In order to achieve cure for HIV-1 infection in patients undergoing suppressive antiretroviral therapy, eradication of all latently infected reservoirs of the virus is required. The focus of HIV cure is predominantly centred on the elimination of latently infected memory T cells, while information on possible elimination of infected macrophages is lacking. Macrophages support continuous virus replication without succumbing to cytopathic effects of HIV-1. Recently, our laboratory has shown a protective role for cellular inhibitor of apoptosis proteins (IAPs) 1/2 in macrophages against Vpr-induced apoptosis. Depletion of cIAP1/2 by Smac mimetics (SM) reverse the IAP-mediated protection and sensitize macrophages to Vpr-induced cell death. My research aims to understand the role IAPs play in apoptotic resistance of HIV-infected macrophages. I hypothesized that ablation of cIAP1/2 by SM may induce apoptosis in HIV-infected macrophages. My results show that SM does not induce cell death in uninfected or healthy macrophages, but induces cell death in chronically infected U1 cells, in vitro infected monocyte-derived macrophages, and ex vivo derived HIV-infected macrophages from HIV-infected individuals. SM induce cell death of infected myeloid cells through apoptosis and not through necroptosis. Moreover, SM-induced apoptosis is independent of TNFα and other endogenously secreted cytokines. In vitro infection of monocyte-derived macrophages leads to the downregulation of RIPK1, RIPK3, and TRAF-1. Interestingly, necrostatin-1-mediated RIPK1- inhibition does not affect viability of healthy macrophages, but in combination with IAP degradation by SM leads to significant induction of apoptosis. This suggests a key role for RIPK1 in SM-induced apoptosis of HIV-infected macrophages. Altogether, the results from this project suggest that modulation of the IAP-associated signalling pathways by SM may be a potential strategy for selective killing of HIV-infected macrophages.

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The aim of the study was to find out if there are features of an image which influence the emotional output to improve the affective pictorial stimuli for advertisements especially in tourism. The present study bases upon emotional pictures of the IAPS which were modified in several ways. In a first step the 18 most desperate images according to Russell's affective space were selected. The second step was the modification from an original picture to a grayscale picture, to a low luminance picture, to a high luminance picture, to a vertical reflected picture, to a high chroma picture and to a blurred picture. In a third step participants were asked to fill out a questionnaire and to rate the evoked emotions using SAM. Results show, that there are important factors of an image which influence the emotional reaction and which could be used to improve the pictorial stimuli for marketing.

Neuroblastoma is the third most common pediatric cancer. Dinutuximab is a recently approved monoclonal antibody targeting GD2, a ganglioside ubiquitously present on neuroblastoma. Recent studies have shown that αGD2 therapy activates PD1-PDL1 signalling, resulting in the inhibition of its full therapeutic potential. The PD1-PDL1 signalling axis is a cellular checkpoint that inhibits immune responses. The blocking of this interaction has been successful in the treatment of numerous cancers, including in combination with anti-GD2 therapy. The Inhibitor of apoptosis (IAP) proteins are commonly upregulated in cancers and prevent cell death through the inhibition of caspases and through the control of NF-κB activity. Smac mimetic compounds (SMCs) have been designed to target IAP activity, thereby promoting cancer cell death. Here, I used the SMC, LCL161, to improve αGD2 antibody treatment against a GD2+ syngeneic neuroblastoma mouse model. I found that murine cell lines NXS2 and N2a were resistant in vitro to LCL161-mediated apoptosis, despite expressing apoptotic components often silenced in neuroblastoma. In vivo, I observed a slight delay in tumour growth induced by LCL161 and I confirmed an in vivo anti-angiogenic effect of LCL161 through ultrasound imaging and necropsy evaluation. I then combined LCL161 and αGD2 antibody (clone ME361-S2a) treatment and reported a delay in NXS2 subcutaneous tumour growth, which was further potentiated with the addition of an αPD-L1 antibody. With optimization, there is potential for SMCs to be used in combination with αGD2 therapy in GD2+ cancers like neuroblastoma.

To address the feasibility of ambulatory emotion recognition, characteristics of biosignals were compared between sitting and controlled walking using different stimulus modalities. Emotional stimulus items were drawn from the International Affective Pictures System and International Affective Digitized Sounds libraries to elicit five basic emotions. To assess which emotion was elicited, participants (n=15) completed self-report scales using the Self-Assessment Manikin and discrete emotion ratings following the presentation of each stimulus item. Autonomic activity was monitored using electrocardiogram, electrodermal activity, and thoracic and abdominal respiration. Multivariate analysis of variance was employed to test for differences in biosignal features and supervised classifiers were trained to predict the elicited emotion using physiological data. The study revealed differences between sitting and walking states but no effect was found for stimulus modality. Self-reported emotions were poorly predicted using our methodology and a discussion of potential directions and recommendations for future research was presented.

To address the feasibility of ambulatory emotion recognition, characteristics of biosignals were compared between sitting and controlled walking using different stimulus modalities. Emotional stimulus items were drawn from the International Affective Pictures System and International Affective Digitized Sounds libraries to elicit five basic emotions. To assess which emotion was elicited, participants (n=15) completed self-report scales using the Self-Assessment Manikin and discrete emotion ratings following the presentation of each stimulus item. Autonomic activity was monitored using electrocardiogram, electrodermal activity, and thoracic and abdominal respiration. Multivariate analysis of variance was employed to test for differences in biosignal features and supervised classifiers were trained to predict the elicited emotion using physiological data. The study revealed differences between sitting and walking states but no effect was found for stimulus modality. Self-reported emotions were poorly predicted using our methodology and a discussion of potential directions and recommendations for future research was presented.

Psicóloga / Introducción: El miedo es una emoción de alto valor adaptativo, siendo limitada su investigación a nivel internacional por la ausencia de estímulos confiables que lo evoquen. El IAPS es el instrumento más utilizado en estudios sobre emociones, basándose principalmente en la aproximación dimensional de las emociones. Objetivo: Identificar un set válido de imágenes evocadoras de miedo del IAPS a través de evaluaciones categóricas (emociones discretas) y dimensionales (valencia, arousal y dominancia) en una muestra chilena, evaluando diferencias por sexo y transculturales. Método: 60 participantes chilenos (30 mujeres), con un promedio de edad de 22,3 años, evaluaron categorial y dimensionalmente 146 imágenes del IAPS. Resultados: Se identificaron seis imágenes que evocan miedo de manera significativa, además de diferencias por sexo y transculturales en la valoración de las imágenes. Conclusiones: Se reafirma importancia de utilizar normas específicas por sexo y país para seleccionar estímulos visuales en estudios sobre emociones, especialmente para el miedo

Orientador: Maria Luiza Cotrim Sator de Oliveira / Resumo: A maioria dos tumores malignos da mama apresenta aspecto morfológico de carcinoma ductal invasivo que pode apresentar diferentes prognósticos. Diante disso, conclui-se que a identificação do padrão morfológico não é suficiente para o estabelecimento do prognóstico, e que existe a necessidade da identificação de novos biomarcadores imunoistoquímicos que indiquem moléculas sinalizadoras importantes para proliferação e sobrevivência das células neoplásicas, e que possam ser alvos de modalidades terapêuticas oncológicas. A ocorrência de alterações dos mecanismos regulatórios genéticos da apoptose pode resultar em uma hiperativação das proteínas antiapoptóticas, com a perda da sensibilidade aos sinais de morte fisiológica, evitando-se a ocorrência da apoptose e resultando, desse modo, na preservação de células geneticamente alteradas com consequente aumento no seu número. Esse é um evento crítico para o início do crescimento tumoral, com repercussões no prognóstico e na resistência ao tratamento oncológico. Dessa forma, o entendimento da maquinaria das vias do processo apoptótico é de crucial importância na avaliação prognóstica do processo neoplásico, pois suas moléculas são importantes biomarcadores da proliferação e sobrevivência celular. Objetivos: Avaliar a relação entre as proteínas inibidoras do processo apoptótico (IAPs) e seu inibidor Smac/DIABLO no carcinoma mamário ductal invasivo, sem outra especificação (SOE). Analisar, também, sua correlação com os marcadores imunoi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Most of malignant breast tumors exhibit morphological aspect of invasive ductal carcinoma, not otherwise specified (NOS) carcinoma, that can present different prognosis. It concludes that the identification of the morphological type of the breast cancer is not enough to establish tumor prognosis. So there is the need to recognize new immunohistochemistry biomarkers that can identify important molecular signaling proteins of neoplastic cell proliferation and survival that can be used as target of oncological therapeutic. Changes in these regulatory mechanisms that favors the hiperactivation of antiapoptotic proteins can set the loss of the physiological signs of cell death avoiding the occurrence of apoptosis and preserving genetic modified cells. This is a critical event to the beginning of tumor growth which may further lead to repercussions in prognosis and cancer resistance to therapy. The knowledge about the different pathways to apoptosis is crucial to evaluate the prognosis of the neoplastic process. Objectives: To study the relation between the inhibitors of apoptosis proteins (IAPs) and their antagonist, the protein SMAC/DIABLO, in invasive ductal breast carcinoma and also evaluate their correlation with the usual prognostic immunohistochemical markers, Apoptotic Index and clinical prognostics factors. Design: TMA paraffin blocks were made with invasive ductal breast carcinoma tissue samples of patients operated at the Botucatu School of Medicine Hospital from 1980 ... (Complete abstract click electronic access below) / Doutor

Novel strategies for the treatment of malignant gliomas are urgently needed. They are characterised by an inherent resistance to both chemo- and radiotherapeutics resulting in unrelenting tumour progression. While the exact mechanisms of treatment resistance remain undefined, it is now recognized that multiple components within the apoptotic pathway are heavily dysregulated in glioma cells and that the over-expression of anti-apoptotic proteins in patient samples correlates with inferior patient survival. The Inhibitor of Apoptosis Proteins (lAPs) represent the final molecular blockade preventing cellular apoptosis and have been identified as a potential rational therapeutic target in gliomas. The work described herein was focused on the development of novel therapeutic strategies that target the lAPs in malignant gliomas, that are readily translatable to the clinic, and that have the potential to improve patient outcomes. The first series of studies examined the hypothesis that targeting the lAPs in conjunction with other conventional and targeted therapies would overcome treatment resistance, and enhance anti-tumour activity. The novel, small molecule, lAP inhibitor LBW242 was shown to successfully target the lAPs in glioma cells and inhibit their ability to bind to and inactivate caspases. However when tested as a single agent in vitro, no stand alone anti-glioma activity of LBW242 was demonstrated. A screen of the activity of LBW242 in combination other pro-apoptotic compounds led to the discovery that lAP inhibition applied in combination with receptor tyrosine kinase (RTK) inhibition led to enhanced caspase activation and induction of apoptosis with a subsequent synergistic anti-glioma effect. The most profound effect was demonstrated with the specific combination of PDGFR and lAP inhibition both in vitro and in vivo as well as in primary patient derived glioma tumourspheres. While multiple RTKs have previously been validated as rational therapeutic targets, the clinical failure of RTK inhibitors in glioma patients has to date remained unexplained. The results in this thesis provide a novel explanation for the resistance of glioma cells to these targeted therapies, and more importantly offer a clinically tractable strategy of overcoming that resistance and improving patient outcomes. The second series of studies investigated the mechanism of synergy between lAP and RTK inhibition. The results showed that PDGFR inhibition does not stimulate apoptosis in glioma cells by previously described pathways. A screen of the entire apoptotic pathway revealed that treatment with imatinib modulates the expression of the anti-apoptotic protein NOL3/ARC. The results showed that imatinib treatment leads to down-regulation of NOL3 and that this effect is critical to the synergy between lAP and PDGFR inhibition. Further analysis suggested a critical role for NOL3 in gliomagenesis and treatment resistance NOL3 was found to be highly expressed in malignant gliomas and with expression levels that are inversely correlated with patient outcomes. A role for NOL3 has not previously been described in malignant gliomas. Finally, a series of studies were undertaken that tested the use of LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide. In vitro assays demonstrated that LBW242 enhanced the pro-apoptotic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple glioma cell lines. Athymic mice bearing established human malignant glioma tumour xenografts treated with LBW242 plus radiation and temozolomide demonstrated a profound and synergistic suppression of tumour growth. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in highly resistant glioma stem cells with a corresponding inhibition of tumour growth. The results indicate a potentially powerful strategy to enhance the therapeutic activity of standard-of-care therapies in glioma patients. Collectively, the findings of the studies in this thesis contribute to a better understanding of the mechanisms of treatment resistance in malignant gliomas, and demonstrate that the pro-apoptotic and anti-glioma effects of radiotherapy, chemotherapy and specific targeted therapies can be enhanced by the addition of a novel, small molecule lAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for glioma patients.

Wounds heal better in skin with terminal hair follicles (large and pigmented) as opposed to those with vellus hair follicles (small and unpigmented), while dermal fibroblasts from different anatomical regions also exhibit phenotypical differences. Tissue repair requires a tight control of cell proliferation, migration and apoptosis, and recent studies have shown the importance of inhibitors of apoptosis proteins (IAPs), which are proteins that prevent the process of apoptosis via their interaction with caspase molecules in wound healing. Oestrogens improve the rate and quality of wound healing, but their relationship with IAPs in human skin has not been studied. Therefore, terminal (scalp) and vellus (facial) hair bearing skin from the same donor was compared in situ and matching primary cultures of dermal fibroblasts were established from terminal (DF(T)) and vellus (DF(V)) hair bearing skin. Using immunofluorescent staining, the expression of IAPs and their antagonists was compared at different stages of the hair cycle following depilation using a murine model and then in terminal and vellus hair bearing human skin. The size and granularity of matching DF(T) and DF(V) cultures was compared by FACS analysis and mRNA and protein expression of Apollon, cIAP2, NAIP and XIAP and their antagonists DIABLO and Xaf1 analysed by qRT-PCR and immunocytochemistry in unwounded and mechanically wounded fibroblast cultures. Differences in proliferation, migration, viability and caspase 3 activity in the presence of 17β-oestradiol and changes in mRNA expression of the oestrogen receptors (GPR30, ERα and ERβ) were compared between the two cell types. IAP protein expression was generally found higher during mid anagen of the hair cycle in murine skin and hair follicles. Overall, expression was slightly higher in human terminal hair bearing skin compared to corresponding vellus hair bearing skin. IAP protein expression was similar in unwounded DF(T) and DF(V) cells with the exception of Apollon which was higher in DF(V) cells. With the exception of XIAP and its direct antagonist Xaf1, mRNA expression was higher in DF(V) cells compared to corresponding DF(T) cells. FACS analysis demonstrated that DF(V) cells were more granular than matching DF(T) cells and proliferated faster. 17β-oestradiol accelerated migration of DF(T) cells only. Mechanical wounding decreased XIAP mRNA in DF(T) and increased it in DF(V) cells, while simultaneously decreasing Xaf1 expression. In unwounded cells, 17β-oestradiol stimulated the expression of XIAP mRNA in both DF(T) and DF(V) cells, but in scratched monolayers, while it also increased expression in DF(T) cells it decreased it in DF(V) cells. A XIAP inhibitor reduced cell viability in both DF(T) and DF(V) cells, which was rescued by 17β-oestradiol in unwounded and mechanically wounded DF(T) cells, but only in unwounded DF(V) cells. 17β-oestradiol decreased caspase 3 activity in the presence of a XIAP inhibitor only in DF(T) cells. These results demonstrate significant differences between dermal fibroblasts cultured from terminal and vellus hair bearing skin of the same individual. The correlation between an increase in XIAP in response to 17β-oestradiol and a higher number of viable cells, along with a reduction in caspase 3 activity suggests that the protective effect of 17β-oestradiol may be modulated via the regulation of XIAP. Further elucidation of these different signalling pathways in dermal fibroblasts from hair bearing skin may lead to improved therapies for chronic non-healing wounds, particularly in postmenopausal females.