Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

An ATM distributed simulator for network management research

Due to the high cost of a large ATM network working up to full strength to apply our ideas about network management, i.e., dynamic virtual path (VP) management and fault restoration, we developed a distributed simulation platform for performing our experiments. This platform also had to be capable of other sorts of tests, such as connection admission control (CAC) algorithms, routing algorithms, and accounting and charging methods. The platform was posed as a very simple, event-oriented and scalable simulation. The main goal was the simulation of a working ATM backbone network with a potentially large number of nodes (hundreds). As research into control algorithms and low-level, or rather cell-level methods, was beyond the scope of this study, the simulation took place at a connection level, i.e., there was no real traffic of cells. The simulated network behaved like a real network accepting and rejecting SNMP ones, or experimental tools using the API node

Shape complexity based on mutual information

Shape complexity has recently received attention from different fields, such as computer vision and psychology. In this paper, integral geometry and information theory tools are applied to quantify the shape complexity from two different perspectives: from the inside of the object, we evaluate its degree of structure or correlation between its surfaces (inner complexity), and from the outside, we compute its degree of interaction with the circumscribing sphere (outer complexity). Our shape complexity measures are based on the following two facts: uniformly distributed global lines crossing an object define a continuous information channel and the continuous mutual information of this channel is independent of the object discretisation and invariant to translations, rotations, and changes of scale. The measures introduced in this paper can be potentially used as shape descriptors for object recognition, image retrieval, object localisation, tumour analysis, and protein docking, among others

Environmental influences on antibody-enhanced dengue disease outcomes

Because an enriched environment (EE) enhances T-cell activity and T-lymphocytes contribute to immunopathogenesis during heterologous dengue virus (DENV) infections, we hypothesised that an EE increases dengue severity. To compare single serotype (SS) and antibody-enhanced disease (AED) infections regimens, serial intraperitoneal were performed with DENV3 (genotype III) infected brain homogenate or anti-DENV2 hyperimmune serum followed 24 h later by DENV3 (genotype III) infected brain homogenate. Compared AED for which significant differences were detected between the EE and impoverished environmental (IE) groups (Kaplan-Meyer log-rank test, p = 0.0025), no significant differences were detected between the SS experimental groups (Kaplan-Meyer log-rank test, p = 0.089). Survival curves from EE and IE animals infected with the AED regimen were extended after corticoid injection and this effect was greater in the EE than in the IE group (Kaplan-Meyer log-rank test, p = 0.0162). Under the AED regimen the EE group showed more intense clinical signs than the IE group. Dyspnoea, tremor, hunched posture, ruffled fur, immobility, pre-terminal paralysis, shock and death were associated with dominant T-lymphocytic hyperplasia and presence of viral antigens in the liver and lungs. We propose that the increased expansion of these memory T-cells and serotype cross-reactive antibodies facilitates the infection of these cells by DENV and that these events correlate with disease severity in an EE.

Role of c-Myc in homeostasis of memory CD8 T cells

RESUME La mémoire immunologique est essentielle durant la vie et permet aux lymphocytes de répondre plus rapidement et efficacement lors d'une deuxième rencontre avec un antigène connu. Les facteurs contrôlant l'homéostasie des cellules T CD8 mémoires in vivo ne sont pas encore bien définis. Cependant, la prolifération homéostatique de ces cellules dans un hôte déplété en cellules hématopoietiques nécessite l'intéraction du TCR avec les molecules du MHC de class I du soi. De plus, le rôle de cytokines, telles que 1'IL-15 et l'IL-7, est essentiel dans ce mécanisme, aussi bien que dans la maintenance des cellules T CD8 mémoires. Puisque la protéine c-Myc - impliquée dans des mécanismes tells que la division, la prolifération, l'apoptose et la differentiation - a été définie comme étant impliquée dans la réponse à différentes cytokines, nous nous sommes intéressés à l'analyse de l'homéostasie des lymphocytes T CD8 mémoires dans des souris déficientes en c-Myc (c_rnycΔORF/+), qui expriment un niveau réduit de cette protéine. Bien que le développement des cellules T dans le thymus soit normal dans les souris c_rnycΔORF/+, nous avons observé une réduction de 2 à 3 fois dans la population des cellules T CD8 de phenotype mémoire (CD44+) dans les organes lymphoïdes de la périphérie de ces souris. Cette différence ne correspond pas à une réduction de prolifération ou d'expression de protéines de survie telles que Bel-2. Cependant, la prolifération homéostatique de cellules T CD8 c_rnycΔORF/+, mais pas T CD4 c_rnycΔORF/+, est reduite de manière dramatique lorsqu'elles sont transférées dans un hôte irradié. De plus, le transfert adoptif de lymphocytes T dans des souris irradiées déficientes en l'IL-15 nous a permis de montrer que la prolifération homéostatique dépendante de l'IL-15 des cellules T CD8 nécessite l'expression de c-Myc. De plus, contrairement aux cellules T CD8 CD44+ de type sauvage, nous avons observé que l'expansion induite par l'IL-15 des cellules T CD8 CD44+ c_rnycΔORF/+ est altérée aussi bien in vivo (en réponse à une injection de polyI:C) et in vitro. Par conséquent, nos résultats identifient c-Myc comme une nouvelle protéine régulatrice de la signalisation par l'IL-15 impliquée dans l'homéostasie des cellules T CD8 CD44+. SUMMARY Immunological memory is essential throughout life and allows memory lymphocytes to respond faster and more efficiently upon re-encounter of a known antigen. Factors controlling homeostasis of memory CD8 T cells under steady-state conditions in vivo are currently not well defined. However, the homeostatic proliferation of memory CD8 T cells in lymphopenic hosts requires the interaction of the TCR with self MHC class I molecules. In addition, cytokines, such as IL-15 and to a lesser extent IL-7, are essential for both homeostatic proliferation and maintenance of memory CD8 T cells. Since c-Myc, a proto-oncogene involved in cell division, proliferation, apoptosis and differentiation, has been widely implicated in responsiveness to cytokines, we were interested in analyzing homeostasis of memory CD8 T cells in c-myc hypomorph (c_rnycΔORF/+) mice, which express reduced levels of c-Myc. Although T cell development in the thymus was normal in c_rnycΔORF/+ mice, we found a selective 2- to 3-fold reduction in the memory-phenotype CD44high CD8 T cell population in the periphery. Reduced numbers of CD44high CD8 T cells did not correlate with decreased steady-state turnover rate or low expression of survival factors such as Bcl- 2. However, homeostatic proliferation of c_rnycΔORF/+ CD8 T cells, but not c_rnycΔORF/+ CD4 T cells, was dramatically reduced upon transfer into sublethally irradiated wild-type recipients. In addition, upon transfer of c_rnycΔORF/+ and c-myc WT cells into IL-15-/- mice, we observed that IL-15-induced homeostatic proliferation of CD8 T cells requires c-Myc. Moreover, in contrast to c-myc WT CD44high CD8 T cells, IL-15-induced expansion of c_rnycΔORF/+ CD44high CD8 T cells was strongly impaired both in vivo (in response to polyI:C injection) and in vitro. Collectively, our data identify c-Myc as a novel downstream regulator of IL-15 signaling involved in homeostasis of memory CD8 T cells.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Imagerie cardiaque non invasive: apport spécifique en clinique des nouvelles modalités (I). Evaluation morphologique [Cardiac imaging: specific clinical role of newly developed non invasive techniques. Part 1: Morphological evaluation].

Echocardiography is the preferred initial test to assess cardiac morphology and ventricular function. Cardiac MRI enables an optimal visualisation of heart muscle without contrast injection, and precise measurement of the ventricular volumes and systolic function. It is therefore an ideal test for patients with poor echocardiographic windows or for the specific evaluation of right heart chambers. Heart CT also remarkably images heart muscle and precisely measures ventricular systolic function after intravenous injection of iodinated contrast. Coronary CT may also, in selected cases, avoid the need for diagnostic coronary angiography. Although very accurate, these imaging modalities are expensive and may be contra-indicated for a particular patient. Their use in clinical practice has to follow the accepted guidelines.

Guidelines for the Conversion of Urban Four-Lane Undivided Roadways to Three-Lane Two-Way Left-turn Lane Facilities, April 2001

Four-lane undivided roadways in urban areas can experience a degradation of service and/or safety as traffic volumes increase. In fact, the existence of turning vehicles on this type of roadway has a dramatic effect on both of these factors. The solution identified for these problems is typically the addition of a raised median or two-way left-turn lane (TWLTL). The mobility and safety benefits of these actions have been proven and are discussed in the “Past Research” chapter of this report along with some general cross section selection guidelines. The cost and right-of-way impacts of these actions are widely accepted. These guidelines focus on the evaluation and analysis of an alternative to the typical four-lane undivided cross section improvement approach described above. It has been found that the conversion of a four-lane undivided cross section to three lanes (i.e., one lane in each direction and a TWLTL) can improve safety and maintain an acceptable level of service. These guidelines summarize the results of past research in this area (which is almost nonexistent) and qualitative/quantitative before-and-after safety and operational impacts of case study conversions located throughout the United States and Iowa. Past research confirms that this type of conversion is acceptable or feasible in some situations but for the most part fails to specifically identify those situations. In general, the reviewed case study conversions resulted in a reduction of average or 85th percentile speeds (typically less than five miles per hour) and a relatively dramatic reduction in excessive speeding (a 60 to 70 percent reduction in the number of vehicles traveling five miles per hour faster than the posted speed limit was measured in two cases) and total crashes (reductions between 17 to 62 percent were measured). The 13 roadway conversions considered had average daily traffic volumes of 8,400 to 14,000 vehicles per day (vpd) in Iowa and 9,200 to 24,000 vehicles per day elsewhere. In addition to past research and case study results, a simulation sensitivity analysis was completed to investigate and/or confirm the operational impacts of a four-lane undivided to three-lane conversion. First, the advantages and disadvantages of different corridor simulation packages were identified for this type of analysis. Then, the CORridor SIMulation (CORSIM) software was used x to investigate and evaluate several characteristics related to the operational feasibility of a four-lane undivided to three-lane conversion. Simulated speed and level of service results for both cross sections were documented for different total peak-hour traffic, access densities, and access-point left-turn volumes (for a case study corridor defined by the researchers). These analyses assisted with the identification of the considerations for the operational feasibility determination of a four -lane to three-lane conversion. The results of the simulation analyses primarily confirmed the case study impacts. The CORSIM results indicated only a slight decrease in average arterial speed for through vehicles can be expected for a large range of peak-hour volumes, access densities, and access-point left-turn volumes (given the assumptions and design of the corridor case study evaluated). Typically, the reduction in the simulated average arterial speed (which includes both segment and signal delay) was between zero and four miles per hour when a roadway was converted from a four-lane undivided to a three-lane cross section. The simulated arterial level of service for a converted roadway, however, showed a decrease when the bi-directional peak-hour volume was about 1,750 vehicles per hour (or 17,500 vehicles per day if 10 percent of the daily volume is assumed to occur in the peak hour). Past research by others, however, indicates that 12,000 vehicles per day may be the operational capacity (i.e., level of service E) of a three-lane roadway due to vehicle platooning. The simulation results, along with past research and case study results, appear to support following volume-related feasibility suggestions for four-lane undivided to three-lane cross section conversions. It is recommended that a four-lane undivided to three-lane conversion be considered as a feasible (with respect to volume only) option when bi-directional peak-hour volumes are less than 1,500 vehicles per hour, but that some caution begin to be exercised when the roadway has a bi-directional peak-hour volume between 1,500 and 1,750 vehicles per hour. At and above 1,750 vehicles per hour, the simulation indicated a reduction in arterial level of service. Therefore, at least in Iowa, the feasibility of a four-lane undivided to three-lane conversion should be questioned and/or considered much more closely when a roadway has (or is expected to have) a peak-hour volume of more than 1,750 vehicles. Assuming that 10 percent of the daily traffic occurs during the peak-hour, these volume recommendations would correspond to 15,000 and 17,500 vehicles per day, respectively. These suggestions, however, are based on the results from one idealized case xi study corridor analysis. Individual operational analysis and/or simulations should be completed in detail once a four-lane undivided to three-lane cross section conversion is considered feasible (based on the general suggestions above) for a particular corridor. All of the simulations completed as part of this project also incorporated the optimization of signal timing to minimize vehicle delay along the corridor. A number of determination feasibility factors were identified from a review of the past research, before-and-after case study results, and the simulation sensitivity analysis. The existing and expected (i.e., design period) statuses of these factors are described and should be considered. The characteristics of these factors should be compared to each other, the impacts of other potentially feasible cross section improvements, and the goals/objectives of the community. The factors discussed in these guidelines include • roadway function and environment • overall traffic volume and level of service • turning volumes and patterns • frequent-stop and slow-moving vehicles • weaving, speed, and queues • crash type and patterns • pedestrian and bike activity • right-of-way availability, cost, and acquisition impacts • general characteristics, including - parallel roadways - offset minor street intersections - parallel parking - corner radii - at-grade railroad crossings xii The characteristics of these factors are documented in these guidelines, and their relationship to four-lane undivided to three-lane cross section conversion feasibility identified. This information is summarized along with some evaluative questions in this executive summary and Appendix C. In summary, the results of past research, numerous case studies, and the simulation analyses done as part of this project support the conclusion that in certain circumstances a four-lane undivided to three-lane conversion can be a feasible alternative for the mitigation of operational and/or safety concerns. This feasibility, however, must be determined by an evaluation of the factors identified in these guidelines (along with any others that may be relevant for a individual corridor). The expected benefits, costs, and overall impacts of a four-lane undivided to three-lane conversion should then be compared to the impacts of other feasible alternatives (e.g., adding a raised median) at a particular location.

Three-phase 18F-fluorocholine PET/CT in the evaluation of prostate cancer recurrence.

AIM: Contribution of 3-phase 18F-fluorocholine PET/CT in suspected prostate cancer recurrence at early rise of PSA. PATIENTS, METHODS: Retrospective analysis was performed in 47 patients after initial treatment with radiotherapy (n=30) or surgery (n=17). Following CT, 10 minutes list-mode PET acquisition was done over the prostate bed after injection of 300 MBq of 18F-fluorocholine. Three timeframes of 3 minutes each were reconstructed for analysis. All patients underwent subsequent whole body PET/CT. Delayed pelvic PET/CT was obtained in 36 patients. PET/CT was interpreted visually by two observers and SUVmax determined for suspicious lesions. Biopsies were obtained from 13 patients. RESULTS: Biopsies confirmed the presence of cancer in 11 of 13 patients with positive PET for a total of 15 local recurrences in which average SUVmax increased during 14 minutes post injection and marginally decreased in delayed scanning. Conversely inguinal lymph nodes with mild to moderate metabolic activity on PET showed a clearly different pattern with decreasing SUVmax on dynamic images. Three-phase PET/CT contributed to the diagnostic assessment of 10 of 47 patients with biological evidence of recurrence of cancer. It notably allowed the discrimination of confounding blood pool or urinary activity from suspicious hyperactivities. PET/CT was positive in all patients with PSA>or=2 ng/ml (n=34) and in 4/13 patients presenting PSA values<2 ng/ml. CONCLUSION: 18F-fluorocholine 3-phase PET/CT showed a progressively increasing SUVmax in biopsy confirmed cancer lesions up to 14 minutes post injection while decreasing in inguinal lymph nodes interpreted as benign. Furthermore, it was very useful in differentiating local recurrences from confounding blood pool and urinary activity.

Improved immunogenicity against HIV-1 clade C antigens by using NYVAC-C with restored replication competence.

In order to improve the immunogenicity of currently available non-replicating pox virus HIV vaccine vectors, NYVAC was genetically modified through re-insertion of two host range genes (K1L and C7L), resulting in restored replicative capacity in human cells. In the present study these vectors, expressing either a combination of the HIV-1 clade C antigens Env, Gag, Pol, Nef, or a combination of Gal, Pol, Nef were evaluated for safety and immunogenicity in rhesus macaques, which were immunized at weeks 0, 4 and 12 either by scarification (conventional poxvirus route of immunization), intradermal or by intramuscular injection (route used in previous vaccine studies).Replication competent NYVAC-C-KC vectors induced higher HIV-specific responses, as measured by IFN- ELISpot assay, than the replication defective NYVAC-C vectors. Application through scarification only required one immunization to induce maximum HIV-specific immune responses. This method simultaneously induced relatively lower anti-vector responses. In contrast, two to three immunizations were required when the NYVAC-C-KC vectors were given by intradermal or intramuscular injection and this method tended to generate slightly lower responses. Responses were predominantly directed against Env in the animals that received NYVAC-C-KC vectors expressing HIV-1 Env, Gag, Pol, Nef, while Gag responses were dominant in the NYVAC-C-KC HIV-1 Gag, Pol, Nef immunized animals.The current study demonstrates that NYVAC replication competent vectors were well tolerated and showed increased immunogenicity as compared to replication defective vectors. Further studies are needed to evaluate the most efficient route of immunization and to explore the use of these replication competent NYVAC vectors in prime/boost combination with gp120 protein-based vaccine candidates. This studies was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

TOPSpro Data Dictionary, July 1, 2005–June 30, 2006

The passage of the Workforce Investment Act (WIA) of 1998 [Public Law 105-220] by the 105th Congress has ushered in a new era of collaboration, coordination, cooperation and accountability. The overall goal of the Act is “to increase the employability, retention, and earnings of participants, and increase occupational skill attainment by participants, and, as a result improve the quality of the workforce, reduce welfare dependency, and enhance the productivity and competitiveness of the Nation.” The key principles inculcated in the Act are: • Streamlining services; • Empowering individuals; • Universal access; • Increased accountability; • New roles for local boards; • State and local flexibility; • Improved youth programs. The purpose of Title II, The Adult Education and Family Literacy Act (AEFLA), of the Workforce Investment Act of 1998 is to create a partnership among the federal government, states, and localities to provide, on a voluntary basis, adult education and literacy services in order to: • Assist adults become literate and obtain the knowledge and skills necessary for employment and self-sufficiency; • Assist adults who are parents obtain the educational skills necessary to become full partners in the educational development of their children; • Assist adults in the completion of a secondary school education. Adult education is an important part of the workforce investment system. Title II restructures and improves programs previously authorized by the Adult Education Act. AEFLA focuses on strengthening program quality by requiring States to give priority in awarding funds to local programs that are based on a solid foundation of research, address the diverse needs of adult learners, and utilize other effective practices and strategies. To promote continuous program involvement and to ensure optimal return on the Federal investment, AEFLA also establishes a State performance accountability system. Under this system, the Secretary and each State must reach agreement on annual levels of performance for a number of “core indicators” specified in the law: • Demonstrated improvements in literacy skill levels in reading, writing, and speaking the English language, numeracy, problem solving, English language acquisition, and other literacy skills. • Placement in, retention in, or completion of postsecondary education, training, unsubsidized employment or career advancement. • Receipt of a secondary school diploma or its recognized equivalent. Iowa’s community college based adult basic education program has implemented a series of proactive strategies in order to effectively and systematically meet the challenges posed by WIA. The Iowa TOPSpro Data Dictionary is a direct result of Iowa’s pro-active efforts in this educational arena.

Increased therapeutic window for the R91W mutant form of Rpe65 compared to Rpe65 null backgroud

Purpose:Given the advances of gene therapy studies to cure RPE65-derived Leber Congenital Amaurosis (LCA) (clinical trials phase I) and the heterogeneity of the targeted patients both genetically and phenotypically, it is of prime importance to examine the rescue efficiency of gene transfer in different mutant contexts. Indeed, half of these mutations are missense mutations, leading to potential residual RPE65 activity. Consequently, we wanted to evaluate the effect on retinal activity and cone survival of lentivirus-mediated gene therapy in the R91W knock-in mouse model expressing the mutant Rpe65R91W gene (Samardzija et al. 2008), a mutation found in LCA patients. Notably we investigated whether if the therapeutic window is prolonged in comparison to null mutations. Methods:An HIV-1-derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. LV-R0.8-RPE65 or GFP was injected into 5-days-old (P5) or 1 month-old R91W mice. Functional rescue was assessed by ERG (1 and 4 months post-injection) and pupillary light response (PLR) recordings and cone survival by histological analysis. Results:Increased light sensitivity was detected by scotopic ERG in animals injected with LV-R0.8-RPE65 at both P5 and 1 month compared to GFP-treated animals or untreated mice. PLR was also improved in some eyes and histological analysis of cone markers showed that the density of cones reached the wild type level in the region of wt RPE65 delivery after treatment at P5. However, the rescue effect of the injection at 1 month was limited and attained 60% of the wild type level, but still more cones were observed in the treated area than in 1 month-old untreated Rpe65R91W mice. Conclusions:We were able to show that lentivirus-mediated Rpe65 gene transfer not only increases retinal activity of the Rpe65R91W mouse and survival of cones after treatment at P5 but also after treatment at 1 month. However even if the treatment at 1 month is more limited (60% of the wild type level) than treatment at P5, the amount of cone markers is increased compared to the proportion found at 1 month of age in untreated animals. This results contrast with the lack of cone rescue by treatment at 1 month of age in Rpe65-/- (Bemelmans et al, 2006). Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window.

Avaliação de necessidades de formação em professores do ensino secundário em Cabo Verde

II Resumo Cabo Verde é um país que tem acompanhado as grandes mudanças políticas, sociais, económicas e educativas, atribuindo um papel preponderante aos professores na educação/formação dos indivíduos. Neste sentido a formação inicial não pode constituir um acto “acabado” que não tem em linha de conta as mudanças que decorrem no seu seio. A formação contínua aparece com um processo de construção da mudança, apoiado no desenvolvimento profissional dos professores. A avaliação de necessidade de formação em professores do ensino secundário em Cabo Verde é o tema do trabalho de investigação, realizado nas escolas secundárias da ilha de Santo Antão, em Cabo Verde, no sentido de identificar questões problemáticas na formação inicial e contínua de professores e as suas implicações na qualidade educativa. Neste sentido, ao longo de 5 capítulos apresentamos os pontos desta investigação, estruturados de forma a se poder acompanhar a sua evolução. No 1º capítulo, apresenta-se a Problemática de Investigação, que constitui a parte inicial da dissertação, pela exposição do quadro conceptual do ensino secundário em Cabo Verde através da contextualização e identificação do problema, da formulação dos objectivos e questões de investigação. No 2º capítulo faz-se o percurso histórico/educativo de Cabo Verde, desde a época colonial, passando pela educação após a independência em 1975, à constituição da Lei de Bases do Sistema Educativo e a Reforma Educativa dos anos 90. No 3º capítulo, intitulado “A Formação Inicial e Continua de Professores em Cabo Verde”, é abordado o enquadramento teórico desta investigação, com referência à contextualização geral da formação e modelos de formação de professores, evoluindo para a realidade Cabo-verdiana, tendo em consideração as instituições de formação de professores e o contexto actual do ensino secundário em Cabo Verde. No 4º capítulo faz-se a apresentação da Metodologia de Investigação, adoptada num estudo extensivo, do qual abordamos a natureza da investigação e caracterizamos a amostra e respondentes. Como técnicas de recolha de dados, são utilizados: o inquérito por questionário (n=77), a professores do ensino secundário e o inquérito por entrevista (n=9), aos directores das escolas secundárias da ilha de Santo Antão e a formadores de professores em São Vicente. Como técnica de análise de dados, são utilizados diversos procedimentos estatísticos e a análise de conteúdo. O 5º capítulo corresponde a apresentação e discussão dos resultados da investigação desses três públicos analisados. Para finalizar, apresenta-se uma conclusão que corresponde a síntese dos resultados obtidos, propostas de sugestões de melhoria e limitações do estudo. Cabo Verde is a country willing to follow the great political, social, economic and educational changes. This important role have attributed to teachers to educate and train individuals. In this sense the initial training cannot be considered as a “finished” task without taking into consideration those changes. The in service training appears as a scientific and pedagogic autonomization towards the growing change. The Evaluation of Secondary School teacher training Needs in Cabo Verde constitutes an investigation work, carried out in Santo Antão's island Secondary schools, aiming to identify problems in initial and continuous teachers' training and their implications in the educational quality. In this sense, along five chapters we will present the points of this investigation, structured to facilitate the understanding of its evolution. In the first chapter we present the Problem of Investigation which is an introductory part of the dissertation, the conceptual theory of the Cabo Verde secondary education through the contextualization and identification of the problem, the formulation of the objectives and investigation issues. In the second chapter we present the Cabo Verde historical/educational background, from the colonial time, going through after independence in 1975, to the constitution of the Basic Educational Law of the System and the nineties Educational Reform. In the third chapter entitled “The Initial and in-service Teacher´s Training in Cabo Verde” brings the theoretical framework of this investigation, where we present a general contextualization of teachers' training and models, developed from the reality of Cabo Verde. In this regard we will consider the teachers' training institutions and the current secondary education context in Cabo Verde. In the fourth chapter we present the Research Methodology, adopted in an extensive study, of which we draw the nature of the investigation and we characterized the sample. We carried out questionnaire (n=77) to teachers, interviews (n=9) to headmasters from Santo Antão's island secondary schools and trainers from São Vicente. We used statistical analyses and content analyses as data analyse techniques. The fifth chapter deals with presentation the discussion of results of those three analyzed publics' investigation. Finally, we present a summary of the obtained results, suggestions for improvement and limitations of study.

Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice.

We have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen monoclonal antibodies (MAb) coupled to fluorescein. The most common clinical application of photodiagnosis has been for the detection of squamous cell carcinomas (SCC) in the upper respiratory tract, but the free dyes used have a poor tumor selectivity. We selected the known MAb E48 directed against SCC and coupled it to a fluorescent dye: indopentamethinecyanin (indocyanin). This dye has an advantage over fluorescein in that it emits a more penetrating fluorescent red signal at 667 nm after excitation with a laser ray of 640 nm. In vitro, an conjugate with an indocyanin:MAb molar ratio of 2, and an additional trace labeling with 125I, showed more than 80% of binding to cells from the SCC line A431. In vivo, when injected i.v. into nude mice bearing xenografts of the same carcinoma line, the MAb E48-(indocyanin)2 conjugate was almost as efficient as the unconjugated MAb E48 in terms of specific tumor localization: 15% of the injected dose per g of tumor at 24 h after injection and a tumor:overall normal tissue ratio of 6-8. There was no selective tumor localization of an irrelevant IgG1-(indocyanin)2 conjugate. Immunophotodetection of the s.c. SCC xenografts on mice given injections of 100 micrograms of MAb E48-(indocyanin), conjugate (representing 1 microgram of indocyanin) was performed at 24 h. Upon laser irradiation, clearly detectable red fluorescence from the indocyanin-MAb conjugate was observed specifically in the SCC xenografts across the mouse skin. In comparison, injection of 100 micrograms of a MAb E48 coupled to 2 micrograms of fluorescein gave a specific green fluorescence signal in the tumor xenografts, which was detectable, however, only after removing the mouse skin. Injection i.v. of a 15 times higher amount of free indocyanin (15 micrograms) gave a diffuse red fluorescence signal all over the mouse body with no definite increase in intensity in the tumor, indicating a lack of tumor selectivity of the free dye. The results demonstrate the possibility of broadening and improving the efficiency of tumor immunophotodiagnosis by coupling to a MAb directed against SCC, a fluorescent dye absorbing and emitting at higher wavelength than fluorescein, and thus having deeper tissue penetration and lower tissue autofluorescence. Such a demonstration opens the way to a new form of clinical immunophotodiagnosis and possibly to the development of a more specific approach to phototherapy of early bronchial carcinomas.

Valved stents for transapical pulmonary valve replacement.

OBJECTIVES: Pulmonary valve insufficiency remains a leading cause for reoperations in congenital cardiac surgery. The current percutaneous approach is limited by the size of the access vessel and variable right ventricular outflow tract morphology. This study assesses the feasibility of transapical pulmonary valve replacement based on a new valved stent construction concept. METHODS: A new valved stent design was implanted off-pump under continuous intracardiac echocardiographic and fluoroscopic guidance into the native right ventricular outflow tract in 8 pigs (48.5 +/- 6.0 kg) through the right ventricular apex, and device function was studied by using invasive and noninvasive measures. RESULTS: Procedural success was 100% at the first attempt. Procedural time was 75 +/- 15 minutes. All devices were delivered at the target site with good acute valve function. No valved stents dislodged. No animal had significant regurgitation or paravalvular leaking on intracardiac echocardiographic analysis. All animals had a competent tricuspid valve and no signs of right ventricular dysfunction. The planimetric valve orifice was 2.85 +/- 0.32 cm(2). No damage to the pulmonary artery or structural defect of the valved stents was found at necropsy. CONCLUSIONS: This study confirms the feasibility of direct access valve replacement through the transapical procedure for replacement of the pulmonary valve, as well as validity of the new valved stent design concept. The transapical procedure is targeting a broader patient pool, including the very young and the adult patient. The device design might not be restricted to failing conduits only and could allow for implantation in a larger patient population, including those with native right ventricular outflow tract configurations.