Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Redox biology of myeloid leukaemias

Internal tandem duplication of FMS-like receptor tyrosine kinase (FLT3-ITD) has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (dsbs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Herein, we observe that the majority of H2O2 in FLT3-ITD-expressing MV4-11 cells colocalises to the endoplasmic reticulum (ER). Furthermore, ER localisation of ROS in MV4-11 cells corresponds to the localisation of p22phox, a small membrane-bound subunit of NOX complex. Furthermore, we show that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, possess higher steady protein levels of p22phox than their wild type FLT3 (FLT3-WT)-expressing counterparts. Moreover, the inhibition of FLT3-ITD, using various FLT3 tyrosine kinase inhibitors, uniformly results in a posttranslational downregulation of p22phox. We also show that depletion of NOX2 and NOX4 and p22phox, but not NOX1 proteins causes a reduction in endogenous H2O2 levels. We show that genomic instability induced by FLT3-ITD leads to an increase in nuclear levels of H2O2. The presence of H2O2 in the nucleus is largely reduced by inhibition of FLT3-ITD or NOX. Furthermore, similar results are also observed following siRNA knockdowns of p22phox or NOX4. We demonstrate that 32D cells transfected with FLT3-ITD have a higher level of DNA damage than 32D cells transfected with FLT3-WT. Additionally, inhibition of FLT3-ITD, p22phox and NOX knockdowns decrease the number of DNA dsbs. In summary, this study presents a novel mechanism of genomic instability generation in FLT3-ITD-expressing AML cells, whereby FLT3-ITD activates NOX complexes by stabilising p22phox. This in turn leads to elevated generation of ROS and DNA damage in these cells.

Traitement de données issues d'un corpus écrit multilingue. Approche agile pour l'analyse du discours eurorégional

L'article présente quelques éléments de la procédure mise en place pour traiter un corpus écrit comportant 617 textes (près de 500 000 mots) relatifs aux eurorégions. Complexe et hétérogène à plusieurs titres (technique, linguistique, éditorial, générique, énonciatif), le corpus pose la difficulté majeure de l’appréhension de données multilingues (français, italien, espagnol, anglais, allemand, néerlandais). Sa manipulation a nécessité une réflexion adaptée et une démarche de modélisation que nous qualifions d’« agile » en raison de son caractère souple et itératif. La plateforme d’analyse élaborée permet de disposer de résultats utiles à l’analyse qualitative ultérieure du discours eurorégional. Elle articule un logiciel d'analyse morphosyntaxique éprouvé (TreeTagger) à des programmes (Perl) et à une base de données (SQLite) développés pour optimiser les requêtes multilingues simultanées et l’exportation automatique des résultats. Les fonctionnalités liées à la localisation contextualisée de mots- pivots, au recueil de dénominations et à la détection de segments répétés nous servent ici de guides pour exprimer les besoins de la recherche, les problèmes rencontrés et les solutions proposées. L'analyse d'observables récurrents, à savoir les notions de décision et de responsabilité, illustre le propos.

Interactions spatiales et auto-organisation des végétations semi-arides

Les recherches récapitulées dans cette thèse de doctorat ont porté sur les causes de l’organisation spatiale des végétations périodiques. Ces structures paysagères aux motifs réguliers, tachetés, tigrés ou labyrinthiques, d’échelle décamétrique à hectométrique, couvrant des étendues considérables sur au moins trois continents, constituent un cas d’école dans l’étude des processus endogènes présidant à l’hétérogénéité du couvert végétal. Ces structures prennent place sur un substrat homogène, mis à part la rétroaction du couvert lui-même, et sont marquées par des écotones abrupts et la persistance d’une proportion considérable de sol nu. Plusieurs modèles ont mis en avant l’existence possible d’un phénomène d’auto-organisation du couvert, qui verrait une structure d’ensemble émerger des interactions locales entre individus. Ces modèles se basent sur le jeu simultané de la consommation de la ressource (compétition) et de l’amélioration de l’un ou l’autre des éléments du bilan de la même ressource par le couvert (facilitation). La condition à l’existence d’une structure d’ensemble spatialement périodique et stable réside dans une différence entre la portée de la compétition (plus grande) et celle de la facilitation. L’apparition de ces structures est modulée par le taux de croissance biologique, qui est le reflet des contraintes extérieures telles que l’aridité, le pâturage ou la coupe de bois. Le modus operandi des interactions spatiales supposées entre individus reste largement à préciser.

Nos recherches ont été menées au sud-ouest de la République du Niger, à l’intérieur et dans les environs du parc Régional du W. Trois axes ont été explorés :(i) Une étude de la dépendance spatiale entre la structure de la végétation (biovolumes cartographiés) et les paramètres du milieu abiotique (relief, sol), sur base d’analyses spectrales et cross-spectrales par transformée de Fourier (1D et 2D). (ii) Une étude diachronique (1956, 1975 et 1996) à large échelle (3000 km²) de l’influence de l’aridité et des pressions d’origine anthropique sur l’auto-organisation des végétations périodiques, basée sur la caractérisation de la structure spatiale des paysages sur photos aériennes via la transformée de Fourier en 2D. (iii) Trois études portant sur les interactions spatiales entre individus :En premier lieu, via l’excavation des systèmes racinaires (air pulsé) ;Ensuite, par un suivi spatio-temporel du bilan hydrique du sol (blocs de gypse) ;Enfin, via le marquage de la ressource par du deutérium.

Nous avons ainsi pu établir que les végétations périodiques constituent bien un mode d’auto-organisation pouvant survenir sur substrat homogène et modulé par les contraintes climatiques et anthropiques. Un ajustement rapide entre l’organisation des végétations périodiques et le climat a pu être montrée en zone protégée. La superficie et l’organisation des végétations périodiques y ont tour à tour progressé et régressé en fonction d’épisodes secs ou humides. Par contre, en dehors de l’aire protégée, la possibilité d’une restauration du couvert semble fortement liée au taux d’exploitation des ressources végétales. Ces résultats ont d’importantes implications quant à la compréhension des interactions entre climat et écosystèmes et à l’évaluation de leurs capacités de charge. La caractérisation de la structure spatiale des végétations arides, notamment par la transformée de Fourier d’images HR, devrait être généralisée comme outil de monitoring de l’état de ces écosystèmes. Nos études portant sur les modes d’interactions spatiales ont permis de confirmer l’existence d’une facilitation à courte portée du couvert végétal sur la ressource. Cependant, cette facilitation ne semble pas s’exercer sur le terme du bilan hydrique traditionnellement avancé, à savoir l’infiltration, mais plutôt sur le taux d’évaporation (deux fois moindre à l’ombre des canopées). Ce mécanisme exclut l’existence de transferts diffusifs souterrains entre sols nu et fourrés. Des transferts inverses semblent d’ailleurs montrés par le marquage isotopique. L’étude du bilan hydrique et la cartographie du micro-relief, ainsi que la profondeur fortement réduite de la zone d’exploitation racinaire, jettent de sérieux doutes quant au rôle communément admis des transferts d’eau par ruissellement/diffusion de surface en tant que processus clé dans la compétition à distance entre les plantes. L’alternative réside dans l’existence d’une compétition racinaire de portée supérieure aux canopées. Cette hypothèse trouve une confirmation tant par les rhizosphères excavées, superficielles et étendues, que dans le marquage isotopique, montrant des contaminations d’arbustes situés à plus de 15 m de la zone d’apport. De même, l’étude du bilan hydrique met en évidence les influences simultanées et contradictoires (facilitation/compétition) des ligneux sur l’évapotranspiration.

/

This PhD thesis gathers results of a research dealing with the causes of the spatial organisation of periodic vegetations. These landscape structures, featuring regular spotted, labyrinthine or banded patterns of decametric to hectometric scale, and extending over considerable areas on at least three continents, constitute a perfect study case to approach endogenous processes leading to vegetation heterogeneities. These patterns occur over homogeneous substratum, except for vegetation’s own feedbacks, and are marked by sharp ecotones and the persistence of a considerable amount of bare soil. A number of models suggested a possible case of self-organized patterning, in which the general structure would emerge from local interactions between individuals. Those models rest on the interplay of competitive and facilitative effects, relating to soil water consumption and to soil water budget enhancement by vegetation. A general necessary condition for pattern formation to occur is that negative interactions (competition) have a larger range than positive interactions (facilitation). Moreover, all models agree with the idea that patterning occurs when vegetation growth decreases, for instance as a result of reduced water availability, domestic grazing or wood cutting, therefore viewing patterns as a self-organised response to environmental constraints. However the modus operandi of the spatial interactions between individual plants remains largely to be specified.

We carried out a field research in South-West Niger, within and around the W Regional Park. Three research lines were explored: (i) The study of the spatial dependency between the vegetation pattern (mapped biovolumes) and the factors of the abiotic environment (soil, relief), on the basis of spectral and cross-spectral analyses with Fourier transform (1D and 2D). (ii) A broad scale diachronic study (1956, 1975, 1996) of the influence of aridity and human induced pressures on the vegetation self-patterning, based on the characterisation of patterns on high resolution remote sensing data via 2D Fourier transform. (iii) Three different approaches of the spatial interactions between individuals: via root systems excavation with pulsed air; via the monitoring in space and time of the soil water budget (gypsum blocks method); and via water resource labelling with deuterated water.

We could establish that periodic vegetations are indeed the result of a self-organisation process, occurring in homogeneous substratum conditions and modulated by climate and human constraints. A rapid adjustment between vegetation patterning and climate could be observed in protected zones. The area and patterning of the periodic vegetations successively progressed and regressed, following drier or wetter climate conditions. On the other hand, outside protected areas, the restoration ability of vegetation appeared to depend on the degree of vegetation resource exploitation. These results have important implications regarding the study of vegetation-climate interactions and the evaluation of ecosystems’ carrying capacities. Spatial pattern characterisation in arid vegetations using Fourier transform of HR remote sensing data should be generalised for the monitoring of those ecosystems. Our studies dealing with spatial interaction mechanisms confirmed the existence of a short range facilitation of the cover on water resource. However, this facilitation does not seem to act through the commonly accepted infiltration component, but rather on the evaporative rate (twice less within thickets). This mechanism excludes underground diffusive transfers between bare ground and vegetation. Inverse transfers were even shown by deuterium labelling. Water budget study and micro-elevation mapping, along with consistent soil shallowness, together cast serious doubts on the traditional mechanism of run-off/diffusion of surface water as a key process of the long range competition between plants. An alternative explanation lies in long range root competition. This hypothesis find support as well in the excavated root systems, shallow and wide, as in isotopic labelling, showing contaminations of shrubs located up to 15 m of the irrigated area. Water budget study also evidenced simultaneous contradictory effects (facilitation/competition) of shrubs on evapotranspiration.

Définition en discours de l'idéal-type eurorégional. Consensus et zones d'ombre autour d'une politique expérimentale

L'article examine comment s'affirme l'idéal-type émergent des eurorégions en Europe. En analysant les discours produits par des institutions, des acteurs économiques et des médias, nous reconstituons la définition du projet eurorégional à partir des diverses positions énonciatives et indépendamment des langues ou de la localisation géographique des eurorégions. D'un côté, les résultats mettent en évidence des métaphores caractéristiques du discours politique européen (la construction, l'expérimentation, le corps) qui contribuent à instaurer l'imaginaire d'un continuum territorial en Europe. D'un autre côté, les résultats dévoilent des zones d'ombre (dissensions, approximations, dispersions, concurrence) qui rendent la définition du projet eurorégional floue et difficile à appréhender pour le citoyen. L'analyse s'appuie sur un corpus authentique et multilingue en vue de déceler des régularités relatives au discours eurorégional. Elle mobilise des résultats textométriques simples mais vérifiables qui servent de repères à l'analyse qualitative.

Particle Dynamics Particulate Carbon And The Oxygen Minimum In An Estuary

Profiles of suspended particulate load and its organic and inorganic carbon contents as well as salinity, dissolved oxygen, ammonia and divalent manganese have been recorded throughout the mixing region of the Tamar Estuary,Southwest England, in late summer when there was pronounced net oxygen consumption. The results indicate that trapping of particulate organic detritus (of both riverine and marine origins) within the high turbidity zone contributes to the localisation and buffering of the seasonal oxygen demand exerted within the low salinity region of the estuary.

Biomonitoring of air pollutants by using lichens (Evernia prunastri) in areas between Kenitra and Mohammedia cities in Morocco.

In this study, Evernia prunastri, a lichen growing in its natural habitat in Morocco was analysed for the concentration of five heavy metals (Fe, Pb, Zn, Cu and Cr) from eleven sites between Kenitra and Mohammedia cities. The control site was Dar Essalam, an isolated area with low traffic density and dense vegetation. In the investigated areas, the concentration of heavy metals was correlated with vehicular traffic, industrial activity and urbanization. The total metal concentration was highest in Sidi Yahya, followed by Mohammedia and Bouznika. The coefficient of variation was higher for Pb and lower for Cu, Zn and Fe. The concentrations of most heavy metals in the thalli differed significantly between sites (p<0.01). Principal component analysis (PCA) revealed a significant correlation between heavy metal accumulation and atmospheric purity index. This study demonstrated also that the factors most strongly affecting the lichen flora were traffic density, the petroleum industry and paper factories in these areas. Overall, these results suggest that the index of atmospheric purity and assessment of heavy metals in lichen thalli are good indicators of the air quality at the studied sites.

K1 of Chevalley groups are nilpotent

Abstract Let F be a reduced irreducible root system and R be a commutative ring. Further, let G(F,R) be a Chevalley group of type F over R and E(F,R) be its elementary subgroup. We prove that if the rank of F is at least 2 and the Bass-Serre dimension of R is finite, then the quotient G(F,R)/E(F,R) is nilpotent by abelian. In particular, when G(F,R) is simply connected the quotient K1(F,R)=G(F,R)/E(F,R) is nilpotent. This result was previously established by Bak for the series A1 and by Hazrat for C1 and D1. As in the above papers we use the localisation-completion method of Bak, with some technical simplifications.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Resonance Raman and lifetime studies on regioselectively deuteriated ruthenium (II) polypyridyl complexes

Two series of ruthenium(II) polypyridyl complexes [Ru(bipy)2(phpytr)]+ and [Ru(bipy)2(phpztr)]+ (where Hphpytr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyridine and Hphpztr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyrazine) are examined by electrochemistry, UV/Vis, emission, resonance Raman, transient resonance Raman and transient absorption spectroscopy, in order to obtain a more comprehensive understanding of their excited state electronic properties. The interpretation of the results obtained is facilitated by the availability of several isotopologues of each of the complexes examined. For the pyridine-1,2,4-triazolato based complex the lowest emissive excited state is exclusively bipy based, however, for the pyrazine based complexes excited state localisation on particular ligands shows considerable solvent and pH dependency.

Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Latin American migration in the central area of Iquique: New fronts of residential location and unequal forms of access to housing

Historically the central area of the city of Iquique has been established as residential space migrants choosing from different backgrounds , however since the late 2000s migration flows are diversified being mostly Latin American immigrants who live in precarious conditions , accessing tugurizados properties , deteriorated in an increasingly growing informal market. The results presented here are derived from quantitative residential location of migrants , as well as the implementation of 13 in-depth interviews . From these results emerge that Latin American migrants access to the same places where once lived internal migrants, however they inhabit a restrictive market , uneven and inadequate living conditions lease, but allows them to articulate residence and proximity to industrial networks , social and popular trade.