<p>The PRLs are a subfamily of dual specificity phosphatases that appear to play important roles in oncogenesis. Much of the current understanding of PRL function has been either correlative, and deduced from observed PRL overexpression in pathological conditions, or from in vitro analysis of signaling pathways following PRL deletion or overexpression. Such studies, necessitated by the general lack of synthetic inhibitors or compounds to probe the substrate specificity and biological interactions of the PRLs, are nonetheless now providing critical insight into potential biological substrates and roles of the PRL phosphatases. The recent identification of PTEN as a substrate for PRL2 provided the foundation for studies to further define the role of PRL2 in oncogenesis and, by analogy, the normal physiological function of PRL2. In the studies described herein, a novel PRL2 conditional knock-out animal was generated and used to validate the PRL2/PTEN interaction in a leukemic phenotype, and further demonstrated that PRL2 inhibition can restore dysregulated PTEN/AKT pathways to significantly attenuate disease progression. Inhibition of PRL2 therefore represents a novel potential therapeutic strategy in the management and treatment of AML. This thesis project also sought to further examine the role of the PRLs in oncogenesis through their regulation and interaction of targets within the TME. Functional analyses revealed that PRL3 was the only PRL to have a prominent role in host response to TME development, and that previously proposed roles for PRL3 in angiogenesis and immune cell recruitment is dependent upon PRL3 expression and activity in cells external to the TME. The study also revealed a previously unrecognized synergism between VEGF and PRL3 in the host in promoting TME angiogenesis. The studies of PRL3 in the TME suggest the potential physiological role of PRL3 in wound healing.</p>

Tese de mestrado. Mestrado Integrado em Engenharia Metalúrgica e de Materiais. Faculdade de Engenharia. Universidade do Porto. 2012

The understanding of the mesorectal spread in rectal cancer has lead to wide acceptance of total mesorectal excision (TME) as the surgical technique of choice for carcinoma in the lower and mid rectum. While oncological results and survival have improved with TME-surgery, morbidity and mortality remain important issues. The most feared complication is symptomatic anastomotic leakage. The aim of this thesis was to focus on the role of the defunctioning stoma, risk factors, and postoperative monitoring in regard to anastomotic leakage in sphincter saving resection of the rectum. Intraoperative adverse events were analysed in a retrospective population based case-control study in which all patients who underwent elective anterior resection in Sweden between 1987 and 1995, and who died within 30 days or during the initial hospital stay (n=140), were compared with patients chosen at random (n=423) who underwent the same operation during the same period, but survived the operation. Intraoperative adverse events were more frequent in those who died, and reconstruction of an anastomosis judged unsatisfactory by the surgeon improved the outcome. In a population based retrospective case-control study, risk factors for symptomatic anastomotic leakage were investigated in randomly chosen sample of patients who underwent anterior resection in Sweden between 1987 and 1995 (n=432). Twelve per cent of the patients developed symptomatic leakage, and 25% of the patients with leakage ended up with a permanent stoma. In multivariate regression analysis, low anastomosis, preoperative radiotherapy, male gender and intraoperative adverse events were independent riskfactors for anastomotic leakage. In a randomised multicentre trial patients operated with sphincter saving TME¨surgery for rectal cancer were randomised to a defunctioning stoma (n=116) or not (n=118). The overall rate symptomatic leakage was 19%. Patienst without a defunctioning stoma leaked in 28% and patients with a defunctioing stoma in 10%, a statistically significant difference (p&lt;0.001) not previously demonstrated in any randomised trial of adequate size. Postoperative monitoring with computed tomography scan (CT-scan) on postoperative day 2 and 7, and C-reactive protein (CRP) daily in 33 patients operated on with anterior resection of the rectum, demonstrated larger pelvic fluid collections in patients with leakage before the leakage was clinically diagnosed. CRP was increased from postoperative day 2 and onwards in patients in whom clinical leakage was diagnosed on median postoperative day 8. In 23 patients who underwent anterior resection of the rectum, intraperitoneal metabolism was investigated using microdialysis technique measuring the carbohydrate metabolites lactate, pyruvate and glucose. Intraperitoneal cytokines IL-6, IL-10 and TNF-α were collected through a pelvic drain and analysed. In patients who developed leakage, the latate/pyruvate ratio was increased near the anastomosis on postoperative day 5 and 6, as well as IL-6 and IL-10 which were increased postoperatively day 1 and 2, while TNF-α was higher on day 1.

La transmission mère-enfant (TME) du VIH-1 est un des enjeux majeurs de la pandémie. Une meilleure compréhension de la réponse des lymphocytes T cytotoxiques CD8+ (LTC) VIH-spécifiques lors de la grossesse facilitera le design de stratégies optimales pour diminuer la TME. Notre objectif est donc de caractériser l’amplitude et la diversité de la reconnaissance antigénique des LTC VIH-spécifiques avant, pendant et après la grossesse chez des femmes infectées par le VIH-1. Nos résultats montrent pour la première fois que l’initiation et la progression de la grossesse, à elles seules, n'ont que peu d’influence sur l’amplitude et la diversité de la reconnaissance antigénique des réponses LTC en termes de production d’IFN‐. Ces résultats indiquent que les femmes infectées par le VIH conservent une immunocompétence durant leur grossesse, du moins dans le contexte d’un traitement antirétroviral efficace. Ceci pourrait éventuellement aider à promouvoir l’immunisation comme stratégie pour prévenir la TME du VIH‐1. / Mother-to-child transmission (MTCT) of HIV-1 is one of the major issues of the pandemic. Characterization of HIV-specific immunity during pregnancy, especially cytotoxic CD8+ T lymphocytes (CTL), will lead to a better understanding of HIV pathogenesis and facilitate design of optimal strategies to prevent MTCT. Our objective is to describe the magnitude and the breadth of antigen recognition of HIV-specific CTL responses before, throughout and after pregnancy in a group of HIV-infected women. Our results revealed for the first time that initiation of pregnancy by itself doesn’t change the magnitude of CTL responses in terms of IFN- production. These findings support the fact that HIV-infected women maintain immunocompetence throughout gestation, at least in the context of effective antiretroviral treatment. These results provide a novel understanding of the dynamics of HIV-specific CTL responses during pregnancy and may help to promote maternal immunization as a strategy to prevent MTCT of HIV-1.

La perte au suivi diminue grandement l’efficacité des interventions de prévention de la transmission mère-enfant du VIH en Afrique subsaharienne. Devrait-on donc choisir des traitements courts ou longs de pTME? Nous avons mené une revue systématique des études sur l’efficacité de traitements avec et sans composante anténatale et évalué l’impact de la perte au suivi sur leur efficacité. Nous avons trouvé qu’après ajustement pour la perte au suivi, les traitements avec composante anténatale ont encore une efficacité plus élevée à prévenir la TME. Les intervenants ne devraient pas craindre l’utilisation de traitements plus longs auprès de populations à risque de perte au suivi. Plus d’études sont nécessaires pour déterminer comment les interventions peuvent être adaptées au virage dans la pTME. / Loss to follow-up greatly decreases efficiency of MTCT interventions in sub-Saharan Africa. Should we therefore choose shorter pMTCT treatments over longer ones? We conducted a systematic review of studies on the efficiency of pMTCT treatments with or without an antenatal component. We found that after adjustment, treatments with an antenatal component still had a higher efficiency at decreasing MTCT despite loss to follow-up. Healthcare providers shouldn’t fear using longer treatments amongst populations at high risk of loss to follow-up. More studies are needed to determine how current interventions can be better adapted to the turnaround in pMTCT interventions.

La transmission mère-enfant (TME) du VIH-1 est un des enjeux majeurs de la pandémie. Une meilleure compréhension de la réponse des lymphocytes T cytotoxiques CD8+ (LTC) VIH-spécifiques lors de la grossesse facilitera le design de stratégies optimales pour diminuer la TME. Notre objectif est donc de caractériser l’amplitude et la diversité de la reconnaissance antigénique des LTC VIH-spécifiques avant, pendant et après la grossesse chez des femmes infectées par le VIH-1. Nos résultats montrent pour la première fois que l’initiation et la progression de la grossesse, à elles seules, n'ont que peu d’influence sur l’amplitude et la diversité de la reconnaissance antigénique des réponses LTC en termes de production d’IFN‐. Ces résultats indiquent que les femmes infectées par le VIH conservent une immunocompétence durant leur grossesse, du moins dans le contexte d’un traitement antirétroviral efficace. Ceci pourrait éventuellement aider à promouvoir l’immunisation comme stratégie pour prévenir la TME du VIH‐1. / Mother-to-child transmission (MTCT) of HIV-1 is one of the major issues of the pandemic. Characterization of HIV-specific immunity during pregnancy, especially cytotoxic CD8+ T lymphocytes (CTL), will lead to a better understanding of HIV pathogenesis and facilitate design of optimal strategies to prevent MTCT. Our objective is to describe the magnitude and the breadth of antigen recognition of HIV-specific CTL responses before, throughout and after pregnancy in a group of HIV-infected women. Our results revealed for the first time that initiation of pregnancy by itself doesn’t change the magnitude of CTL responses in terms of IFN- production. These findings support the fact that HIV-infected women maintain immunocompetence throughout gestation, at least in the context of effective antiretroviral treatment. These results provide a novel understanding of the dynamics of HIV-specific CTL responses during pregnancy and may help to promote maternal immunization as a strategy to prevent MTCT of HIV-1.

La perte au suivi diminue grandement l’efficacité des interventions de prévention de la transmission mère-enfant du VIH en Afrique subsaharienne. Devrait-on donc choisir des traitements courts ou longs de pTME? Nous avons mené une revue systématique des études sur l’efficacité de traitements avec et sans composante anténatale et évalué l’impact de la perte au suivi sur leur efficacité. Nous avons trouvé qu’après ajustement pour la perte au suivi, les traitements avec composante anténatale ont encore une efficacité plus élevée à prévenir la TME. Les intervenants ne devraient pas craindre l’utilisation de traitements plus longs auprès de populations à risque de perte au suivi. Plus d’études sont nécessaires pour déterminer comment les interventions peuvent être adaptées au virage dans la pTME. / Loss to follow-up greatly decreases efficiency of MTCT interventions in sub-Saharan Africa. Should we therefore choose shorter pMTCT treatments over longer ones? We conducted a systematic review of studies on the efficiency of pMTCT treatments with or without an antenatal component. We found that after adjustment, treatments with an antenatal component still had a higher efficiency at decreasing MTCT despite loss to follow-up. Healthcare providers shouldn’t fear using longer treatments amongst populations at high risk of loss to follow-up. More studies are needed to determine how current interventions can be better adapted to the turnaround in pMTCT interventions.

L'intégration du traitement et de la prévention des maladies chroniques non transmissibles (MNT) au sein des soins de santé primaires représente le principal défi à venir pour la santé publique et les systèmes de santé dans les pays à faible et moyen revenu comme l’Afrique du Sud. Il constitue le principal objectif de cette thèse. L’expérience de l'intégration de la prévention de la transmission du VIH de la mère à l'enfant (PTME) dans les soins de santé primaires (SSP) peut apporter des leçons importantes pour l'intégration de la prévention du diabète chez les femmes souffrant de diabète gestationnel récent (DSG) dans les SSP. Il a été estimé que le DSG touche plus de 9,1 % des grossesses en 2018 en Afrique du Sud. Le DSG augmente le risque de développer ultérieurement du diabète de type 2 (DT2). Le DSG multiplie par plus de 7 le risque de développer un DT2 ainsi que les risques de troubles métaboliques pour les bébés des femmes qui en sont atteintes. Cette thèse explore comment appliquer les leçons tirées de l’intégration de la PTME pour intégrer le dépistage du DSG et les initiatives de prévention du DT2 dans les soins de santé primaires de routine en Afrique du Sud. Le cadre conceptuel adapté pour cette thèse permet ainsi de comprendre les aspects de l’intégration au niveau du patient et du système de santé, englobant les contextes, les mécanismes et la mise en œuvre de l’intégration d’interventions préventives dans les services existants. L'étude s'inscrit dans le cadre du projet IINDIAGO, « Intervention intégrée du système de santé visant à réduire les risques de diabète de type 2 chez les femmes défavorisées après un diabète gestationnel en Afrique du Sud ». La thèse présente d’abord une revue narrative de l’impact de la PTME sur les services et les systèmes de soins de santé en Afrique subsaharienne (Article 1). Les résultats de cette revue montrent que la PTME a eu l’impact positif et négatif sur d’autres services de soins de santé et que son intégration dans les systèmes de santé est de plus en plus privilégiée. L’article 2 est une étude qualitative analysant l’histoire et l’expérience locales de l’intégration de la PTME dans les SSP de routine en Afrique du Sud de différents points de vue. Bien qu’elle ait constaté un fort soutien en faveur de l’intégration parmi tous les répondants, cette étude a fait état de multiples obstacles à la pleine intégration de la PTME dans les SSP, le post-partum en particulier. Les articles 3 et 4 ont utilisé les méthodes mixtes et révélé que l’intégration des services dans les SSP de routine, à base communautaire, pour dépister universellement le DSG et pour prévenir ou retarder le DT2 après le DSG, était perçue comme faisable, acceptable et nécessaire de toute urgence en Afrique du Sud. L’article 6 (dont le protocole est l’article 5) présentait une revue systématique et une méta-analyse sur la prise en charge intégrée du DSG et du DT2 dans le contexte de la multimorbidité en Afrique. Les 13 études incluses dans cette étude ont montré que la gestion intégrée du DSG et du DT2 dans le cadre de la multimorbidité était mise en œuvre avec succès, mais qu’elle nécessitait une formation et une supervision adéquates des infirmières, et la fourniture d’équipements et de médicaments additionnels au sein des systèmes de santé nationaux en Afrique. Les conclusions de cette thèse suggèrent que, bien qu’elle n’ait pas toujours été retenue, en raison de défis structurels et opérationnels, l’intégration complète plutôt que partielle des services de santé est considérée comme souhaitable et réalisable par les femmes, les travailleurs de la santé, les gestionnaires et les experts. L’intégration complète pourrait être idéale pour dépister, diagnostiquer et soigner les maladies chroniques, y compris le DSG et le DT2, au sein des SSP de routine et selon l’approche de la PTME dont les leçons d’intégration n’ont pas été adaptées à ce prochain défi de santé publique. / Integrating chronic, non-communicable diseases (NCDs) and their prevention into primary health care is the next major challenge for public health and health systems in low and middle-income countries like South Africa and is the primary focus of this thesis. The experience of integration of Prevention of Mother-to-Child Transmission (PMTCT) of HIV into primary health care (PHC) may have important lessons for integrating prevention of diabetes among women with recent gestational diabetes (GDM) into PHC. GDM was estimated to affect more than 9.1% of pregnancies in 2018 in South Africa. GDM increases the risk of developing subsequent type 2 diabetes (T2DM) more than 7-fold as well as increasing the risks of metabolic disorders for the babies of women who had GDM. This thesis conducted a systematised narrative synthesis, a systematic review and a convergent mixed methods study using primarily qualitative methods in South Africa (focus on Cape Town, Western Cape) to explore how to apply lessons from PMTCT integration in order to integrate GDM screening and T2DM prevention initiatives into routine PHC in South Africa. The adapted conceptual framework for this thesis enables to understand both patient-level and health system-level aspects of integration and encompassing the contexts, mechanisms and implementation for integrating preventive interventions in the existing services. The study was nested in the IINDIAGO project, “Integrated health system intervention aimed at reducing type 2 diabetes risks in disadvantaged women after gestational diabetes in South Africa”. The thesis first presents a narrative review of the impact of PMTCT on health care services and systems in sub-Saharan Africa (Paper 1). This review findings show that PMTCT has had positive and negative impacts on other health care services and that its integration into health systems is increasingly favored. Paper 2 qualitatively documented the local history and experience of PMTCT integration into routine PHC in South Africa from different perspectives. Though it found strong support for integration among all respondents (N=20), this study reported multiple barriers for the full integration of PMTCT into PHC, especially in postpartum. Papers 3 and 4 used mixed methods and highlighted that integrating services within routine, community-based PHC to universally screen GDM and to prevent or delay of T2DM after GDM, was perceived as feasible, acceptable and urgently needed in South Africa – but that it is not currently occurring at a satisfactory level, despite international and national guidelines. The fifth article is a published protocol for Paper 6, a systematic review and meta-analysis on the integrated management of GDM and T2DM in the context of multimorbidity in Africa. This was a study in which all 13 included studies showed that integrated management of GDM and T2DM within multimorbidity was successfully implemented but it required adequate training and supervision of nurses, provision of additional equipment and drugs to the existing resources within national health systems in Africa. This thesis concludes that although not always opted for, due to structural and operational challenges, the full instead of partial integration of health services to screen, diagnose and care for chronic diseases including GDM and T2DM into routine PHC, following the PMTCT approach, was seen as both desirable and feasible by women, health workers, managers, and experts. However, the lessons learned through the history of PMTCT and its integration have not been adapted to this next public health challenge.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>

<p>  </p> <p>The phosphatases of regenerating liver are a subfamily of protein tyrosine phosphatases that consist of PRL1, PRL2 and PRL3. The overexpression of PRLs promote cell proliferation, migration and invasion and contribute to tumorigenesis and metastasis to aggravate survival outcome. Although there is increasing interest in understanding the implication of these phosphatases in tumor development, currently, limited knowledge is available about their mechanism of action and the efficacy of PRL inhibition in <em>in vivo</em> tumor models, the tumor extrinsic role of PRLs that allow them to impact tumor development, as well as <em>in vivo</em> physiological function of PRLs that could implicate them in diseases other than cancer. The work presented here aims to address these limitations.</p> <p><br></p>