga de Llibres On-Line KIWIX

L'objectiu del present Projecte final de carrera consisteix en la realització d'un dissenye implementació d'un lloc web de venta de llibres al detall.El sistema mostra a l'usuari una sèrie de productes. Tots aquest productespoden ser adquirits per l'usuari prèvia identificació i validació de les dades.