Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Influence du climat, de la disponibilité des ressources et de la taille des populations sur la phénologie et les patrons de migration du caribou migrateur, "Rangifer tarandus"

De nombreuses populations migratrices sont actuellement en déclin. Les changements climatiques entrainent des modifications dans les habitats des espèces migratrices et la phénologie des processus naturels, lesquels se répercutent sur la migration, une période critique pour ces espèces. Comprendre comment les variables environnementales et climatiques affectent la phénologie et les patrons de migration est donc crucial. Ma thèse s’intéresse à l’impact du climat, des ressources alimentaires et de la compétition sur les migrations printanières et automnales des caribous migrateurs, Rangifer tarandus, des troupeaux Rivière-George (TRG) et Rivière-aux-Feuilles (TRF) du Nord-du-Québec et du Labrador. Le premier volet de ma thèse propose une approche objective, basée sur la détection des changements dans la structure des déplacements saisonniers, pour identifier les dates de départ et arrivée en migration. Validée à l’aide de trajets simulés, elle a été appliquée aux migrations printanières et automnales de femelles caribous. Le second volet porte sur l’impact des conditions environnementales sur la phénologie des migrations de printemps et d’automne. Il montre que la phénologie de la migration est principalement affectée par les conditions climatiques rencontrées lors de la migration, les conditions d’enneigement affectant notamment les coûts des déplacements. Au printemps, les caribous subissent des conditions défavorables lorsque la fonte des neiges est précoce. À l’automne, ils semblent ajuster leurs déplacements et migrent plus vite quand la neige débute tôt pour limiter les coûts de déplacement dans une neige profonde. Le troisième volet porte sur les patrons de migration à l’automne et montre que ceux-ci sont affectés essentiellement par une compétition intra- et inter-troupeaux pour les aires d’hivernages. Les caribous du TRG répondent à une augmentation de la compétition sur les aires les plus proches de l’aire de mise bas, liée à une taille de population élevée, en migrant préférentiellement vers les aires les plus éloignées. L’utilisation des aires hivernales par les caribous du TRF est, quant à elle, contrainte par la présence et l’abondance du TRG, cette contrainte diminuant à mesure que le TRG décline et abandonne les migrations vers les aires d’hivernages communes aux deux troupeaux. Cette thèse améliore notre compréhension de l’influence des facteurs environnementaux sur la phénologie et les patrons de migration du caribou migrateur. Ces connaissances sont très utiles pour comprendre l’impact des changements climatiques et établir les plans de conservation pour les espèces migratrices.

Wind Farm Facilities in Germany Kill Noctule Bats from Near and Far

Over recent years, it became widely accepted that alternative, renewable energy may come at some risk for wildlife, for example, when wind turbines cause large numbers of bat fatalities. To better assess likely populations effects of wind turbine related wildlife fatalities, we studied the geographical origin of the most common bat species found dead below German wind turbines, the noctule bat (Nyctalus noctula). We measured stable isotope ratios of non-exchangeable hydrogen in fur keratin to separate migrants from local individuals, used a linear mixed-effects model to identify temporal, spatial and biological factors explaining the variance in measured stable isotope ratios and determined the geographical breeding provenance of killed migrants using isoscape origin models. We found that 72% of noctule bat casualties (n = 136) were of local origin, while 28% were long-distance migrants. These findings highlight that bat fatalities at German wind turbines may affect both local and distant populations. Our results indicated a sex and age-specific vulnerability of bats towards lethal accidents at turbines, i.e. a relatively high proportion of killed females were recorded among migratory individuals, whereas more juveniles than adults were recorded among killed bats of local origin. Migratory noctule bats were found to originate from distant populations in the Northeastern parts of Europe. The large catchment areas of German wind turbines and high vulnerability of female and juvenile noctule bats call for immediate action to reduce the negative cross-boundary effects of bat fatalities at wind turbines on local and distant populations. Further, our study highlights the importance of implementing effective mitigation measures and developing species and scale-specific conservation approaches on both national and international levels to protect source populations of bats. The efficacy of local compensatory measures appears doubtful, at least for migrant noctule bats, considering the large geographical catchment areas of German wind turbines for this species.

Estimating Bat and Bird Mortality Occurring at Wind Energy Turbines from Covariates and Carcass Searches Using Mixture Models

Environmental impacts of wind energy facilities increasingly cause concern, a central issue being bats and birds killed by rotor blades. Two approaches have been employed to assess collision rates: carcass searches and surveys of animals prone to collisions. Carcass searches can provide an estimate for the actual number of animals being killed but they offer little information on the relation between collision rates and, for example, weather parameters due to the time of death not being precisely known. In contrast, a density index of animals exposed to collision is sufficient to analyse the parameters influencing the collision rate. However, quantification of the collision rate from animal density indices (e.g. acoustic bat activity or bird migration traffic rates) remains difficult. We combine carcass search data with animal density indices in a mixture model to investigate collision rates. In a simulation study we show that the collision rates estimated by our model were at least as precise as conventional estimates based solely on carcass search data. Furthermore, if certain conditions are met, the model can be used to predict the collision rate from density indices alone, without data from carcass searches. This can reduce the time and effort required to estimate collision rates. We applied the model to bat carcass search data obtained at 30 wind turbines in 15 wind facilities in Germany. We used acoustic bat activity and wind speed as predictors for the collision rate. The model estimates correlated well with conventional estimators. Our model can be used to predict the average collision rate. It enables an analysis of the effect of parameters such as rotor diameter or turbine type on the collision rate. The model can also be used in turbine-specific curtailment algorithms that predict the collision rate and reduce this rate with a minimal loss of energy production.

A Triple-Isotope Approach to Predict the Breeding Origins of European Bats

Despite a commitment by the European Union to protect its migratory bat populations, conservation efforts are hindered by a poor understanding of bat migratory strategies and connectivity between breeding and wintering grounds. Traditional methods like mark-recapture are ineffective to study broad-scale bat migratory patterns. Stable hydrogen isotopes (delta D) have been proven useful in establishing spatial migratory connectivity of animal populations. Before applying this tool, the method was calibrated using bat samples of known origin. Here we established the potential of delta D as a robust geographical tracer of breeding origins of European bats by measuring delta D in hair of five sedentary bat species from 45 locations throughout Europe. The delta D of bat hair strongly correlated with well-established spatial isotopic patterns in mean annual precipitation in Europe, and therefore was highly correlated with latitude. We calculated a linear mixed-effects model, with species as random effect, linking delta D of bat hair to precipitation delta D of the areas of hair growth. This model can be used to predict breeding origins of European migrating bats. We used delta C-13 and delta N-15 to discriminate among potential origins of bats, and found that these isotopes can be used as variables to further refine origin predictions. A triple-isotope approach could thereby pinpoint populations or subpopulations that have distinct origins. Our results further corroborated stable isotope analysis as a powerful method to delineate animal migrations in Europe.

De gata borralheira a Cinderela :nova espacialidade decorrente do desenvolvimento turístico, diferenciada pelo estilo de vida em Ponta Negra, Natal, RN

The transformations economical, social and politics in you finish them decades of the century XX brought changes that didn't just limit to the production system. The flexible accumulation took many workers lost her/it their workstations and they look for her/it new survival forms, migrating for administrative activities, of services rendered and for the tourist activity of small and medium load. The State has been investing in the implantation of plans of tourist development in order to create favorable conditions for the reproduction of the tourist activity in Brazil, mainly in the Northeast. A space when it starts to present a predominant economical activity suffers a restructuring in their social and economical relationships. The restructuring of these relationships takes to the construction of a new espacialidade. In the city of Christmas, in Rio Grande do Norte, the neighborhood of Black Tip is the most representative of the public investments for the tourist development. After intense process of tourist urbanization, Black Tip passed interfering in the global context consolidating as the tourist locus in the city. The tourist urbanization of the neighborhood took to the transformation of the space in merchandise that is sold and consumed as such. The recreation of fragments of other cultures brought by social actors, resulting from migratory processes stimulated by the tourist development, it has been presenting ruled social relationships in the informational technology, consumption of global goods and in the fragmentation of the urban space characterized by the internationalization and cosmopolitização. That process has been masking the inequalities partners and cultural as well as the territorial appropriation for an economical elite. The spaces are being appropriate for investors of the tourist section, private investors, agents and real estate producers, where the inequality is not just economical, but also cultural. The local population, mainly of the urban fraction of the Town of Black Tip, it doesn't participate of the productive process in function of the little or any professional qualification and he/she doesn't also have access to the consumption process. To the native ones it remains the fight for the preservation of his/her cultural identity and for the survival

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

Integração de alunos imigrantes e refugiados no Instituto Federal de Brasília - IFB

Dissertação apresentada para obtenção a grau de mestre na área de Educação Social e Intervenção Comunitária

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Social relations and dynamics shaping the implementation of the voluntary guidelines on small-scale fisheries (SSF guidelines) in South Africa

Integral to achieving the SSF Guidelines goal of targeting the most vulnerable and marginalized persons and eliminating discrimination is the need to have adequate understanding of the power relations and intersectionalities that shape access to and control over marine and other resources according to gender, age, race, ethnicity, labour and migratory status, disability, geographic location and other characteristics relevant in each national contexts. This monograph identifies and explores the key social relations and dynamics in the SSF fisheries sector in South Africa impacting the implementation of the SSF Guidelines. The monograph will be useful for researchers, scientists, fishworker organizations, environmentalists and anyone interested in the protection of marine biodiversity and the promotion of sustainable fisheries management.

CONSTRUCCIÓN DE ESPACIOS DE INFORMACIÓN PARA GARANTIZAR EL ACCESO AL CONOCIMIENTO EN LAS POBLACIONES INDÍGENAS: EL CASO DE COSTA RICA

Esta ponencia se basa en el proyecto CENTRO DE CONOCIMIENTO PARA GRUPOS INDÍGENAS CENTROAMERICANOS (GEIC), coordinado por la Escuela de Bibliotecología, Documentación e Información (EBDI) de la Universidad Nacional de Costa Rica, (UNA), su objetivo es “proponer la construcción de espacios de información para la población indígena, a partir del proyecto CENTRO DE CONOCIMIENTO PARA GRUPOS INDÍGENAS CENTROAMERICANOS y con esta ponencia se pretende presentar las lecciones aprendidas durante 2003-2007, años de recolección y análisis de datos, de establecimiento de relaciones, de coordinar actividades y ejecutar acciones tendientes a garantizar el cumplimiento al derecho de acceso a la información de las poblaciones indígenas costarricenses.El objetivo de GEIC fue crear un Centro de Conocimiento sobre/de Grupos Étnicos Indígenas Centroamericanos que sirviera de eje central para la consolidación del desarrollo de procesos tendientes a fortalecer la temática y el desarrollo de los grupos étnicos indígenas centroamericanos.El proyecto inició en el 2003, pero al no contar con personal permanente, se interrumpe su proceso hasta enero de 2004, con el desarrollo de la primera etapa, que comprende implementar cinco objetivos en Costa Rica. En posteriores etapas se espera integrar a los demás países centroamericanos.La población indígena costarricense corresponde a 63,876 personas, representando el 1.6% de la población nacional; existen ocho grupos socioculturales indígenas distintos, Cabécares, Bribris, Ngäbe, Térrabas, Borucas, Huetares, Malekus y Chorotegas, habitan en 24 territorios y hablan en 6 idiomas indígenas. A ellos se deben sumar poblaciones indígenas migratorias como los Miskitos de Nicaragua y Ngäbes de Panamá que trabajan en la producción agrícola en distintas zonas del país. El Proyecto GEIC, buscó la ejecución de la propuesta de creación de una unidad de información especializada en asuntos indígenas, en Shiroles Talamanca, para esto se realizó una investigación diagnóstica en la zona, determinando los recursos disponibles: tecnológicos, humanos, económicos y educacionales. En la actualidad se está gestionando y buscando financiamiento en instituciones locales, nacionales, e internacionales para cubrir los costos, aspecto que resulta un poco difícil por la falta de valoración de la importancia de la información en las comunidades indígenas.Otra actividad paralela a ésta es la construcción de un portal disponible en la dirección: http://www.una.ac.cr/bibliotecología/proyectogeic, y que fue avalado por las comunidades indígenas, con la participación de los y las protagonistas. En esa oportunidad se les explicó cada sección del mismo y se les solicitó sus observaciones y comentarios al respecto para involucrarlos(as) y se sintieran apropiados(as) de la misma.

When males are more inclined to stay at home :Insights into the partial migration of a pelagic seabird provided by geolocators and isotopes

Partial migration has never been studied in pelagic seabirds, but investigating old unresolved questions in new contexts can provide useful fresh insights. We used geolocators and stable isotopes to investigate this phenomenon in a migratory pelagic seabird, the Cory’s shearwater (Calonectris diomedea). Although most birds migrated to the southern hemisphere, 8.1% of studied birds (N = 172) remained close to the breeding colony (Selvagem Grande, Madeira, Portugal), foraging within the Canary current. Almost all resident birds were males, while age or body size did not predict migratory status. Despite displaying a high repeatability (R = 0.72) in the choice of wintering area, residency was not a fixed strategy and individuals could switch between migrating and staying in the Canary current in different years. The predictions resulting from the “body size” and the “social dominance” hypotheses, in which larger individuals or dominant individuals, respectively, remain closer to the breeding areas, were not supported by our data. Resident males were able to occupy the nesting burrows much earlier than migratory males and arrival time in this species is known to affect the probability of engaging in a reproductive attempt. The selective pressure to arrive early at the colony is therefore the most likely explanation for the maintenance of this partial migration system.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

La actividad laboral de las familias africanas y latinoamericanas reagrupadas en la España mediterránea

La investigación se basa en los resultados de una encuesta realizada en 2009-2010, por el equipo investigador, a 348 inmigrados reagrupantes africanos y a 457 latinoamericanos que residen en las provincias litorales entre Girona y Almería. La información que se ofrece está referida a los aspectos laborales de estos dos colectivos continentales, tanto a escala del conjunto territorial indicado como para tres subáreas incluidas en él: Cataluña litoral, Comunidad Valenciana y Murcia-Almería; según los temas, se trata por separado a los reagrupantes y a los subgrupos familiares que conviven en España (reagrupantes, cónyuges, hijos). El estudio se centra en las estructuras de empleo por sectores económicos. También se estudian las redes migratorias, tan decisivas en la orientación de estos flujos hacia España, la regulación laboral de estos trabajadores, la repercusión de la crisis actual en los empleos de las familias reagrupadas (paro, número de ocupados por familia e ingresos), la satisfacción laboral de los miembros de las familias (horas de trabajo, problemas laborales y salariales) y la estabilidad laboral (antigüedad en el empleo, cursos de formación profesional, trabajo actual y expectativas del inmigrante). El recorrido por los temas laborales indicados, vinculados a los distintos grupos continentales y familiares y a las diferentes escalas territoriales, concluyen en mostrar situaciones económicas y sociales muy diferentes entre africanos y latinoamericanos, y entre los que residen en Cataluña litoral y en Murcia-Almería, en lo que intervienen, entre otras causas, los distintos «capitales» personales aportados por los dos colectivos continentales para acceder al empleo (formación, idioma, …) y las diferentes estructuras económicas de los territorios estudiados. Los africanos y los que residen en las provincias meridionales son los que presentan peores situaciones socioeconómicas.