Rozprawy doktorskie na temat „Invasive phenotype”

Thioredoxin is a small ubiquitous oxido-reductase found in all species. The highly conserved active site, which facilitates thioredoxins redox activity, contains two redox active cysteine residues. Thioredoxin has numerous protein substrates to which it donates H+ ions and it can also function as a free radical scavenger. Through these activities thioredoxin is able to influence the redox state of not only its protein targets, but also the entire cellular environment. Thioredoxin has been implicated in many biological functions, and one mechanism by which it influences these functions is through interactions with a number of transcription factors including NF-kappa-B and p53. Thioredoxin also has numerous extracellular biological roles. It has been shown that thioredoxin is actively secreted from a number of normal and transformed cell lines including fibroblasts and activated B and T cells. This study investigates the role of thioredoxin in embryonic implantation and cancer cell metastasis, two physiological functions which rely on the same basic processes. Thioredoxin expression has previously been shown to be increased in many cancers. However it has not yet been established whether this increase is a causative or a side effect of the cancerous phenotype. Similarly thioredoxin expression has previously been shown to be increased during different phases of the oestrus cycle and pregnancy. This thesis describes the role of thioredoxin in embryonic implantation using a marmoset model. A thioredoxin cDNA was isolated and subsequently sequenced. Preliminary antibody experiments indicated that the anti human thioredoxin monoclonal antibodies available in our laboratory would recognise marmoset thioredoxin. Subsequently immunocytochemistry using anti human thioredoxin antibodies was carried out on sectioned marmoset uterus and embryonic tissue. The results indicated that thioredoxin is expressed by cells at the embryonic-maternal interface of early implantation sites. Further studies demonstrated that thioredoxin is also expressed and secreted by cultured blastocysts in vitro. This thesis also describes the role of thioredoxin in cancer cell metastasis. Results of this study indicate that thioredoxin is actively involved in facilitating the invasive phenotype of breast cancer cells. The two cell lines utilised were MCF-7, a well differentiated, relatively non-invasive breast cancer cell line; and MDA-MB-231, a poorly differentiated, highly invasive breast cancer cell line. The cell lines were transfected with thioredoxin sense, antisense and 1SS (encodes thioredoxin with both active cysteine residues mutated to serine residues and is thus redox inactive) constructs. The results demonstrate that when endogenous thioredoxin levels are increased, i.e. transfected with a sense thioredoxin construct, the invasive breast cancer cell line MDA-MB-231 becomes more invasive, conversely when endogenous levels are decreased, i.e. transfected with antisense or 1SS constructs, the invasive capacity of these cells decreases. However, when the endogenous level of thioredoxin was manipulated in the relatively non-invasive cell line MCF-7 very little effect was observed. Results also indicate that thioredoxin has the ability to act as a chemoattractant for actively invading breast cancer cells. Both of these functions appear to be dependent on thioredoxin's redox activity. Additional studies described in this thesis have shown that thioredoxin is involved in the regulation of Sp1 in vitro. Sp1 is a transcription factor known to regulate the transcription of a number of genes whose products are intimately involved in the invasive phenotype. The results in this study suggest that Sp1 DNA binding is regulated by thioredoxin such that when reduced by the enzyme its binding to DNA is facilitated. Results also indicate that Sp1 may regulate the transcription of thioredoxin by binding to Sp1 sites within the thioredoxin promoter.

Thioredoxin is a small ubiquitous oxido-reductase found in all species. The highly conserved active site, which facilitates thioredoxins redox activity, contains two redox active cysteine residues. Thioredoxin has numerous protein substrates to which it donates H+ ions and it can also function as a free radical scavenger. Through these activities thioredoxin is able to influence the redox state of not only its protein targets, but also the entire cellular environment. Thioredoxin has been implicated in many biological functions, and one mechanism by which it influences these functions is through interactions with a number of transcription factors including NF-_B and p53. Thioredoxin also has numerous extracellular biological roles. It has been shown that thioredoxin is actively secreted from a number of normal and transformed cell lines including fibroblasts and activated B and T cells. This study investigates the role of thioredoxin in embryonic implantation and cancer cell metastasis, two physiological functions which rely on the same basic processes. Thioredoxin expression has previously been shown to be increased in many cancers. However it has not yet been established whether this increase is a causative or a side effect of the cancerous phenotype. Similarly thioredoxin expression has previously been shown to be increased during different phases of the oestrus cycle and pregnancy. This thesis describes the role of thioredoxin in embryonic implantation using a marmoset model. A thioredoxin cDNA was isolated and subsequently sequenced. Preliminary antibody experiments indicated that the anti human thioredoxin monoclonal antibodies available in our laboratory would recognise marmoset thioredoxin. Subsequently immunocytochemistry using anti human thioredoxin antibodies was carried out on sectioned marmoset uterus and embryonic tissue. The results indicated that thioredoxin is expressed by cells at the embryonic-maternal interface of early implantation sites. Further studies demonstrated that thioredoxin is also expressed and secreted by cultured blastocysts in vitro. This thesis also describes the role of thioredoxin in cancer cell metastasis. Results of this study indicate that thioredoxin is actively involved in facilitating the invasive phenotype of breast cancer cells. The two cell lines utilised were MCF-7, a well differentiated, relatively non-invasive breast cancer cell line; and MDA-MB-231, a poorly differentiated, highly invasive breast cancer cell line. The cell lines were transfected with thioredoxin sense, antisense and 1SS (encodes thioredoxin with both active cysteine residues mutated to serine residues and is thus redox inactive) constructs. The results demonstrate that when endogenous thioredoxin levels are increased, i.e. transfected with a sense thioredoxin construct, the invasive breast cancer cell line MDA-MB-231 becomes more invasive, conversely when endogenous levels are decreased, i.e. transfected with antisense or 1SS constructs, the invasive capacity of these cells decreases. However, when the endogenous level of thioredoxin was manipulated in the relatively non-invasive cell line MCF-7 very little effect was observed. Results also indicate that thioredoxin has the ability to act as a chemoattractant for actively invading breast cancer cells. Both of these functions appear to be dependent on thioredoxin's redox activity. Additional studies described in this thesis have shown that thioredoxin is involved in the regulation of Sp1 in vitro. Sp1 is a transcription factor known to regulate the transcription of a number of genes whose products are intimately involved in the invasive phenotype. The results in this study suggest that Sp1 DNA binding is regulated by thioredoxin such that when reduced by the enzyme its binding to DNA is facilitated. Results also indicate that Sp1 may regulate the transcription of thioredoxin by binding to Sp1 sites within the thioredoxin promoter.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

The infiltrative growth pattern in colorectal cancer has been demonstrated to confer a poor prognosis particularly in early stage disease. The phenotype demonstrates histological features of aggression and with reference to the contrasting pushing phenotype provides an in vivo model for invasive behaviour which has hitherto not been exploited in the context of molecular biology.

The purpose of this study was to determine the differences in DNA damage responses (DDRs) between immortalized normal prostate epithelial cells (PrEC) and prostate cancer cells (DU145). To study the DDRs, two protein markers were used. The first, phosphorylated KRAB Associated Protein 1 (pKAP-1) activates target genes involved in the DDR. The second, phosphorylated H2AX (pH2AX) is required for the assembly of damaged chromatin as well as for activation of checkpoints proteins. Ionizing Radiation (IR) was used to induce DNA damage. Flow Cytometry and Western Blot analyses were used to measure the levels of the proteins when exposed to various doses of IR (0, 2, 4, 8 Gy). In both cell lines, there was a clear dose dependent increase in pH2AX and pKAP-1, however error bars showed that with the pH2AX protein, only the later doses showed significant differences. Fluorescence staining was used to examine the localization of the proteins. Both cell lines showed punctate staining of pH2AX in the nuclei at 8Gy and no staining in unirradiated cells. With pKAP-1, the PrEC cells had punctate staining in the nuclei with 8Gy of radiation whereas the DU145 cells had a diffuse pattern in the nuclei.

In the current study, we initially investigated the molecular mechanisms driving tumour cell migration and invasion in vitro using in-house generated invasive CRC cell line models, with which we reported an association between HGF-independent overexpression and hyper-phosphorylation of c-MET with a mesenchymal, migratory and invasive phenotype. Upon identification of this association, we subsequently demonstrated that RNAi-mediated ablation of c-MET abolished the basal migratory and invasive potential of CRC cell lines, illustrating a key role for c- MET expression in regulation of these phenotypes. Using a molecular pathology based methodology incorporating RNAscope®, a novel quantitative mRNA in situ hybridisation technique, we translated our observations in vivo, whereby we demonstrated that the MET transcript is significantly upregulated in tumour budding foci at the invasive edge of stage III CRC tumours. Upon evaluation of the role of the TME in dictating tumour cell phenotype using an in vitro coculture methodology, we highlighted a role for colonic myofibroblasts in promoting a migratory and invasive phenotype, and minor resistance to MEK1/2 inhibition in KRASMT CRC cell lines. Importantly, subsequent molecular characterisation revealed HG F as a major fibroblast-derived factor that was promoting these phenotypes through phosphorylation of c-MET, whereby neutralisation of HGF could partially abolish the pro-invasive fibroblast-derived signal. Finally, in attempting to assess the role of c-MET as a prognostic biomarker in early stage CRC, we demonstrated an apparent disparity between the prognostic value of MET gene expression and cMET protein expression, whereby only the former could predict poor outcome. Collectively, our study provides pre-clinical rationale for further investigation of therapeutic inhibition of the HGF/cMET pathway in the adjuvant setting to circumvent metastasic dissemination and therefore halt recurrence.

The invasive and destructive properties of pannus tissue contribute to the loss of cartilage and bone in rheumatoid arthritis (RA). To further analyze the process of cartilage degradation, we have developed an in vitro model which allows for observations of cellular degradation and invasion of a cartilage matrix isolated from porcine knee joints. This matrix contains collagen type II, proteoglycans and glycosaminoglycans, and is similar in composition to human cartilage. Synovial fibroblasts isolated from the pannus tissue of patients with aggressive RA, based on clinical evaluation, demonstrated a highly invasive phenotype in this in vitro model; whereas synovial fibroblasts isolated from pannus tissue of less aggressive RA, based on clinical evaluation, showed a less invasive phenotype. Further characterization of cell-cartilage matrix interactions reveal that RA and normal synovial fibroblasts adhere to a similar degree to cartilage matrix, but this adhesion can be inhibited with arginine-glycine-aspartic acid (RGD) peptides, thus implicating integrins in this attachment process. During the subsequent process of spreading on cartilage matrix, highly invasive RA synovial fibroblasts maintained a round phenotype for a longer duration than normal synovial fibroblasts. Furthermore, with respect to the degradative ability of these cells, the level of expression and secretion of key metalloproteinases was measured, and interstitial collagenase activity was shown to correlate with the RA phenotype, while no expression was detected in normal synovial fibroblasts. By comparison, there was no differential expression of stromelysin, gelatinase A and gelatinase B found in normal synovial fibroblasts versus RA synovial fibroblasts. Normal synovial fibroblast invasion was augmented by culturing these cells in the presence of 5 U/ml IL-1β or 18 U/ml TGFβ whereas, PDGF at 100 ng/ml did not affect normal synovial fibroblast invasion. In addition, normal synovial fibroblasts cultured in 150 U/ml TNFα demonstrated a significant decrease in their ability to invade cartilage matrix. Collectively, these in vitro data suggest that highly invasive synovial fibroblasts correlate with advanced RA clinical disease. Moreover, this invasive phenotype may be due, in part, to decreased spreading ability on the cartilage matrix in addition to interstitial collagenase activity. This model allows for further molecular characterization of the invasive properties of the synovial fibroblast.

L’huître creuse du Pacifique Crassostrea gigas est une espèce d’intérêt économique en France. L’extension de cette espèce pendant le dernier siècle a rapidement conduit au développement de populations sauvages. C. Gigas est, à présent, considérée comme une espèce invasive dans de nombreuses régions du globe. L’objectif principal de ce travail est de comprendre les capacités adaptatives de cette espèce, à l’aide d’une approche génétique et physiologique à différentes échelles spatiales et temporelles. A macro-échelle (le long des côtes françaises) et à micro-échelle (bas et haut d’estran), une différenciation génétique significative a été observée entre les population adultes à l’aide de marqueurs exoniques et allozymiques. A contrario, cette différence n’a pas décelée par les microsatellites. Dans trois sites bretons, une structuration génétique significative a été observée entre les adultes et les jeunes recrues des années 2005 et 2006, e également entre ces recrues et des naissains cultivés. Dans la seconde partie de cette étude, plusieurs indicateurs physiologiques de santé et de la « fitness » ont été estimés sur des adultes des trois sites cités précédemment. De fortes différences significatives ont été observées entre des individus de sexe et de stade de gamétogenèse différent, au cours de l’année 2005. Cependant, aucune différence majeure des performances physiologiques n’a été révélée entre les adultes à micro- et macro-échelles spatiales. En conclusion, l’ensemble de ces résultats suggère un fort potentiel invasif de C. Gigas, résultant d’une plasticité phénotypique et surtout d’une sélection locale appliquée sur sa forte diversité génétique
The Pacific oyster Crassostrea gigas is an important commercial species in France. The wide distribution of this species during the last decade quickly led to the developmeht of “wild” populations mainly in rocky intertidal areas. C. Gigas is now considered an invasive species in various parts of the world. The main objective of this work was to characterize the adaptative capabilities of C. Giqas in Brittany using genetic and physiological approaches at different spatial and temporal scales. Using exonic markers, significant genetic structures were observed between populations of adult oysters along the French coasts. In contrast, this difference was not observed using microsatellites. The study at the micro-scale, demonstrated no significant difference in allelic frequencies between oysters at high and low tidal heights, except at the MPI locus via allozymes analysis. At three sites f rom Brittany, significant genetic structure was observed between adult and juvenile oysters recruited in 2005 and 2006 and also between these same juveniles and cultivated spat. In the second part of this study, several physiological indicators of health and fitness-related traits were assessed on adults from the three sites described above. Strong significant differences were found between sex and gametogenesis stages throughout the year 2005. In contras no differential physiological performance was illustrated between adults at macro and micro-spatial scales. In conclusion, aIl of these results suggest a high potential of invasion for as that correspond to the compound of phenotypic plasticity and all above local selection applied to its high genetic diversity

Changes in the microenvironment have been implicated in the transition of pre-invasive ductal carcinoma in-situ (DCIS) to invasive breast cancer. Normal myoepithelial cells have a tumour suppressor phenotype but they are altered in DCIS and ultimately lost with transition to invasive cancer. A consistent change in DCIS is up-regulation of the integrin αvβ6 in myoepithelial cells. Preliminary observations identified a correlation between myopeithelial αvβ6 and an increased peri-ductal inflammatory infiltrate. The hypothesis of this study is that the altered myoepithelial phenotype influences the peri-ductal inflammatory environment, which in turn mediates a pro-apoptotic effect on myoepithelial cells contributing to their loss. To investigate this, the inflammatory infiltrate was characterised in a series of DCIS tissue in relation to αvβ6 status. This demonstrated significantly higher levels of CD4+ve and FOXP3+ve T cells around αvβ6+ve DCIS ducts compared to αvβ6-ve ducts (P=<0.01), suggesting an increase in Treg cells. In-vivo, Matrigel plugs containing injected into the flanks of female C57/Blk6 normal mice generated influx of higher levels of CD4+ve cells (p=0.005) and FOXP3+ T cells (p=0.007) in the presence of αvβ6+ve myoepithelial cells compared to αvβ6-ve cells, supporting the findings in human tissue samples. Since Treg cells produce TRAIL that can induce apoptosis, we investigated the influence of αvβ6 on myoepithelial cells on the levels of TRAIL in T cells and the hypothesis that αvβ6-positive myoepithelial ells may be more susceptible to TRAIL-induced apoptosis, leading to loss of the myoepithelial barrier. Firstly, levels of TRAIL in Jurkat and primary T cell populations co-cultured with β4 (ii) or β6 myoepithelial cells were measured. This demonstrated a higher level of TRAIL in primary T cells co-cultured β6 myoepithelial cells compared to those co-cultured with β4 myoepithelial cells. β6+ve and β6-ve myoepithelial cells were exposed to TRAIL, and this demonstrated that TRAIL enhanced apoptosis, measured by cleaved PARP, in β6+ve cells. Furthermore, these cells showed loss of the anti-apoptotic protein Galectin-7, and knockdown of Galectin-7 in normal β6-ve myoepithelial cells rendered them more susceptible to TRAIL-induced apoptosis. In DCIS tissues, an inverse relationship between αvβ6 and Galectin-7 in myoepithelial cells was demonstrated, and Cytokine Array analysis showed that αvβ6+ve myoepithelial cells express higher levels of IL-16, which has a role in Treg cell recruitment. Taken together these results suggest that expression of αvβ6 by myoepithelial cells in DCIS generates a tumour-promoter peri-ductal inflammatory infiltrate through altered cytokine release, is associated with reduced galectin-7 expression and enhances myoepithelial cell apoptosis in response to TRAIL. This provides a potential mechanism by which myoepithelial cells may be lost during evolution of DCIS and so contribute to progression to invasive disease.

Cardiovascular disease remains a major cause of mortality and morbidity and is underpinned by Oxidative stress, within which, inactivation of nitric oxide (NO) by superoxide (SO) and other reactive oxygen species is characteristic. Two major enzyme systems are implicated within oxidative stress; NAD(P)H oxidase and endothelial nitric oxide synthase (eNOS). eNOS generates NO while at the same time, and within the same cells, NAD(P)H plays a powerful role in the generation of SO. Evidence is accumulating that polymorphisms of the genes encoding these enzyme systems may play an important role in the pathophysiology of CAD. Additionally there has been much recent interest in both biochemical markers of oxidative stress and low grade chronic inflammation as well as a non invasive vascular phenotype, pulse wave analysis. This thesis reports a series of studies (utilising the techniques described in chapter 2) which aimed to ascertain:- The reproducibility of pulse contour analysis as a non invasive intermediate cardiovascular phenotype (Chapter 3). Whether common single nucleotide polymorphisms of the p22phox gene CYBA and the endothelial nitric oxide synthase gene, NOS3, have an effect upon arterial compliance in patients with coronary artery disease (Chapters 4,5 and 6). In healthy volunteers, free of cardiovascular disease whether a relationship existed between markers of low grade inflammation and arterial stiffness (Chapter 7). Chapter 3: The reproducibility of diastolic pulse wave contour analysis and its relation to systolic pulse contour analysis. This clinical study demonstrated that both large (C1) and small artery (C2) compliance values were reproducible and that there was a significant correlation between both Augmentation Index (AIx) and C1and AIx and C2 in healthy volunteers and though there was no association between AIx and C1 in patients with coronary artery disease AIx did correlate with C2 in this population. Chapter 4: The effect of the G894T SNP of the NOS3 gene upon arterial stiffness in patients with coronary artery disease. There was no association observed between this polymorphism and blood pressure or large artery compliance however ANOVA revealed a statistically significant association for TT homozygosity and small artery compliance. The highest small artery compliance was seen in the patients homozygous for the G allele, an intermediate value observed in heterozygotes and the lowest value demonstrated in patients homozygous for the T allele. Multiple regression analysis, examining the possible contribution of confounders showed that only small artery compliance was significant when NOS3 G894T genotype was assigned as the dependent variable. Chapter 5: The C242T single nucleotide polymorphism of the CYBA gene and blood pressure and arterial compliance in patients with coronary artery disease. We sought to examine the influence of the C242T SNP of CYBA upon vascular compliance and blood pressure using the dominant allele model. The presence of the 242T allele was associated with significantly higher systolic blood pressure. Patients homozygous for the C allele had lower systolic blood pressure than heterozygotes and patients homozygous for the T allele. There was no statistically significant effect upon diastolic blood pressure but there was however a significant association observed between the 242T allele and pulse pressure. Chapter 6: Combined analysis of NOS3 G894T and CYBA C242T genotypes upon arterial stiffness. In order to contrast the arterial stiffness between the favourable versus the non-favourable genotypes patients homozygous for the NOS3 G allele and homozygous for the CYBA C allele were compared with those homozygous for the NOS3T allele and possessing the CYBA 242T allele. The former displayed higher large and small artery compliance than the latter group. Multiple regression analysis, examining the possible contribution of confounders showed that only the large and small artery compliance values contributed significantly when genotype was assigned as the dependent variable. Chapter 7 Chronic low grade inflammation and insulin resistance and arterial compliance in healthy volunteers. Within healthy volunteers multiple regression analysis showed that small artery compliance was significantly associated with IL 6, CRP and ICAM. Augmentation index showed only an association with ICAM1. There was no significant correlation between Adiponectin levels and either of the arterial stiffness parameters studied. Conclusions Diastolic pulse wave contour analysis is a reproducible assessment of arterial stiffness with the potential to represent a high fidelity non invasive vascular phenotype. Small artery compliance is correlated with Augmentation Index and although the measurements are not analogous they both represent useful means of acquiring quantitative data concerning arterial stiffness. The 242T allele of the p22phox gene, CYBA, is associated with decreased large but not small artery compliance and increased systolic and pulse pressure. Homozygosity for a common NOS3 polymorphism (894 GT) was associated with decreased small artery compliance but not with large artery compliance or blood pressure. The markers of chronic inflammation Interleukin 6, ICAM and hsCRP but not Adiponectin, a marker of Insulin resistance, predict small artery compliance in healthy individuals apparently free of vascular disease.

Invasion physiology is an emerging field that endeavors to understand the influence of physiological traits on the establishment of non-native species in novel environments. The invasive European green crab,Carcinus maenas, is one of the world's most successful aquatic invaders, and is currently distributed across temperate marine ecosystems globally. The work presented here explored the thermal physiology of this species, and has highlighted several physiological traits that have likely influenced establishment success. Intraspecific comparisons of crabs sampled from the northern and southern edges of their recipient, or invaded range on the west coast of North America have identified both organismal and cellular physiological difference with respect to upper and lower thermal tolerances. Crabs sampled from British Columbia, Canada (BC) had a significantly lower mean upper thermal tolerance threshold and heat shock protein synthesis, Hsp70, compared to their warm acclimated conspecifics sampled from California (CA). These differential physiologic responses may be rooted in the disparate natural thermal habitats that each population occupies within their respective environments. The ability of this species to extend its current range limits was also investigated. Range expansion to the south has been limited, and is likely restricted by this species lack of adaptation to warmer temperatures. Because range expansion has been chiefly northward, characterizing this species' response to cold stress can identify whether colder temperatures poleward may limit further range expansion. Cold tolerance capacity was determined in the laboratory, and crabs sampled from Vancouver Island, British Columbia were able to withstand the over-wintering thermal regime that occurs in Sitka, Alaska, a site that is currently beyond the range limits of this species. Furthermore, intraspecific assessments found that the cold acclimated BC population exposed to cold shock significantly down regulated protein levels of cyclin D1, cell cycle modulator. Distinct differences in carapace width (CW) were detected along the thermal gradient present in the green crabs' range. This variation in body size was utilized to the test the temperature size rule hypothesis for ectotherms. Simply stated, the temperature size rule is the tendency for ectotherms to develop slower but mature to a larger body sizes at cooler temperatures. The results supported this hypothesis as crabs sampled from the warm portion of the range were found to be smaller than crabs sampled from the colder portion of the range. This pattern was detected along the native range as well. Differences in body size have the potential to influence the scope of invasion; larger individuals are generally more fecund and longer lived, which can increase both the intensity and frequency of larval dispersal that could further propel range expansion. The physiologic properties that the green crab possesses which may influence invasion success were examined using peer-reviewed literature with the aim of determining if these physiological traits confer invasion success across taxa. This analysis tested four hypotheses: 1) Broad geographic temperature tolerances (thermal width) confer a higher upper thermal tolerance threshold when comparing invasive and native species. 2) The upper thermal extreme experienced in nature is correlated with upper thermal tolerance threshold. 3) Protein chaperone expression, a cellular mechanism underlying thermal tolerance threshold, is greater in invasive organisms than in native ones. 4) Acclimation to higher temperatures can promote a greater range of thermal tolerance for invasives compared to natives. These preliminary results generally support the four stated hypotheses, and provide a solid foundation for further studies to explore and identify physiologic traits that facilitate invasion success. Overall, these studies investigated the thermal physiology ofCarcinus maenasfrom an invasive metapopulation and have brought about significant advances in our understanding of what physiologic traits correlate to invasion success in this species. In addition, the data presented here can aid resource managers in identifying habitats, based on thermal tolerance measurements that fit the criteria for invasion. Understanding how invasive organisms vary with respect to thermal tolerance can aid our understanding the patterns and processes of species invasions.

La tesis doctoral que presento es el resultado del trabajo realizado en el Servicio de Neumología del Hospital de la Santa Creu i Sant Pau de Barcelona y del Institut de Recerca Biomèdica de Sant Pau (IIB Sant Pau), siguiendo las normativas de la Universitat Autònoma de Barcelona para su presentación como compendio de publicaciones. El estudio de la inflamación bronquial en el asma, se ha convertido en una herramienta valiosa para el diagnóstico, control y predicción de respuestas terapéuticas. En los últimos años ha aumentado el interés en los métodos que permitan evaluar de una forma no invasiva la inflamación de la vía aérea. Entre los métodos no invasivos descritos usados en la práctica clínica para evaluar la inflamación de la vía aérea figuran: el recuento de células inflamatorias en el esputo inducido y la fracción exhalada de óxido nítrico (FeNO). La temperatura del aire exhalado y la nariz electrónica se plantean como nuevas herramientas de medición de la inflamación bronquial y del control del asma. La línea de investigación desarrollada tuvo como objetivo fundamental mejorar los conocimientos sobre los fenotipos inflamatorios del asma a través de métodos no invasivos. Está basada en tres proyectos. El primero, es un proyecto clínico cuyos resultados muestran que existen un alto porcentaje de pacientes con disociación entre los resultados de la FeNO y de los eosinófilos en el esputo inducido y que cursan con características clínicas e inflamatorias diferenciales. Los otros dos proyectos permiten conocer la utilidad en el asma de dos nuevos métodos no invasivos como lo son, la temperatura del aire exhalado (TAE) y el reconocimiento de patrones de compuestos orgánicos volátiles mediante la “nariz electrónica”. Estos trabajos aportaron como resultados principales que en el caso de la TAE, no parece que éste sea un método que proporcione una información clínica útil puesto que no se encontró ninguna correlación entre este método y el grado de control del asma, la gravedad de la enfermedad, la obstrucción bronquial, o la inflamación bronquial. Por otro lado, los resultados del tercer estudio sí que fueron alentadores, puesto que de manera significativa, el uso de la nariz electrónica en un entorno clínico habitual permitió discriminar con fiabilidad los diferentes fenotipos inflamatorios bronquiales en pacientes con asma. Así pues, los resultados de esta tesis sirven como fundamento del estudio y aplicabilidad de diversos métodos no invasivos en el asma.
This doctoral thesis is the result of the work done in the Service of Respiratory Medicine of the Hospital de la Santa Creu i Sant Pau in Barcelona and the Institute of Biomedical Research of Sant Pau (IIB Sant Pau), following the regulations of the Universitat Autònoma de Barcelona. The study of bronchial inflammation in asthma has become a valuable tool for its diagnosis, monitoring and prediction of therapeutic responses. In recent years, there has been an increased interest in methods of noninvasive evaluation of the airway inflammation. The described non-invasive methods used in the clinical practice to assess airway inflammation include the inflammatory cell counts in induced sputum and the fractional exhaled nitric oxide (FeNO). The exhaled breath temperature and the electronic nose device are considered to be new tools for measuring airway inflammation and control of asthma. The line of the developed research had as a main goal to improve the knowledge of inflammatory phenotypes of asthma through non-invasive methods. It is based on three projects. The first (1) is a clinical project that shows a high percentage of patients with dissociation between the results of the FeNO and eosinophils in induced sputum, presenting clinical and inflammatory differential characteristics. The other two projects provide an insight into the utility of two non-invasive diagnostic methods: 2) the exhaled breath temperature (EBT) and 3) the recognition of the patterns formed by organic volatile compounds using the electronic nose device. The second study does not support the usefulness of the EBT plateau, because no correlation was found between EBT and control of asthma, severity of disease, bronchial obstruction or bronchial inflammation. Furthermore, the results of the third study were encouraging since the using of an e-nose device in a regular clinical setting can reliably discriminate different inflammatory asthma phenotypes among patients with persistent asthma. Thus, the results of this thesis disclosed the applicability of various non-invasive methods performed in routine clinical practice.

The malignant phenotype, by definition, is composed of two facets, consisting of the tumor's ability to proliferate as well as its ability to invade the surrounding normal tissue. We propose that in malignant gliomas the protein kinase C (PKC) family of enzymes, which has been shown to be overexpressed in these tumours, plays a pivotal role in both their proliferative and invasive capacity. In the first genes of experiments where the expression of the alpha isoform of PKC was significantly decreased following antisense oligonucleotide treatment, we were able to report the novel finding that this inhibition not only inhibits glioma proliferation in a dose-dependent manner but that this inhibition of growth was the consequence of programmed cell death (1). In the second focus of this thesis, we addressed the postulate that the high PKC activity present in glioblastoma cells may also contribute to the invasive nature of this tumor by modulating the expression of proteolytic enzymes, the matrix metalloproteinases, (MMPs). In contrast to previous studies implicating MMP-9, our data demonstrated that MMP-2 was the metalloproteinase most closely associated with glioma invasion. Furthermore, we were also able to report that the regulation of MMP-2, and thus glioma invasion, could be modulated by PKC (2). In summary, this work presents evidence to support a role for PKC in both the proliferative and invasive phenotypes which characterize malignant gliomas. The translational nature of this research is readily illustrated by the emergence of clinical trials in which PKC and MMPs constitute the principal chemotherapeutic targets.

Background: Temperature has a fundamental effect on organisms because it alters the speed of biochemical reactions and thus metabolism. This influence scales up to have ecosystem wide effects as the life history strategies of individual species differ in response to temperature. With the prospect of increasing global temperatures ecosystem functions could be interrupted. In order to predict the consequences of changing environmental conditions it is important to first establish how fitness related traits are affected by changing thermal conditions. Aims: The aim of this thesis was to develop a detailed understanding of the thermal niche of an invasive, tropical freshwater fish species. Methods: Using ecologically realistic conditions this thesis investigates the effect of environmental variation within and between generations on behavioural, growth, physiological, and reproductive characteristics of the Trinidadian guppy (Poecilia reticulata). Results: The results provide an insight into the fundamental thermal niche of a widely used model fish species as well as detailed measures of how thermal change alters phenotypic characteristics. Guppies are demonstrated to have a broad thermal tolerance and be phenotypically responsive to changing environmental conditions. The results also suggest that environmental characteristics of the guppy's habitat make an important contribution to the differences observed between populations of guppies in Trinidad. Conclusions: Water temperature in the guppy's natural environment varies widely over a daily cycle and I suggest that this is partly responsible for the guppy becoming phenotypically plastic and thermally tolerant. Furthermore, phenotypic flexibility is an important characteristic that will enable guppies to withstand some climate warming and continue to expand their invasive range poleward. Using experimental conditions which resemble those in the natural environment is important for developing accurate model parameters. These are necessary for predicting the ecosystem effects of environmental variation and for adaptive mitigation or pre-emptive management of range extensions by invasive species.

Severe malaria caused by Plasmodium falciparum (P. falciparum) remains a leading cause of death in many low and middle income countries. The intraerythrocytic reproduction cycle of the parasite is responsible for all the symptoms and mortality of malaria. The merozoite, first invade a red blood cell (RBC) in the circulation, then grows, develops and multiplies within it by clonal division. Merozoite invasion is a complex process involving dynamic interactions between ligands in the merozoite coat and receptors on the red blood cell membrane. Therefore, filming the complete malaria invasion processes may shed the light on its mechanism. The rationale of this work is that learning how the various ligand-receptor interactions affect invasion phenotypes will lead us to a better understanding of the key biological and biophysical aspects of parasite growth in the blood. The work described has firstly involved the development of an optimised imaging platform for recording egress-invasion sequences. I used live cell microscopy to understand this stage of malarial infection better, by monitoring egress-invasion sequences in live cultures under controlled conditions and addressing the morphology and kinetics of erythrocyte invasion by P. falciparum. In addition, the erythrocyte invasion phenotypes of the various P. falciparum strains were systematically investigated for the first time by live cell microscopy. Furthermore, to better understand genetic recombination affecting erythrocyte invasion phenotypes, progeny from the 7G8 x GB4 cross was compared to their parents. In order to investigate specific receptor-ligand interactions and their distinct functional characterisations at each distinct stage, the enzymes that cleave receptors on the erythrocytes and antibodies targeting ligands on the merozoites were studied and their effects observed using the live-imaging platform. In the results, the functions of ligands on the merozoites demonstrated for the first time distinct and sequential functions of proteins during erythrocyte invasion, which could potentially guide the design of more effective malaria vaccines. In addition, I have designed microfluidic devices for studying blood stage malaria. Polydimethylsiloxane (PDMS) microfluidic devices are optically transparent, non-toxic and have biocompatible features. Building on previous work, I made specific microfluidic devices for achieving a high throughput of egress-invasion observations. Infected red blood cells were delivered into a microfluidic device channel containing cage-like "nests". The nests were designed to selectively trap these stiff, egress-ready cells, in order to obtain streams of merozoites on maturation. Uninfected RBCs were delivered from another input into a long serpentine channel co-flowing with the egressed merozoites. The results indicated that, during P. falciparum erythrocyte invasion under flow conditions, the morphological effect on erythrocytes and the kinetic properties show significant differences to those in static conditions. In addition, with optimised flow rates, it is possible to reach higher throughput of egress-invasion observations than static conditions. Both the static and flow experiments carried out in this study highlight important mechanisms and processes of malaria invasion, and represent new ways of studying blood stage malaria. Precise and high throughout recording of single-event host-pathogen interaction events will allow us to address a new area of fundamental biological questions in future work.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Bióloga Ambiental.
Las invasiones biológicas son una de las principales amenazas para la biodiversidad en todo el mundo; una forma de entender por qué algunas especies son más invasoras que otras, es analizando sus rasgos fenotípicos (o rasgos funcionales) relevantes para el desempeño, pero en un contexto biogeográfico, o sea comparar estos rasgos entre individuos provenientes de rangos nativos y los rangos invadidos. Esta comparación puede ser considerada un experimento biogeográfico y puede servir también para estudiar el potencial evolutivo de las especies enfrentadas a nuevos ambientes. Para este trabajo se compararon dos regiones invadidas (Chile y Nueva Zelanda) con su correspondiente región nativa (California) en una especie de hierba originaria de California, Eschscholzia californica. Los rasgos a comparar fueron a) Tamaño de semilla b) Fecundidad c) Presión de propágulo y las comparaciones fueron California vs Chile y California vs Nueva Zelanda, considerando dos poblaciones locales sometidas a diferentes temperaturas ambientales (10°C y 12°C). Los resultados han mostrado diferencias entre las regiones sugiriendo que las plantas producen semillas más grandes en las regiones invadidas, teniendo relación con su origen como planta ornamental lo que podría estar dándole ventajas para invadir, para la región nativa la fecundidad pareciera ser mayor y presión de propágulo no muestras diferencias, sin embargo, las diferencias locales son aparentemente más importantes. Finalmente, el análisis de componentes principales mostró más cercanía entre nueva Zelanda y California que California y Chile.
Biological invasions constitute a biodiversity threat worldwide; One way to understand why some species are more invasive than others are to analyse their phenotypic traits (or functional traits) relevant for fitness ideally between invaded range in relation to native ranges. These comparisons can be considered a biogeographical experiment which can help to elucidate the evolutionary potential of species facing to new environments. in this study, we examined two invaded regions (Chile and New Zealand) and the native range of Eschscholzia californica, a perennial grass original to California. We compared traits such as Seed size, Fecundity and Propagule pressure and the comparisons were California vs Chile and California vs New Zealand; at each region, we considered two local populations which differ in the thermal environment: (10°C and 12°C). Our results have shown differences between the regions suggesting that the plants produce bigger seeds in the invaded regions, In opposition to our expectations, at the native range fecundity was higher and for propagule pressure there were no differences. Local differences between populations, seem to be more important to take account to phenotypic variability. Finally, the principal components analysis showed more proximity between New Zealand and California than California and Chile.
Septiembre 2019

Exotic species often invade new areas and displace native species. The problems associated with such invasions are well known, but for many exotic species, experimental work has not yet been done to predict which, and under what conditions they may become a problem. Two greenhouse experiments were devised to investigate the plasticity, shade tolerance, and phenotypic differences of full-siblings from 3 populations of Cardamine impatiens, a Eurasian species potentially invasive in North America. Potted plants were subjected to 0, 54, 76, or 91% shade created by neutral density shade cloth application. In addition, the impact of a cold pre-treatment of seedlings on the growth and reproductive output of C. impatiens plants was examined.

In our first experiment, we subjected Cardamine impatiens to non-shaded cages, 54%, or 76% shade intensity. Plants died very quickly, so LD50 data were used as a relative measure of fitness, and relative growth indices were calculated over time. Other relative measures of fitness included canopy area, leaf area, number of leaves, number of leaves per canopy area, and final plant weight. Plants in cages with no shade treatment grew faster than those in cages with shade cloth and final plant weight decreased as shade treatment percentage increased. In each population, the number of leaves increased over time and the number of leaves per canopy area decreased over time under shade treatments. Our second experiment involved the application of 54%, 76%, and 91% shade intensity. The additional shade treatment of 91% was applied to determine the extent of plant tolerance and plasticity in response to light reduction. Due to high plant mortality in our first experiment, we treated Cardamine impatiens with a 4 week cold period prior to treatment, which simulates its biennial growth form in its natural western Virginia region habitat. Since this second experiment took place later in the year, day length was extended to more accurately duplicate the conditions during the first experiment. LD50 calculations were not necessary, and 7 of the 135 plants produced seed. Relative measures of fitness included canopy area, leaf area, number of leaves, number of leaves per canopy area, and final plant weights. As in experiment one, the number of leaves per plant increased over time, final plant weight decreased as shade treatment increased, and the number of leaves per canopy area decreased as shade treatment increased. From these two experiments, we determined that Cardamine impatiens is a species that exhibits phenotypic plasticity and therefore may pose a threat as an invasive species. C. impatiens is able to grow and exhibit plasticity of plant architecture under the conditions of very low light. The number of leaves per canopy area decreased as shade increased, suggesting that C. impatiens is highly adaptable to low light conditions, and therefore may be exhibiting phenotypic plasticity by reallocating its resources by producing fewer leaves while maintaining canopy area. This data along with other C. impatiens traits such as high levels needed for seed production, its persistence in seed banks, along with a lack of known major enemies, indicates that they have a great capacity to invade a wide variety of habitats. We also determined that a cold treatment is necessary in order for C. impatiens to obtain optimal growth and reproduction.
Master of Science

Invasive species often encounter climatic conditions that differ significantly from those of their native range. These environmental shifts may trigger phenotypic responses, resulting through some combination of adaptation and plasticity, that enable the invader to persist under novel thermal regimes. In this dissertation, I examine phenotypic changes in a tropical lizard that has successful invaded a cooler temperate climate, specifically examining traits that may promote survival and reproduction in their new range. First, I examined physiological traits, as I predicted greater cold tolerance would be necessary to survival in the invasive range. I found that invasive populations tolerated lower temperatures, exhibited greater maximum sprint speeds, and had higher metabolic rates than native populations. Next, I examined how life-history traits may change in the invasive range in order to facilitate reproduction under shorter breeding and growing seasons. I found that compared to native females, invasive females had shorter interlaying intervals and produced eggs that hatched more quickly. Once I quantified changes physiological and life-history traits that may have aided in successful establishment, I executed a common garden study to determine whether changes were the result of adaptation or plasticity. I found that differences in critical thermal minimum, metabolic rate, interlaying interval, and incubation period were maintained in lab-reared offspring, while measures of sprint speed converged. My results provide evidence that life history and physiology can evolve rapidly during invasion. These findings are useful to understanding contemporary evolution, and also provide valuable insight on how species respond to environmental shifts, both during invasions and as a result of climate change. �
Doctor of Philosophy
When species invade a new area, they often face different climates that make can make survival and reproduction challenging. In response, species may alter traits in order to adjust to new temperatures and conditions. In this dissertation, I examine trait changes in a tropical lizard that has successfully invaded a cooler temperate climate, specifically examining traits that may help them to survive and reproduce in their new range. First, I examined physiological traits, as I predicted greater cold tolerance would be necessary to survival in the invasive range. I found that invasive populations tolerated lower temperatures, could sprint faster, and had higher metabolism than native populations. Next, I examined how reproductive traits may change in the invasive range in order to facilitate reproduction under shorter breeding and growing seasons. I found that compared to native females, invasive females had less time between egg lays and produced eggs that hatched more quickly. Once I assessed how traits may have changed in the new range, I determined whether changes resulted from evolution or not. I found that differences in low temperature tolerance, metabolic rate, the time between egg lays, and incubation period were the result of evolution, while sprint speed did not seem to be the result of evolution. My results provide evidence that traits can evolve rapidly during invasion, allowing invasive species to persist and spread in new areas.

All species on Earth occupy limited geographic space. More than a century of observational, experimental, and theoretical work investigating the factors that drive species distributions have demonstrated the importance of the interactions between abiotic, biotic, and demographic factors in determining why species are found where they are. However, it is still unclear when and where these factors interact to set species range limits. Filling the existing knowledge gaps is imperative for the accurate predictions of how species will respond to global change, and particularly for invasive species, many of which are expected to benefit from global change. Here, I sought to investigate the mechanisms that enable, as well as limit, the range expansion of the globally invasive plant Sorghum halepense (L.) Pers. (Johnsongrass). I performed a series of field and laboratory experiments to study population and range dynamics throughout Johnsongrass's North American distribution, and test for the effects of climate, local habitat, and competition on multiple functional traits. I found Johnsongrass consistently demonstrated impressive performance across varying environments, often growing more than 3 m tall, producing hundreds of flowering culms within a single growing season, and maintaining positive population growth rates, even under intense competition with resident weeds. I also found evidence that seed germination has adapted to varying climates encountered during Johnsongrass's range expansion resulting in a shift in the germination temperature niche from warmer to cooler as Johnsongrass spread from warmer climates in the south to more temperate climates in higher latitudes. This shift in the germination temperature niche may have been an important contributing factor in the range expansion of Johnsongrass by enabling the optimization of seed germination in varying climates. On the other hand, results from a field study suggested a possible trade-off between flowering time and growth in populations originating from the range periphery (i.e., range boundary) which may be limiting, or slowing, continued range expansion of Johnsongrass. Together, the outcomes of this work contribute to our understanding of the factors involved in the distribution of species, which is a fundamental goal of Ecology, and essential to accurately predict how invasive species will respond to global change.
Doctor of Philosophy
Invasive species threaten our natural ecosystems, our agricultural systems, and even our infrastructure, and we spend billions of dollars each year attempting to control them and reduce their negative impacts. Climate change, habitat destruction, and other forms of global change, will benefit many of these species, magnifying their impacts and promoting their invasion into new territories. Because of the damaging effects of invasive species, and the costs to control them, it is imperative that we are able to predict how they will respond to global change so that we can improve plans to reduce their impact and spread. First, we need to understand the processes that promote their invasion across large swaths of land. Just as importantly, we must study the processes that prevent their invasion of certain areas. Here, I investigated some of the processes that have facilitated, as well as hampered, the spread of the invasive plant Johnsongrass. For this work, I used Johnsongrass plants originating from different habitats, including regions where Johnsongrass is highly invasive and those where Johnsongrass is very rare. I found Johnsongrass originating from regions where it is highly invasive were able to grow very large and produce thousands of seeds that were able to germinate under a range of conditions. These traits may have contributed to the invasion success of this species. However, I found a different pattern for plants that originated from regions where Johnsongrass is rare. These plants reached reproductive age earlier and grew smaller across all environmental conditions, potentially due to the less hospitable climates of these range edges. These findings allow us to project into future climate change scenarios, because it is likely that, as temperatures warm, invasive species will be able to invade new regions, where they will impact the work of conservationists, natural resource professionals, agricultural produces, and other land managers.

Biological invasions are a major threat to biodiversity world wide with annual economic costs up to 1.4 trillion dollars. The round goby (Neogobius melanostomus) is a particularly fierce invader that threatens ecological function of the Baltic Sea. Individual variation in behavioral traits that remain constant through time and context have been identified as crucial factors for explaining different parts of the invasion process. For example, asocial behavior facilitates dispersal from high density populations and comes with fitness benefits in low conspecific density. The latter is especially relevant, in an invasion context, following the initial colonization of a novel environment when population density usually is low. This thesis investigates the role of individual variation in phenotypic traits on species invasions. The main focus is on the effects of sociability, activity and boldness, but also including aggression and physiological stress tolerance, on dispersal tendency and selection at invasion fronts. To do this, we studied four round goby populations in the Baltic Sea, two of the most recently established and two of the oldest populations. In 2012 we demonstrated that asocial, active and bold round gobies are overrepresented at invasion fronts. Two years later we showed that dispersal from the new populations was led by individuals with high activity levels, while in all populations larger individuals dispersed. We also determined the length of the socalled lag-phase, between colonization and spread, in both newly established populations. The end of the lag-phase is hypothesized being triggered by high population density in the harbors leading to dispersal and subsequen colonization of the surrounding areas by small asocial individuals. In our final experiment, we present evidence of stress coping styles in round gobies, in which more aggressive individuals are also more stress tolerant and vice versa. Though we found no connection between stress coping and population age, we found that mortality was unaffected by population density and that the gobies became more aggressive and stress tolerant when kept in high density. To conclude, we have shown that: 1) individuals with high levels of activity, boldness and asociality are common at invasion fronts; 2) a lag phase occurs between colonization and spread in round goby invasions; 3) asocial individuals drive the spread from high density populations at the invasion front and; 4) round gobies adapt to high densities with high aggression and stress tolerance.

Le Virus de l'Immunodéficience Humaine de type 1 (VIH-1) infecte les macrophages. Contrairement aux lymphocytes T CD4+, les macrophages résistent aux effets cytotoxiques du virus et représentent un réservoir pour ce pathogène. Dans ces cellules, le virus est produit et stocké dans un compartiment intracellulaire spécifique appelé VCC (Virus-Containing Compartment). Ce compartiment à pH neutre, transitoirement connecté à la membrane plasmique, reste cependant très peu caractérisé. Par ailleurs, le VIH-1 induit une perturbation des fonctions des macrophages, permettant ainsi le développement de bactéries opportunistes, telles que des souches particulières de Salmonella Typhimurium. Nous avons étudié en particulier des souches de Salmonella Typhimurium invasives non typhiques qui se sont développées, chez des patients séropositifs. Les objectifs de ma thèse ont donc été d'étudier, dans les macrophages primaires humains, les facteurs moléculaires impliqués dans la production du VIH-1 et le développement de souches invasives de Salmonella Typhimurium. Dans un premier temps, j'ai contribué à étudier les effets de l'infection par le VIH-1 sur les fonctions des macrophages. Leur fonction majeure est la phagocytose qui est un mécanisme de défense contre les pathogènes permettant leur internalisation et leur dégradation. Il avait déjà été montré au laboratoire que l'étape d'internalisation était en partie inhibée par le facteur de virulence Nef dans les macrophages infectés. Dans ce travail, nous avons montré que l'infection de ces cellules par le VIH-1 inhibe également la maturation des phagosomes, mais via la protéine virale Vpr. Nous avons aussi mis en évidence que le VIH-1 conduit le macrophage dans en état de pré-activation, mais empêche la cellule de répondre à un stimulus ultérieur comme une surinfection bactérienne. Dans un second temps, j'ai participé à l'étude des coinfections entre VIH-1 et les bactéries Salmonella Typhimurium invasives, qui ont émergé avec l'infection par le virus, en comparaison avec des souches de référence. Ce travail nous a permis de montrer que les bactéries, pour leur survie, n'exploitent pas le compartiment viral dans les macrophages co-infectés. J'ai ensuite observé que la souche invasive de Salmonella Typhimurium induit moins de mort cellulaire par pyroptose qu'une souche de référence. J'ai alors déterminé les voies de signalisation en amont de cette mort cellulaire qui est associée à un mécanisme inflammatoire. Ainsi, j'ai mis en évidence que la souche invasive de Salmonella détourne les mécanismes de pyroptose et survit mieux dans les macrophages, ce qui pourrait expliquer la dissémination observée chez les patients. Enfin, j'ai initié l'étude de nouveaux facteurs cellulaires impliqués dans la production virale par les macrophages. À la suite d'une analyse transcriptomique sur des macrophages primaires humains infectés ou non par le VIH-1, nous avons identifié un nombre important de transporteurs membranaires appelés SLC (Solute Carrier) dont l'expression est modulée par l'infection. Après la sélection de candidats, j'ai pu mettre en évidence que certains de ces SLC étaient importants pour la production virale par les macrophages. En conclusion, l'ensemble de ces travaux contribue à définir comment le VIH-1 infecte les macrophages et diminue leurs fonctions d'activation et de clairance, et comment se développent des bactéries opportunistes pathogènes
Human Immunodeficiency Virus type 1 (HIV-1) infects macrophages. In contrast to CD4+ T cells, macrophages are resistant to the cytotoxic effects of the virus and represent a reservoir for the pathogen. In these cells, the new virions are produced and stored in a specific intracellular compartment called Virus-Containing Compartment (VCC). This non-acidic compartment, transiently connected to the plasma membrane, remains poorly characterized. In addition, HIV-1 induces an alteration of macrophage function, allowing the development of opportunistic bacteria, such as specific strains of Salmonella Typhimurium. In particular, we studied invasive non-typhoidal Salmonella Typhimurium (iNTS) strains that developed in HIV-positive patients. The aims of my thesis have been to identify the molecular factors involved in the production of HIV-1 in primary human macrophages and to study the development of the invasive strains of Salmonella Typhimurium. First, I participed in studying the effects of HIV-1 infection on macrophage function. Their main role is phagocytosis, which is a defense mechanism enabling internalization and degradation of pathogens. It has previously been shown in the host laboratory that in HIV-1 infected macrophages, the internalization step is partially inhibited by the virulence factor Nef. In this work, we have shown that the infection of these cells by HIV-1 also inhibits the maturation of phagosomes, in this case, via the viral protein Vpr. Further, we have demonstrated that HIV-1 leads to a pre-activation state of the macrophage, while preventing the cell from responding to subsequent stimuli, such as bacterial superinfection. Secondly, I studied the coinfections between HIV-1 and an invasive strain of Salmonella Typhimurium that was compared to reference strains. This work demonstrated that bacteria do not hijack the viral compartment for their survival in co-infected macrophages. Additionally, the invasive strain of Salmonella Typhimurium was observed to induce less cell death by pyroptosis than a reference strain. The signaling pathways upstream of this cell death were determined to be associated with an inflammatory mechanism. Hence, it was demonstrated that the invasive strain of Salmonella hijacks the mechanism of pyroptosis to survive in macrophages. This may explain the dissemination observed in patients. Finally, a study of new cellular factors involved in viral production in macrophages was conducted. Following a transcriptomic analysis of human primary macrophages infected, or not, with HIV-1, we identified a large number of membrane transporters called SLC (Solute Carrier) whose expression was modulated by the infection. After selecting some of the candidates for further study, I have demonstrated that some of these SLCs are important for viral production in macrophages. In conclusion, this work contributes to defining how HIV-1 infects macrophages and disturbs their activation and clearance functions, and how opportunistic pathogenic bacteria develop

Hieracium lepidulum is an invasive weed in New Zealand. It colonises a wide range of habitats including pine plantations, scrubland, native Nothofagus forest, and mid-altitude to alpine tussock grassland, where it is competing with indigenous species. Understanding the breeding systems and population genetic structure of H. lepidulum is important for biocontrol, and aids in the understanding of evolutionary colonisation processes. H. lepidulum is a triploid, diplosporous, obligate apomict. This type of reproduction through clonal seed does not involve meiosis or fertilisation, and theoretically populations should contain very low levels of genetic variation, the only source being somatic mutation. Common garden experiments and microsatellite markers were used to determine the population genetic structure of H. lepidulum populations in the Craigieburn Range, Canterbury. Both experiments revealed that populations, sampled from three replicate altitudes within three geographically-separated locations, contained no genetic variation; individuals all possessed the same microsatellite genotype. These results strongly suggest that the Craigieburn Range H. lepidulum individuals reproduce solely by apomixis and populations belong to the same clonal lineage. Populations were also examined for their response to two abiotic environmental ‘stresses’, drought and shade. H. lepidulum populations’ exhibited high drought tolerance, yet appeared to be shade-intolerant. Low levels of reproduction in light-limiting habitats will prevent the invasion of H. lepidulum into closed-canopy forest habitats. H. lepidulum appears to have overcome the reduction in fitness associated with apomictic reproduction by phenotypic plasticity, fixed heterozygosity and polyploidy – all associated with increased vigour, fitness, and the ability to occupy broader ecological niches. This study’s results are hopeful for the development of biocontrol programs involving genotype-specific pathogens but suggest that grazing management may not succeed. The data will be useful for future comparisons of genetic structure during the course of H. lepidulum invasions and will contribute to the management of this invasive weed.

Dans un contexte d’expansion des espèces invasives, leur survie et succès sont conditionnés par leur capacité à s’adapter. En France, Ludwigia grandiflora (jussie) a envahi bon nombre de biotopes aquatiques et son déploiement récent dans les prairies humides a conduit à l’apparition de deux morphotypes, l’un aquatique et l’autre dit « terrestre ». L’objectif de cette thèse visait à mieux comprendre les capacités d’acclimatation de la jussie au milieu terrestre en explorant les sources de flexibilité que sont les mécanismes génétiques et épigénétiques. Les réponses des morphotypes aquatique et terrestre à différentes contraintes hydriques ont été évaluées via l’observation des traits morphologiques et développementaux, des dosages de métabolites et de phytohormones. La piste épigénétique a été abordée par l’utilisation d’une drogue hypométhylante, la zébularine. Ces travaux ont montré que L. grandiflora adapte son développement et son métabolisme avec des valeurs de biomasses élevées etLe morphotype terrestre présente des valeurs de traits plus importants que ceux du morphotype aquatique, quelle que soit la condition. Cependant, la plasticité phénotypique est plus importante chez le morphotype aquatique. Enfin, l’épigénétique via la méthylation de l’ADN semble impliquée dans la transition du morphotype aquatique vers le milieu terrestre. Nos résultats suggèrent une implication de la méthylation de l’ADN et de la plasticité phénotypique dans la réponse de la jussie au changement de milieu. Le morphotype terrestre ayant des capacités supérieures au morphotype aquatique, sa
Abstract: In the context of the expansion of invasive species, their survival is conditioned by their ability to adapt. In France, Ludwigia grandiflora has invaded aquatic biotopes and its recent deployment in wet meadows has led to the emergence of two morphotypes, one aquatic and the other called “terrestrial”. The aim of this thesis was to get a better understanding of water primrose acclimation capacities to terrestrial environment through exploring genetic and epigenetic sources of flexibility. The responses of two morphotypes to different water stresses were evaluated by observing physiological traits. The epigenetic pathway was addressed by the use of a hypomethylant drug. This work showed that L. grandiflora adapts its development and metabolism according to environmental conditions.The terrestrial morphotype shows higher trait values than those of the aquatic morphotype, regardless of the condition. However, phenotypic plasticity is higher in the aquatic morphotype. Finally, our results suggest the involvement of DNA methylation and phenotypic plasticity in the response of water primrose to environmental change. Since the terrestrial morphotype has higher capacities than the aquatic morphotype, its management must become a priority. The acquisition of genomic resources in L. grandiflora will make it possible to search for genetic and epigenetic markers of acclimation to the terrestrial environment

Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.

Le commerce mondial et les mouvements humains accroissent les probabilités de transport à longue distance de propagules, et leur introduction dans de nouvelles aires géographiques. Dans certains cas, des espèces récemment établies peuvent devenir dominantes dans la communauté envahie. Malgré les menaces sur les communautés natives et le fonctionnement des écosystèmes, les invasions biologiques constituent des expériences naturelles qui permettent d’étudier les processus éco-évolutifs en temps réel, notamment l’impact de nouvelles interactions biotiques sur la composition et la dynamique des communautés, l’adaptation rapide à de nouvelles conditions environnementales, ou la dispersion en limite de répartition. Les îles océaniques sont particulièrement sensibles aux invasions biologiques en raison de la faible diversité de leurs communautés natives. Dans les terres australes françaises, le carabique marcheur Merizodus soledadinus, natif de Patagonie, a été accidentellement introduit à Kerguelen en 1913. La présente étude vise à comprendre les principaux mécanismes à l’origine du succès invasif de cet insecte aux Iles Kerguelen. Un large ensemble de méthodes ont été utilisée pour explorer les traits écologiques de M. soledadinus, des populations à la molécule. Les analyses génétiques confortent l’hypothèse historique d’un unique évènement d’introduction dans un seul site des Iles Kerguelen. Les populations échantillonnées le long du gradient d’invasion ne montrent pas de structuration génétique. Les traits phénotypiques mesurés montrent une forte différentiation entre les individus selon le temps de résidence des populations, confirmant l’hypothèse de tri spatial des populations au cours de l’expansion géographique. Nous avons démontré que l’expansion géographique et la sélection d’habitats par M. soledadinus est principalement gouvernée par la disponibilité en eau, comme le suggère par la forte sensibilité des adultes au stress hydrique. En parallèle, la colonisation d’habitats en altitude dépend des conditions thermiques, qui semblent être contraignantes pour cet insecte à partir de 200m d’altitude. La colonisation d’habitats d’altitude progresse pourtant, probablement assistée par le changement climatique. Pour finir, les adultes M. soledadinus sont longévifs et actifs toute l’année. Les connaissances apportées sur l’écologie de M. soledadinus et sur la dynamique de son expansion géographique suggèrent la poursuite de la colonisation de l’archipel par ce prédateur. L’ensemble de ces connaissances pourraient être utiles à la paramétrisation d’un modèle d’expansion géographique, qui permettrait de définir les routes de dispersion et les taux d’expansion, dans l’objectif d’assister les mesures de gestion par les agents de la Réserve naturelle des Terres Australes et Antarctiques Françaises
Global trade and human movements increase the likelihood of long-distance transportation of propagules and their subsequent introduction into new geographic regions. In some instances, newly established species can become dominant in invaded communities, at the expense of native species. Besides threatening invaded communities and ecosystem functions, biological invasions constitute natural experiments that allow to study eco-evolutionary processes in real time, including the occurrence of new biotic interactions affecting community composition, rapid adaptation to novel environmental conditions, or dispersal evolution at range margins. Because of their impoverished native communities, oceanic islands’ ecosystems are particularly sensitive to biological invasions, and the French subantarctic islands are no exception. For instance, the flightless predatory carabid beetle Merizodus soledadinus is native from the southern tip of South America, and has been accidentally introduced to the Kerguelen Islands in 1913. In the present work, we aimed at understanding the main mechanisms underlying the invasive success of this insect at the Kerguelen Islands. Using a vast array of methodologies, ecological features of M. soledadinus were investigated with analytical procedures scaling from population to molecule through the individual level. Genetic investigations support the historically-based hypothesis of a single introduction event at a unique location of the Kerguelen Islands. No genetic structure was observed among individuals sampled from different populations along the invasion gradient. We tested the hypothesis of spatial sorting of populations during range expansion, by exploring phenotypic changes among individuals sampled along the invasion gradient. The measured phenotypic traits revealed major differentiation of adults according to the residence time of their populations, confirming the occurrence of spatial sorting of populations during geographic expansion. We also demonstrated that the geographic expansion of M. soledadinus, and microhabitat selection, are primarily governed by the availability of water resources, as suggested by the high sensitivity to water stress of adults of this ground beetle. In parallel, colonisation of altitudinal habitats is governed by thermal conditions, which seem to be physiologically constraining from 200m asl onwards. As the altitudinal distribution of M. soledadinus still extends, we concluded that ongoing climatic changes play a pivotal role in this expansion. Finally, adults of this ground beetle are long-lived and active year-round. The ecological knowledge of M. soledadinus characteristics and spatial expansion dynamics suggest that the colonisation process of the Kerguelen archipelago by this species will continue. Altogether, these data could be used for parametrising range expansion models that would delineate dispersal pathways and expansion rates, in the objective to assist stakeholders’ management decisions

Au cours de ce travail j'ai developpe une technique de pcr quantitative competitive pour le dosage d'arnm de cytokines. Differents standards internes de cytokines ont ete construits et ont permis la mesure de l'expression d'arnm de cytokines dans un modele de tumeurs invasives du col de l'uterus. Nous avons montre que les concentrations intratumorales d'arn messager d'il-6 etaient plus elevees dans les lesions malignes que dans les dysplasies ou tissus sains. Des etudes immunohistochimiques ont revele une origine stromale vraisemblablement macrophagique a cette production d'il-6. Nous avons ensuite transfecte des lignees derivees de cancer du col de l'uterus avec l'adnc de l'il-17 une cytokine dont l'une des proprietes est d'induire la secretion d'il-6. Comme attendu la production d'il-6 par les lignees exprimant l'il-17 est augmentee par rapport aux lignees parentales. Ces tumeurs exprimant l'il-17 sont caracterisees par une vitesse de croissance acceleree et un infiltrat macrophagique plus marque par rapport a des tumeurs controles. Dans les deux modeles etudies, l'augmentation d'expression d'il-6 et l'infiltration macrophagique sont donc correlees a un caractere aggressif de de la maladie. Dans une deuxieme partie de ce travail nous avons mis en evidence chez des patientes atteintes de tumeurs du col de l'uterus, une association entre des concentrations intratumorales basses d'arnm d'ifng et un pronostic clinique defavorable. Ces travaux montrent donc que la determination des concentrations intratumorales d'arnm de cytokines represente une nouvelle approche pour analyser des mecanismes d'evolutivite tumorale et permet de definir de nouveaux types de marqueurs tumoraux.

Phenotypic plasticity is the adaptive response of a genotype to presenting different types of phenotypes in response to environmental changes. These adaptive responses can contribute to a particular species being able to occupy a particular habitat or habitats and thus expand its range. This thesis studies some aspects of the mechanisms of invasion, establishment and expansion of the invasive species Alburnus alburnus in different basins of the Iberian Peninsula and provide more information for a better understanding of its biology and mechanisms of invasion. On the other hand, it also aims to compare the morphology, metabolism and critical swimming speed of two fartet populations, one captive and one wild and to use this information to apply it to captive breeding programs and improve the management of reintroductions of captive-bred individuals into their natural habitat
La plasticitat fenotípica és la resposta adaptativa d’un genotip a presentar diferents tipus de fenotips en resposta a canvis ambientals. Aquestes respostes adaptatives poden contribuir a que una espècie determinada pugui ocupar un hàbitat o habitats determinats i d’aquesta manera, expandir la seva àrea de distribució. Aquesta tesi estudia alguns aspectes dels mecanismes d’invasió, establiment i expansió de l’espècie invasora Alburnus alburnus en diferents conques de la Península Ibèrica i aportar més informació per a una millor comprensió de la seva biologia i mecanismes d'invasió. D’altra banda, també té com objectiu comparar la morfologia, metabolisme i velocitat crítica de natació de dues poblacions de fartet, una criada en captivitat i una altra de salvatge i utilitzar aquesta informació per aplicar-la a programes de cria en captivitat i millorar la gestió de les reintroduccions d'individus criats en captivitat en el seu hàbitat natural
Programa de Doctorat en Ciència i Tecnologia de l'Aigua

Les invasions biologiques sont un composant du changement global et ont des impacts dramatiques sur les écosystèmes, les agrosystèmes et la santé humaine. Néanmoins, ces processus biologiques particuliers offrent la possibilité d'étudier l'évolution phénotypique et génétique en un sur des temps écologiques. En effet, les invasions biologiques impliquent de fortes contraintes environnementales et démographiques sur les populations, et de forts effets sous-jacents de la sélection et de la dérive. Pourtant, les espèces envahissantes sont parmi les colonisateurs les plus prolifiques de la nature, et surprennent par leur capacité à répondre à ces contraintes. Le potentiel évolutif et adaptatif des populations envahissantes a été à de nombreuses reprises proposé comme facteur facilitant le succès de ces invasions. Qu'il s'agisse de processus génétiques d'adaptations (i.e. des changements de fréquences d'allèles) ou plastiques (i.e. un ajustement par plasticité phénotypique en réponse à un stimulus environnemental), la capacité de réponse à la sélection des espèces envahissantes les placent au centre des études de la biologie évolutive moderne. Ici, nous utilisons la récente invasion mondiale de la drosophile à ailes tachetées Drosophila suzukii pour étudier en détail les mécanismes de la réponse à la sélection potentiellement impliqués dans le succès de cette invasion. L'analyse des patrons de variations moléculaires neutre, nous ont permit de retracer l'histoire complexe de cette invasion mondiale, et d'évaluer par la suite la divergence phénotypique et l'évolution de la variation génétique quantitative en comparant les populations ancestrales de D. suzukii à leurs populations dérivées. Nous avons pu ainsi estimer les effets de la sélection et de la dérive génétique au cours de cette invasion, et discuter leur importance au regard de l'évolution de la forme de l'aile dans cette espèce. Enfin des protocoles expérimentaux d'analyse de la plasticité phénotypique ainsi que des méthodes de modélisation de niche climatique nous permettent de discuter l'influence de la fluctuation des conditions environnementales sur le succès de cette invasion
Biological invasions are a component of global change and have dramatic effects on ecosystems, agrosystems and human health. Nonetheless, these peculiar biological processes offer a great opportunity for the study of rapid phenotypic and genetic evolution, at an ecological timescale. Biological invasions often involve environmental and demographic constraints on populations, as well as strong effects of selection and drift. However, these species are among the most successful colonialists in nature, and their ability to respond to these constraints is remarkable. The evolutionary and adaptive potential of invasive populations have been proposed as facilitating factors of the success of invasions. Processes of genetic (i.e. changes in allele frequencies) and plastic (i.e. adjustment to environmental fluctuation through phenotypic plasticity) involved in the success of biological invasions are at the center of modern evolutionary biology. Here, we use the recent spread of the spotted-wing Drosophila suzukii to study the underlying mechanisms of response to selection potentially involved in the success of this global invasion. Analyzing patterns of neutral genetic variation allowed us to decipher the complex history of this worldwide invasion, and subsequently evaluate phenotypic divergence and evolution of quantitative genetic variation among ancestral and derived populations. We thus estimated the effects of selection and drift throughout this invasion and discuss their importance regarding the evolution of wing shape in this species. Finally, experimental protocols on the analysis of phenotypic plasticity as well as Species Distribution Modeling methods allowed us to discuss the influence of environmental fluctuations on the success of this invasion

Orientador: Massanori Takaki
Banca: Victor José Mendes Cardoso
Banca: Sergius Gandolfi
Banca: Débora Leonardo dos Santos
Banca: Dalva Maria Silva Matos
Resumo: As espécies invasoras são reconhecidas como um dos mais importantes e difíceis fatores que influenciam a conservação dos ecossistemas nativos. Na restauração de ambientes degradados, estas espécies podem afetar profundamente a trajetória do recobrimento vegetal pelas espécies nativas e, conseqüentemente, a composição do ecossistema. Um fator importante para o sucesso de algumas espécies vegetais invasoras é a plasticidade fenotípica, dandolhes grande capacidade de aclimatação em diversas condições ambientais. O conhecimento dos fatores ambientais e das características da planta que contribuem para o sucesso das espécies invasoras é de grande importância para predizer a habilidade de invasão e para esforços de manejo. Neste contexto, o objetivo principal deste estudo foi conhecer algumas características fisiológicas e ecológicas de Clausena excavata Burm. f., uma espécie de árvore exótica reconhecida como invasora em algumas regiões no mundo, no intuito de compreender o sucesso da espécie como invasora e contribuir com informações relevantes para possíveis tentativas de controle. Primeiramente, foram avaliados os efeitos da luz e da temperatura em sua germinação (condição controlada) e o efeito da luz na emergência de suas plântulas (condição natural, a pleno sol e sob a copa das árvores). As sementes germinaram tanto na presença como na ausência de luz, nas temperaturas de 20 a 35°C, sem diferença entre a porcentagem de sementes germinadas nas diferentes temperaturas. Sementes mantidas a 20°C, em ambas as condições de luz, germinaram mais lentamente em relação às demais temperaturas. Independente da temperatura, na presença de luz as sementes apresentaram uma germinação muito mais sincronizada do que aquelas mantidas no escuro. Tais resultados mostraram que as sementes de C. excavata são fotoblásticas neutra. Em campo, a emergência... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The invasive species are recognized one of more important and difficult factors that prejudice the conservation of native ecosystems. In the restoration of degraded environment, these species can to affect profoundly the trajectory of recovery by native species and, therefore, the ecosystems composition. An important factor to the success of invasive species is their phenotypic plasticity, which gives them a great ability to adjust in several environmental conditions. The knowledge of environmental factors and plant characteristics that contribute to the success of invasive species is very important to predict the invasion capacity and to manage efforts. In this context, the main objective of this study was to know some physiological and ecological characteristics of Clausena excavata Burm. f., a nonnative tree species recognized as invasive somewhere of the world, with intention to understand the success of this species as invasive and to contribute with relevant informations for a possible management. At first, were evaluated the effects of light and temperature on their seeds germination (controlled condition) and the effect of light on seedling emergence (natural condition, at full sun and shade). The seeds germinated in presence and absence of light, from 20 to 35°C, without difference among the germination percentage at different temperatures. Seeds maintained in 20°C, at both light conditions, germinated slowly than others temperatures. Independent of temperature, in light the seeds showed a germination more synchronized than the seeds kept in darkness. Those results showed that C. excavata have neuter photoblasics seeds. On the field, the emergence occurred at both environments, but at full sun all seeds produced a seedling. The seedling emergence occurred with same rate and synchronization index on two tested conditions. Thereafter, the seedlings were observed with purpose... (Complete abstract click electronic access below)
Doutor

La compréhension des mécanismes déterminant le succès des espèces invasives est une étape essentielle dans la gestion des invasions biologiques actuelles et la prédiction des futurs risques d'invasion. En adoptant un cadre d'étude conceptuel intégrant les processus écologiques et évolutifs, l'objectif de ce travail était d'analyser les déterminants de la colonisation de l'ambroisie à feuilles d'armoise en France. Tout d'abord, l'étude des interactions biotiques et abiotiques a permis de montrer la capacité de tolérance de l'ambroisie à l'herbivorie et au stress hydrique. L'ambroisie est capable de tolérer la défoliation grâce à une croissance compensatoire efficace, sans que sa reproduction en soit affectée. Cette forte tolérance à l'herbivorie est maintenue chez les populations introduites, malgré la faible pression des ennemis naturels dans la zone d'introduction. En condition de stress hydrique, l'ambroisie produit une biomasse racinaire supérieure aux espèces présentes dans les communautés qu'elle envahit. De plus, les différences dans les valeurs moyennes pour les traits mesurés suggèrent une occupation différente par l'ambroisie des niches écologiques disponibles. La comparaison en jardins communs de populations de l'aire d'origine avec des populations de l'aire d'introduction isolées et issues du foyer central d'invasion a montré que l'adaptation de l'ambroisie à son environnement reposait principalement sur la plasticité phénotypique plutôt que sur la différenciation des traits. Les études de génétiques quantitatives ont mis en évidence un potentiel évolutif élevé pour les traits liés à la germination. Les traits liés à la morphologie, à la phénologie et à la physiologie de la plante montrent une variance additive et une héritabilité plus faibles et donc un potentiel évolutif moindre. En revanche, la variation dans les normes de réaction indique un potentiel évolutif important de la plasticité phénotypique. La tolérance au stress hydrique et à l'herbivorie sont des facteurs qui potentiellement augmentent la capacité de l'ambroisie à coloniser une large gamme d'habitat. De plus, la plasticité phénotypique et le potentiel évolutif important peuvent favoriser une augmentation ou un déplacement de la niche écologique de l'espèce et ainsi favoriser l'expansion de son aire de répartition

Carcinomas de mama são heterogêneos e consistem de diversos tipos celulares. Perfis de expressão gênica usando DNA microarrays identificaram quatro subtipos moleculares fundamentais baseados na expressão de receptores hormonais (estrógeno e progesterona) e de fator de crescimento epidérmico (HER2) (luminal tipo A, luminal tipo B, tumores expressando somente HER2 e triplos negativos) refletindo a heterogeneidade molecular dos carcinomas. Sugeriu-se que esta heterogeneidade advém da presença de células tronco tumorais com a capacidade de se diferenciar ao longo de vias divergentes e outros estudos sugeriram que a presença destas células tronco tumorais pode ser evidenciada pela análise fenotípica de CD44 e CD24. Nosso objetivo foi detectar a freqüência de CD24 e CD44 isolados ou combinados, analisados por imunoistoquímica e sua associação com os subtipos moleculares e com diversos marcadores biológicos em 95 casos de carcinoma ductal infiltrativo organizados em um microarranjo tissular (TMA). Realizamos determinações imunoistoquímicas de CD44, CD24, citoqueratinas (CK5, CK6, CK18), claudina 7 e Ki67. Subgrupos moleculares foram definidos pela expressão imunoistoquímica de RE, RP e HER2. Resultados: Os tumores apresentaram uma maior freqüência dos grupos luminais (49,5%) atribuído à alta expressão de RP ou RE (47,4%), e freqüência menor de tumores triplo negativos (21,5%) e HER2 (9,5%). Os fenótipos CD44+CD24- e CD44-/CD24+ estavam respectivamente presentes em 8,4% e 16,8% dos tumores e o fenótipo duplamente positivo foi predominante (45,3%). Ausência de ambas as proteínas foi evidente em 6,3% dos tumores. Tumores com fenótipo CD44+CD24- (definido como um marcador de células tronco tumorais por estudos in vitro) foram mais comuns em tumores triplos negativos mas não demonstraram nenhum tipo de associação com características clinico-patológicas e demais marcadores. Este fenótipo não foi expresso nos tumores HER2 positivos. O fenótipo duplamente positivo CD44+CD24+ mostrou-se mais freqüente nos subtipos luminais ou com alta expressão de HER2. Os fenótipos (CD44-CD24+ e CD44-CD24-) não mostraram associação com os subgrupos. Tumores expressando CD24+ isolado, com grande freqüência deste marcador (74,7%), mostraram significativa associação com positividade do RE, RP e Ki67 e uma significância marginal com marcadores de diferenciação luminal (CK18 e claudina 7, p = 0,14). Nenhuma associação foi observada com tumores CD44+ quando analisado isoladamente. A expressão de claudina 7 e Ki67 não mostrou associação com os subgrupos e a expressão de CK5 apresentou uma tendência a uma maior negatividade nos subtipos luminais e uma freqüência maior de positividade nos tumores HER2 e triplo negativos. De outro lado, associação da freqüência da expressão positiva de CK18 nos subgrupos luminais foi estatisticamente significativa (p = 0,003). Para se determinar se CD24+ e CD44+ e seus subtipos combinados poderiam afetar a sobrevida global e o intervalo livre da doença preparamos curvas de sobrevida de acordo com Kaplan-Meier que foram analisadas estatisticamente (log rank test). A mediana do período de seguimento das pacientes do nosso estudo foi de 4,8 anos (0,36 10,9 anos). Estas análises não demostraram influência dos fenótipos CD44+CD24- ou CD44+ sobre a sobrevida global ou intervalo livre de doença, mas observamos uma tendência a um prognóstico mais favorável. Interessantemente tumores HER2 positivos não expressaram este fenótipo, sugerindo que outros marcadores de células tronco caracterizam estes tumores. O fenótipo CD44-CD24+ mostrou-se mais freqüente nos tumores luminais, mas não apresentou correlação com marcadores clínico-patológicos ou biológicos analisados. Não houve diferenças significativas com respeito a sobrevida global ou intervalo livre de doença . A expressão de CD24+ isolado associou-se a expressão dos marcadores de diferenciação celular e a uma diminuição do intervalo livre de doença. A sobrevida livre de doença (10 anos) indicou uma percentagem de 94,1% para CD24- e 72,1% para os pacientes CD24+ enquanto a sobrevida global foi de 84,2% para os pacientes CD24- e 72,1% para os pacientes CD24+. Citoqueratinas (CK5, CK18) e Ki67 não influenciaram a sobrevida e o intervalo livre de doença. No entanto a expressão positiva de claudina 7, embora não associada à sobrevida global, foi estatisticamente associada ao decréscimo do intervalo livre da doença (p = 0,05). Conclusão: As características dos tumores CD44+CD24- e sua tendência a associação um prognóstico mais favorável parecem não estar de acordo com as propriedades descritas na literatura para células tronco e enfatizam a necessidade de outros marcadores. A determinação da freqüência de CD44+ e claudina 7 positiva pode contribuir para a análise do prognóstico em carcinoma de mama
Background: Breast carcinomas consist phenotypically of diverse cells and exhibit intra tumoral heterogeneity being stratified in several subgroups based in gene expression profiles or histochemical biomarkers. It was suggested that this heterogeneity is derived in part from the transformation of different subsets of cancer stem cells (CSC) in each intrinsic subgroup. The presence of CSC can be evidenced by phenotypic analysis of CD44 e CD24. This study aimed to identify the CD24 and CD44 immunophenotype within invasive ductal breast carcinoma (IDC) subtypes and determine its influence on prognosis as well as its association with the expression of Ki67, citokeratins (CK5, CK6 and CK18) and claudin-7. Methods: Immuno expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with intrinsic subgroups defined as luminal A (ER+, PR+, HER2-), luminal B (ER and or PR+, HER2+), HER2 subtype (ER-, PR-, HER2+) and triple negative (ER-, PR-, HER2-), and the other markers and prognosis was analyzed. Results: CD44+CD24- and CD44-CD24+ were respectively presents in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD44+CD24+ was determined in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44+CD24- phenotype was more common in the basal subgroups but the frequency of this subtype has not been associated with clinical characteristic or biological markers. The phenotype was absent in HER2 tumors whereas luminal tumors are enriched in CD44-CD24+ and CD44+CD24+ cells which did not show associations with clinical/biological markers features. There was also no significant association of the subtypes with the event free (DFS) and overall survival (OS) but the CD44+CD24- phenotype showed a more favorable prognostic as compared to CD44-CD44+ phenotype that showed a worse prognosis (p = 0.26) (median follow up, 4.8 years) CD44+ alone was evident in 57.9%, while CD24+ was positive in 74.7% of the tumors, the latter showing a significant association with ER, PR and Ki67 and a marginal association with CK18 and claudin-7. Expression of claudin-7 and Ki67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found. CD44+ was not significantly associated with OS (p = 0.684) and DFS (p = 0.386) whereas CD24+ expression was also no significantly associated with OS (p = 0.32) but was associated with a decrease in DFS (p = 0.07). CK5, CK18 and Ki67 expression had no influence in OS or DFS, however claudin-7 positive although not statistically associated with OS, was associated with reduced DFS (p = 0.05). Conclusions: The heterogeneity of cells with several CD44CD24 expression may indicate the presence of different stem cell populations. Ocurrence of CD44+CD24- phenotype is more common in triple negative tumors and lower in tumors of luminal type and absent in HER2 tumors. Although not associated significantly with patho-biological markers or OS and DFS, the CD44+CD24- phenotype has a tendency to be a favorable prognostic marker in breast cancer raising the possibilty that the putative tumorigenic ability may no be restricted to cells of this phenotype. The presence of CD44-CD24+ may indicat a worse prognosis. CD24+ was associated with ER, PR, Ki67and showed a marginal association with CK18 and claudin-7. CD24 and Claudin-7 positivity were the only biological markers associated with reduced DFS. These two investigated markers can be used to improve the assessement of prognosis in breast cancer

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As espécies invasoras são reconhecidas como um dos mais importantes e difíceis fatores que influenciam a conservação dos ecossistemas nativos. Na restauração de ambientes degradados, estas espécies podem afetar profundamente a trajetória do recobrimento vegetal pelas espécies nativas e, conseqüentemente, a composição do ecossistema. Um fator importante para o sucesso de algumas espécies vegetais invasoras é a plasticidade fenotípica, dandolhes grande capacidade de aclimatação em diversas condições ambientais. O conhecimento dos fatores ambientais e das características da planta que contribuem para o sucesso das espécies invasoras é de grande importância para predizer a habilidade de invasão e para esforços de manejo. Neste contexto, o objetivo principal deste estudo foi conhecer algumas características fisiológicas e ecológicas de Clausena excavata Burm. f., uma espécie de árvore exótica reconhecida como invasora em algumas regiões no mundo, no intuito de compreender o sucesso da espécie como invasora e contribuir com informações relevantes para possíveis tentativas de controle. Primeiramente, foram avaliados os efeitos da luz e da temperatura em sua germinação (condição controlada) e o efeito da luz na emergência de suas plântulas (condição natural, a pleno sol e sob a copa das árvores). As sementes germinaram tanto na presença como na ausência de luz, nas temperaturas de 20 a 35°C, sem diferença entre a porcentagem de sementes germinadas nas diferentes temperaturas. Sementes mantidas a 20°C, em ambas as condições de luz, germinaram mais lentamente em relação às demais temperaturas. Independente da temperatura, na presença de luz as sementes apresentaram uma germinação muito mais sincronizada do que aquelas mantidas no escuro. Tais resultados mostraram que as sementes de C. excavata são fotoblásticas neutra. Em campo, a emergência...
The invasive species are recognized one of more important and difficult factors that prejudice the conservation of native ecosystems. In the restoration of degraded environment, these species can to affect profoundly the trajectory of recovery by native species and, therefore, the ecosystems composition. An important factor to the success of invasive species is their phenotypic plasticity, which gives them a great ability to adjust in several environmental conditions. The knowledge of environmental factors and plant characteristics that contribute to the success of invasive species is very important to predict the invasion capacity and to manage efforts. In this context, the main objective of this study was to know some physiological and ecological characteristics of Clausena excavata Burm. f., a nonnative tree species recognized as invasive somewhere of the world, with intention to understand the success of this species as invasive and to contribute with relevant informations for a possible management. At first, were evaluated the effects of light and temperature on their seeds germination (controlled condition) and the effect of light on seedling emergence (natural condition, at full sun and shade). The seeds germinated in presence and absence of light, from 20 to 35°C, without difference among the germination percentage at different temperatures. Seeds maintained in 20°C, at both light conditions, germinated slowly than others temperatures. Independent of temperature, in light the seeds showed a germination more synchronized than the seeds kept in darkness. Those results showed that C. excavata have neuter photoblasics seeds. On the field, the emergence occurred at both environments, but at full sun all seeds produced a seedling. The seedling emergence occurred with same rate and synchronization index on two tested conditions. Thereafter, the seedlings were observed with purpose... (Complete abstract click electronic access below)

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

碩士
國立成功大學
藥理學研究所
92
This study is aimed to identify the specific growth factors which are critically involved in cervical cancer cell invasion and proliferation, and test the hypothesis that adhesion receptors of integrin family and growth factor receptor signaling may cooperate functionally to promote cancer invasion and proliferation. The results demonstrate that insulin like growth factor-1 (IGF-1) is a potent stimulator of cervical cancer cell invasion and proliferation. The IGF-1-stimulating effects are completely inhibited by antagonistic antibody against IGF-1 receptor (IGF-1R), whereas the effects was unaffected by either IgG or blocking antibody to insulin receptor (IR). Blocking ligand occupancy of alphav beta3, but not alpha2, alpha3, alpha4, alpha6, beta1, beta4 or alpha2 beta1 integrin, result in attenuation of cervical cancer cell response to IGF-1 stimulation. The immunofluorescent stains of surgical specimens show that cervical cancer cell with a strong tendency to invade or metastasize have higher expression of IGF-1/IGF-1R than those with a low ability to do so. Furthermore, IGF-1R overexpression correlates with poor clinical outcome in early stage of cervical cancer. Blockade of alphav beta3 integrin and IGF-1R signalings may provide novel strategies for the treatment of invasive phenotypes of cervical cancer. The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with　membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral K-Clcotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose-and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase (PI3K) and　mitogen-activated protein kinase (MAPK) cascades leading to the activation of Akt and　extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2MAPK and PI3K signaling pathways are differentially required for IGF-1-stimulated biosynthesis of KCC polypeptides. IGF-1 stimulated cellular invasiveness and proliferation are abolished by the KCC inhibitor, 50 mM DIOA. It demonstrates that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n=28), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and invasion of cervical cancer cells.

Tese de mestrado, Microbiologia Clínica e Doenças Infecciosas Emergentes, Universidade de Lisboa, Faculdade de Medicina, 2020
The introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) for children in Portugal resulted in significant changes in the serotype distribution of the pneumococcal population responsible for invasive pneumococcal disease (IPD) not only in children, but also in adults, consistent with herd protection. A 23-valent pneumococcal polysaccharide vaccine (PPV23) is also available in Portugal since 1996, although adult uptake is believed to be low. The effect of broader PCV13 uptake in children in adult IPD is currently unknown. We aimed to characterize the pneumococcal population causing adult IPD (≥18 years old) in Portugal after the introduction of PCV13 in the Nacional Immunization Plan for children, in 2015. A total of 1608 Streptococcus pneumoniae isolates were recovered from adult IPD in 61 hospitals in Portugal between 2015 and 2017. These strains were characterized phenotypically (serotyping and antimicrobial susceptibility testing) and by molecular methods (MLST). The results obtained were compared with those from previous studies to assess the effect of PCV13 introduction in Nacional Immunization Plan in the studied population. Among the 1608 isolates, 56 different serotypes were found. The most common serotypes were serotype 8 (17.8%, n=287), 3 (14.7%, n=236), 22F (7.4%, n=119), 14 (6.9%, n=111), 19A (6.2%, n=100) and 9N (4.1%, n=66). Most of the cases corresponded to serotypes exclusively found in the PPV23 (42.4%, n=681). The PCV13 serotypes were responsible for 37.6% (n=605) of the cases, and 7-valent pneumococcal conjugate vaccine (PCV7) serotypes accounted for a small fraction of the cases (14.0%, n=225). Penicillin and erythromycin non-susceptibility were detected in 14.9% (n=239) and 16.4% (n=263) of the bacterial isolates, respectively. PCV7 and serotypes exclusively found in PCV13 were responsible for 51.0% (n=122) and 15.5% (n=37) of the penicillin non-susceptible isolates and 37.6% (n=99) and 19.0% (n=50) of the erythromycin resistant isolates, respectively. Regarding all collection, 31.2% (n=501) of the strains were non-susceptible to at least one of the tested antibiotics, where serotypes 14 and 19A were the most common. MLST analysis was performed in 726 strains, where 56 different clonal complexes were found. PCV13 serotypes presented a high genetic diversity, while serotypes exclusively found in PPV23 and non-vaccine serotypes (NVT) presented a lower genetic diversity. Thirteen major clonal complexes (CC) were defined in this study that accounted for 86.4% (n=627) of the isolates: CC156, CC180, CC433, CC378, CC97, CC235, CC439, CC199, CC260, CC315, CC994, CC30 and CC191. Eleven of the 43 PMEN clones were at least double-locus-variant of 33.6% (n=244) of the isolates, however no significant association was established between clonal complexes and penicillin and erythromycin non-susceptible isolates. Serotyping was also performed using in silico methods, where both approaches (SeroBa and PneumoCAT) were found to be good alternatives to conventional methods for the determination of S. pneumoniae serotypes. Comparison with previous studies revealed an increase in the proportion of serotypes 8 and 12F, while serotypes 1, 12B and 7F decreased. Non-susceptible serotypes 8 and 22F isolates increased, whereas non-susceptible 19A isolates decreased. Although not significant, serotype 3 also presented an increase in the proportion of non-susceptible isolates to some of the antimicrobials tested. Regarding genetic lineages, sequence type (ST) 53 (serotype 8) increased, while ST191 (serotype 7F) and ST276 (serotype 19A) decreased. Even with the introduction of PCV13 in the National Immunization Plan, serotype 3 is still one of the dominant serotypes in adult IPD in Portugal, along with serotypes 14 and 19A (all serotypes present in PCV13). The increase in serotypes that do not belong to PCV13, especially serotype 8, 22F and 9N is also of concern, particularly serotypes 8 and 22F where antimicrobial non-susceptible isolates increased. With these, our data suggests that, in a situation of higher vaccination coverage, PCV13 serotypes are still significant causes of adult IPD, especially serotypes 3. However, serotypes not present in PCV13 have been showing to be an important cause of adult IPD, particularly serotypes 8 and 22F, reinforcing the continuous need for surveillance studies.
Streptococcus pneumoniae é um dos agentes patogénicos mais comuns do ser humano, responsável por elevadas taxas de mortalidade e morbilidade. Este microrganismo coloniza, tipicamente, a nasofaringe das crianças, podendo tornar-se patogénico, levando ao desenvolvimento de doenças pneumocócicas invasivas como a pneumonia, meningite e bacteriemia, ou não invasivas como otite média ou sinusite. Tem como principais grupos de risco crianças, indivíduos imunocomprometidos e idosos. Com a introdução das vacinas pneumocócicas conjugadas, observou-se um decréscimo na incidência da doença pneumocócica invasiva em crianças, assim como alterações na distribuição dos serotipos, também na população adulta. Este decréscimo deveu-se sobretudo à redução da incidência dos serotipos incluídos nestas vacinas. Contudo, observou-se um aumento na proporção de serotipos não vacinais, assim como a persistência de alguns serotipos vacinais (como foi o caso do serotipo 3 que se encontra presente na vacina pneumocócica conjugada 13-valente). A vacina pneumocócica conjugada 7-valente (PCV7, inclui os serotipos 4, 6B, 9V, 14, 18C, 19F e 23F) foi introduzida em Portugal em 2001. Apesar de disponível apenas no sector privado, a cobertura desta vacina foi aumentado gradualmente ao longo dos anos, tendo-se estimado uma cobertura vacinal de 75% das crianças com ≤2 anos em 2008. Tal como nas crianças, nos adultos observou-se uma redução da proporção de doença pneumocócica invasiva causada por serotipos abrangidos pela vacina PCV7. No entanto, os serotipos 1, 3, 7F e 19A emergiram como causas importantes de doença pneumocócica invasiva nos adultos pós-PCV7 (estudo de 2006 a 2008). Em meados de 2009, a vacina pneumocócica conjugada 10-valente (PCV10, engloba todos os serotipos presentes na vacina PCV7, mais os serotipos 1, 5 e 7F) ficou disponível, seguindo-se em 2010 a disponibilização da vacina pneumocócica conjugada 13-valente (PCV13, abrange todos os serotipos presentes na vacina PCV10, mais os serotipos 3, 6A e 19A), ambas no sector privado. A introdução desta última vacina em Portugal resultou numa alteração da distribuição dos serotipos da população de S. pneumoniae responsáveis por doença pneumocócica invasiva em adultos, apesar de estes permanecerem como importantes causas de doença pneumocócica invasiva nos adultos, consistente com o efeito de proteção de grupo. A vacina pneumocócica polissacarídica 23-valente (PPV23, integra todos os serotipos presentes na vacina PCV13, à exceção do serotipo 6A, mais os serotipos 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F e 33F) encontra-se disponível em Portugal desde 1996, ainda assim estima-se que a cobertura desta vacina seja baixa. Em 2015, a vacina PCV13 foi introduzida no plano nacional de vacinação das crianças, esperando-se, deste modo, uma maior cobertura vacinal, o que pode levar a alterações na população deste microrganismo causador de doença pneumocócica invasiva em criança e, consequentemente, em adultos. Assim, este estudo teve como objetivo caracterizar a população de Streptococcus pneumoniae causadora de doença invasiva pneumocócica nos adultos (indivíduos com ≥18 anos) em Portugal, após a introdução da vacina PCV13 no plano nacional de vacinação para as crianças em 2015. Um total de 1608 estirpes de Streptococcus pneumoniae responsável por doença invasiva pneumocócica em adultos foram recebidas por 61 hospitais de todo o país entre 2015 e 2017. Estas estripes foram caracterizadas fenotipicamente (através de serotipagem e teste de suscetibilidade aos antimicrobianos) e genotipicamente (através de métodos moleculares, obtendo-se o perfil de multilocus sequence typing por sequenciação total do genoma). Os resultados obtidos foram ainda comparados com os de estudos anteriores, de forma a avaliar o efeito da introdução da vacina PCV13 no plano nacional de vacinação na população em estudo. De entre as 1608 estirpes de pneumococos, foram encontrados 56 serotipos diferentes, sendo que os mais frequentes foram os serotipos: 8 (17.8%, n=287), 3 (14,7%, n=236), 22F (7,4%, n=119), 14 (6,9%, n=111), 19A (6,2%, n=100) e 9N (4,1%, n=66). Grande parte dos casos pertenceram a serotipos exclusivos da vacina PPV23 (42,4%, n=681). Os serotipos da vacina PCV13 foram responsáveis por 37,6% (n=605) dos casos em estudo, sendo que serotipos da vacina PCV7 foram responsáveis por uma pequena fração dos casos em estudo (14,0%, n=225). A não suscetibilidade à penicilina e à eritromicina foi detetada em 14,9% (n=239) e 16,4% (n=263) das estirpes estudadas, respetivamente. Serotipos da vacina PCV7 e serotipos exclusivos da vacina PCV13 foram responsáveis por 51,0% (n=122) e 15,5% (n=37) dos casos de não suscetibilidade à penicilina e 37,6% (n=99) e 19,0% (n=50) dos casos de resistência à eritromicina, respetivamente. Em relação à coleção total de estirpes, 31,2% (n=501) das estirpes apresentaram não suscetibilidade a pelos menos um dos antimicrobianos testados, onde os serotipos 14 e 19 foram os mais comuns. A analise dos perfis de multilocus sequence typing foi realizada em 50% das estipes que apresentaram mais do que 10 estirpes do mesmo serotipo, tendo-se obtido um total de 726 estirpes sequenciadas, onde foram detetados 56 complexos clonais diferentes. Serotipos da vacina PCV13 apresentaram uma elevada diversidade genética, enquanto que os serotipos exclusivos da vacina PPV23 e serotipos não vacinais apresentaram uma diversidade genética baixa. Treze complexos clonais principais foram definidos neste estudo, englobando 86,4% (n=627) das estirpes sequenciadas: complexo clonal (CC) 156, CC180, CC433, CC378, CC97, CC235, CC439, CC199, CC260, CC315, CC994, CC30 e CC191. Onze dos 43 clones PMEN foram identificados como sendo, pelo menos, double-locus-variants de 33,6% (n=244) das estirpes sequenciadas. No entanto, não se estabeleceu nenhuma associação significativa, depois da correção para testes múltiplos, entre complexos clonais e não suscetibilidade à penicilina e à eritromicina. Determinaram-se, ainda, os serotipos através de métodos in silico, onde se observou que ambos os softwares SeroBa e PneumoCAT são boas alternativas aos métodos convencionais para a determinação dos serotipos de S. pneumoniae. Em relação à analise comparativa com estudos anteriores, observou-se um aumento na proporção dos serotipos 8 e 12F, ao passo que os serotipo 1, 12B e 7F apresentaram um decréscimo significativo. A proporção de serotipos 8 e 22F não suscetíveis a antimicrobianos aumentou quando comparada com a proporção do estudo anterior, enquanto que a proporção de serotipo 19A não suscetível a antimicrobianos diminuiu significativamente. Apesar de não significativo após a correção para testes múltiplos, o serotipo 3 também apresentou um aumento na sua proporção de estirpes não suscetíveis a determinados antimicrobianos testados. Em relação à analise comparativa das linhagens genéticas, sequence type (ST) 53 (constituído maioritariamente por estirpes do serotipo 8) aumentou em proporção, ao passo que os ST191 (constituído sobretudo por estirpes do serotipo 7F) e ST276 (constituído principalmente por estirpes do serotipo 19A) diminuíram, todos de modo significativo. Deste modo, observou-se que mesmo com a introdução da vacina PCV13 no plano nacional de vacinação, o serotipo 3 continua a ser um dos serotipos mais frequente como causa de doença invasiva pneumocócica em adultos em Portugal, juntamente com os serotipos 14 e 19A (todos estes serotipos estão presentes na vacina PCV13). O aumento da proporção de serotipos não abrangidos pela vacina PCV13, nomeadamente os serotipos 8, 22F e 9N, são de especial preocupação, principalmente o caso dos serotipos 8 e 22F onde se observou um aumento na proporção de estirpes não suscetíveis a antimicrobianos. Assim, podemos concluir com este estudo que numa situação de elevada cobertura vacinal, os serotipos da vacina PCV13 ainda são causas importantes de doença pneumocócica invasiva nos adultos, em particular o serotipo 3. Contudo, os serotipos não abrangidos por esta vacina têm-se mostrado, também, relevantes causas de doença pneumocócica invasiva em adultos em Portugal, especialmente os serotipos 8 e 22F, reforçando desta forma a continua necessidade de realizar estudos de vigilância epidemiológica.