As vias amiloidogênica e não-amiloidogênica, representadas pelas enzimas secretases como a enzima clivadora do sítio beta da APP (BACE) e a enzima conversora do fator de necrose tumoral-alfa (TACE) respectivamente, são responsáveis pela clivagem da proteína precursora amiloide (APP). Alterações no processamento da APP associadas ao acúmulo do peptídeo β-amiloide (Aβ) parecem estar relacionadas aos déficits cognitivos observados na doença de Alzheimer (DA), no entanto, estudos avaliando a maquinaria de processamento da APP durante o envelhecimento fisiológico são raros. O Aβ é formado através da clivagem da proteína precursora amiloide (APP) pela enzima BACE. Por outro lado, a APP pode ser clivada por secretases como a TACE gerando APPα, o qual é considerado neuroprotetor. Alguns estudos têm sugerido um envolvimento de vesículas denominadas exossomos no transporte de proteínas como o peptídeo Aβ além de um papel dos exossomos durante o estresse oxidativo e no processo de envelhecimento. No entanto, estudos avaliando a relação entre exossomos e marcadores oxidativos no envelhecimento ainda não foram realizados. Além disso, apesar de diversas evidências demonstrarem os efeitos benéficos do exercício físico, os efeitos exercidos sobre a modulação da atividade das secretases, especificamente TACE e BACE, e sobre o perfil dos exossomos durante o envelhecimento fisiológico têm sido pouco investigados. Portanto, o objetivo deste trabalho foi avaliar os efeitos do exercício físico sobre a atividade das secretases e sobre o perfil de exossomos circulantes em ratos durante o envelhecimento. Ratos Wistar de 3, 21 e 26 meses de idade foram divididos em sedentários e exercitados, o protocolo de exercício consistiu em 20 min/ dia durante 14 dias e após a última a sessão de exercício todos os animais foram submetidos ao teste da esquiva inibitória. As estruturas cerebrais e o sangue troncular foram coletados 1h (período da tarde) e 18 h (período da manhã) após a última sessão de exercício com o objetivo de avaliar os efeitos transitórios e tardios do protocolo de exercício. O hipocampo e o córtex pré-frontal foram dissecados e utilizados para quantificar o conteúdo de APP e avaliar a atividade das enzimas TACE e BACE. Os exossomos foram isolados do soro e utilizados para quantificar CD63, atividade da acetilcolinesterase (AChE), conteúdo de espécies reativas, atividade da superóxido dismutase (SOD) e conteúdo de Aβ. / The amyloidogenic and non-amyloidogenic pathways, represented by secretases named β-site APP cleaving enzyme(BACE) and tumor necrosis factor-alpha converting enzyme (TACE), respectively, are responsible for amyloid protein precursor (APP) processing. APP cleavage modifications leading to increased β-amyloid (Aβ) peptide levels seem to be related to cognitive decline observed in Alzheimer disease (AD), however, works evaluating the APP processing machinery in the normal aging process are rarely studied. The Aβ is formed through APP cleavage by BACE enzyme, named amyloidogenic pathway. On the other hand, APP can be cleaved by a non-amyloidogenic pathway through secretase enzymes such as TACE producing APPα, which is a neuroprotective product. Some evidences suggested that extracellular vesicles named exosomes could carry proteins including the Aβ peptide between different cells. Yet, exosomes appear to be linked to oxidative stress and aging process. However, studies evaluating the relationship between exosomes and oxidative stress marks in the aging were not yet performed. The beneficial exercise impact in the aging process are widely described, nevertheless, its effects on secretase activities, specifically BACE and TACE, and exosome profile during normal aging remains understood. Therefore, the aim of this study was to evaluate the exercise effects on secretase activities and circulating exosomes profile in the aging process. Wistar rats (3-, 21- and 26-month-old) were divided into sedentary and exercised groups; the exercise protocol consisted in a daily moderate treadmill exercise (20 min each day during 2 weeks). After the last exercise sessions, all animals were subjected to inhibitory avoidance task. To identify transitory and delayed exercise effects, specifically 1 and 18 hours after the last exercise training session, hippocampi and prefrontal cortices as well as blood were obtained at different times of day, respectively, in the afternoon and early morning. The brain areas were used to quantify the APP content and BACE and TACE activities. The circulating exosomes were isolated from serum and used to quantify CD63, reactive species and Aβ content, besides AChE and superoxide dismutase (SOD) activities.

Prostate cancer is the most common cancer in men, with the incidence rapidly increasing in Europe over the past two decades. Reliable biomarkers for prostate cancer are currently unavailable. Thus, there is an urgent need for improved biomarkers to diagnose prostate cancer at an early stage and to determine the best treatment options. Higher expression of transforming growth factor-β (TGFβ) has been reported in patients with aggressive cancer. TGFβ is a multifunctional cytokine that acts as a tumor suppressor during early tumor development, and as a tumor promoter at later stages of cancer. TGFβ signals through the canonical Smad or non-Smad cascade via TGFβ type II and type I receptors. The TGFβ signaling cascade is regulated by various post-translational modifications of its key components. The present investigation aimed to identify a potential function of TRAF6 in TGFβ-induced responses in prostate cancer. The first two articles of this thesis unveil the proteolytic cleavage of TGFβ type I receptor (TβRI), and the biological importance of the liberated TβRI intracellular domain (TβRI-ICD) in the nucleus. We found that tumor necrosis factor receptor-associated factor 6 (TRAF6) polyubiquitinates TβRI, which leads to cleavage of TβRI by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta (PKCζ)-dependent manner. Following ectodomain shedding, TβRI undergoes a second cleavage by presenilin 1 (PS1), which liberates TβRI-ICD. TβRI-ICD translocates to the nucleus, where it regulates its own expression as well as expression of the pro-invasive gene Snail1, thereby promoting invasion. We further found that TβRI-ICD associates with Notch intracellular domain (NICD) to drive expression of the pro-invasive gene Snail1, as well as Notch1 ligand Jag1. The third article provides evidence that TRAF6 promotes Lys63-linked polyubiquitination of TβRI at Lys178 in a TGFβ-dependent manner. TβRI polyubiquitination was found to be a prerequisite for TβRI nuclear translocation, and thus for regulation of the genes involved in cell cycle, differentiation, and invasion of prostate cancer cells. In the fourth article we investigated the role of the pro-invasive gene Snail1 in TGFβ-induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells.

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients. TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling. The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model. In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner. In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells. In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.

As vias amiloidogênica e não-amiloidogênica, representadas pelas enzimas secretases como a enzima clivadora do sítio beta da APP (BACE) e a enzima conversora do fator de necrose tumoral-alfa (TACE) respectivamente, são responsáveis pela clivagem da proteína precursora amiloide (APP). Alterações no processamento da APP associadas ao acúmulo do peptídeo β-amiloide (Aβ) parecem estar relacionadas aos déficits cognitivos observados na doença de Alzheimer (DA), no entanto, estudos avaliando a maquinaria de processamento da APP durante o envelhecimento fisiológico são raros. O Aβ é formado através da clivagem da proteína precursora amiloide (APP) pela enzima BACE. Por outro lado, a APP pode ser clivada por secretases como a TACE gerando APPα, o qual é considerado neuroprotetor. Alguns estudos têm sugerido um envolvimento de vesículas denominadas exossomos no transporte de proteínas como o peptídeo Aβ além de um papel dos exossomos durante o estresse oxidativo e no processo de envelhecimento. No entanto, estudos avaliando a relação entre exossomos e marcadores oxidativos no envelhecimento ainda não foram realizados. Além disso, apesar de diversas evidências demonstrarem os efeitos benéficos do exercício físico, os efeitos exercidos sobre a modulação da atividade das secretases, especificamente TACE e BACE, e sobre o perfil dos exossomos durante o envelhecimento fisiológico têm sido pouco investigados. Portanto, o objetivo deste trabalho foi avaliar os efeitos do exercício físico sobre a atividade das secretases e sobre o perfil de exossomos circulantes em ratos durante o envelhecimento. Ratos Wistar de 3, 21 e 26 meses de idade foram divididos em sedentários e exercitados, o protocolo de exercício consistiu em 20 min/ dia durante 14 dias e após a última a sessão de exercício todos os animais foram submetidos ao teste da esquiva inibitória. As estruturas cerebrais e o sangue troncular foram coletados 1h (período da tarde) e 18 h (período da manhã) após a última sessão de exercício com o objetivo de avaliar os efeitos transitórios e tardios do protocolo de exercício. O hipocampo e o córtex pré-frontal foram dissecados e utilizados para quantificar o conteúdo de APP e avaliar a atividade das enzimas TACE e BACE. Os exossomos foram isolados do soro e utilizados para quantificar CD63, atividade da acetilcolinesterase (AChE), conteúdo de espécies reativas, atividade da superóxido dismutase (SOD) e conteúdo de Aβ. / The amyloidogenic and non-amyloidogenic pathways, represented by secretases named β-site APP cleaving enzyme(BACE) and tumor necrosis factor-alpha converting enzyme (TACE), respectively, are responsible for amyloid protein precursor (APP) processing. APP cleavage modifications leading to increased β-amyloid (Aβ) peptide levels seem to be related to cognitive decline observed in Alzheimer disease (AD), however, works evaluating the APP processing machinery in the normal aging process are rarely studied. The Aβ is formed through APP cleavage by BACE enzyme, named amyloidogenic pathway. On the other hand, APP can be cleaved by a non-amyloidogenic pathway through secretase enzymes such as TACE producing APPα, which is a neuroprotective product. Some evidences suggested that extracellular vesicles named exosomes could carry proteins including the Aβ peptide between different cells. Yet, exosomes appear to be linked to oxidative stress and aging process. However, studies evaluating the relationship between exosomes and oxidative stress marks in the aging were not yet performed. The beneficial exercise impact in the aging process are widely described, nevertheless, its effects on secretase activities, specifically BACE and TACE, and exosome profile during normal aging remains understood. Therefore, the aim of this study was to evaluate the exercise effects on secretase activities and circulating exosomes profile in the aging process. Wistar rats (3-, 21- and 26-month-old) were divided into sedentary and exercised groups; the exercise protocol consisted in a daily moderate treadmill exercise (20 min each day during 2 weeks). After the last exercise sessions, all animals were subjected to inhibitory avoidance task. To identify transitory and delayed exercise effects, specifically 1 and 18 hours after the last exercise training session, hippocampi and prefrontal cortices as well as blood were obtained at different times of day, respectively, in the afternoon and early morning. The brain areas were used to quantify the APP content and BACE and TACE activities. The circulating exosomes were isolated from serum and used to quantify CD63, reactive species and Aβ content, besides AChE and superoxide dismutase (SOD) activities.

As vias amiloidogênica e não-amiloidogênica, representadas pelas enzimas secretases como a enzima clivadora do sítio beta da APP (BACE) e a enzima conversora do fator de necrose tumoral-alfa (TACE) respectivamente, são responsáveis pela clivagem da proteína precursora amiloide (APP). Alterações no processamento da APP associadas ao acúmulo do peptídeo β-amiloide (Aβ) parecem estar relacionadas aos déficits cognitivos observados na doença de Alzheimer (DA), no entanto, estudos avaliando a maquinaria de processamento da APP durante o envelhecimento fisiológico são raros. O Aβ é formado através da clivagem da proteína precursora amiloide (APP) pela enzima BACE. Por outro lado, a APP pode ser clivada por secretases como a TACE gerando APPα, o qual é considerado neuroprotetor. Alguns estudos têm sugerido um envolvimento de vesículas denominadas exossomos no transporte de proteínas como o peptídeo Aβ além de um papel dos exossomos durante o estresse oxidativo e no processo de envelhecimento. No entanto, estudos avaliando a relação entre exossomos e marcadores oxidativos no envelhecimento ainda não foram realizados. Além disso, apesar de diversas evidências demonstrarem os efeitos benéficos do exercício físico, os efeitos exercidos sobre a modulação da atividade das secretases, especificamente TACE e BACE, e sobre o perfil dos exossomos durante o envelhecimento fisiológico têm sido pouco investigados. Portanto, o objetivo deste trabalho foi avaliar os efeitos do exercício físico sobre a atividade das secretases e sobre o perfil de exossomos circulantes em ratos durante o envelhecimento. Ratos Wistar de 3, 21 e 26 meses de idade foram divididos em sedentários e exercitados, o protocolo de exercício consistiu em 20 min/ dia durante 14 dias e após a última a sessão de exercício todos os animais foram submetidos ao teste da esquiva inibitória. As estruturas cerebrais e o sangue troncular foram coletados 1h (período da tarde) e 18 h (período da manhã) após a última sessão de exercício com o objetivo de avaliar os efeitos transitórios e tardios do protocolo de exercício. O hipocampo e o córtex pré-frontal foram dissecados e utilizados para quantificar o conteúdo de APP e avaliar a atividade das enzimas TACE e BACE. Os exossomos foram isolados do soro e utilizados para quantificar CD63, atividade da acetilcolinesterase (AChE), conteúdo de espécies reativas, atividade da superóxido dismutase (SOD) e conteúdo de Aβ. / The amyloidogenic and non-amyloidogenic pathways, represented by secretases named β-site APP cleaving enzyme(BACE) and tumor necrosis factor-alpha converting enzyme (TACE), respectively, are responsible for amyloid protein precursor (APP) processing. APP cleavage modifications leading to increased β-amyloid (Aβ) peptide levels seem to be related to cognitive decline observed in Alzheimer disease (AD), however, works evaluating the APP processing machinery in the normal aging process are rarely studied. The Aβ is formed through APP cleavage by BACE enzyme, named amyloidogenic pathway. On the other hand, APP can be cleaved by a non-amyloidogenic pathway through secretase enzymes such as TACE producing APPα, which is a neuroprotective product. Some evidences suggested that extracellular vesicles named exosomes could carry proteins including the Aβ peptide between different cells. Yet, exosomes appear to be linked to oxidative stress and aging process. However, studies evaluating the relationship between exosomes and oxidative stress marks in the aging were not yet performed. The beneficial exercise impact in the aging process are widely described, nevertheless, its effects on secretase activities, specifically BACE and TACE, and exosome profile during normal aging remains understood. Therefore, the aim of this study was to evaluate the exercise effects on secretase activities and circulating exosomes profile in the aging process. Wistar rats (3-, 21- and 26-month-old) were divided into sedentary and exercised groups; the exercise protocol consisted in a daily moderate treadmill exercise (20 min each day during 2 weeks). After the last exercise sessions, all animals were subjected to inhibitory avoidance task. To identify transitory and delayed exercise effects, specifically 1 and 18 hours after the last exercise training session, hippocampi and prefrontal cortices as well as blood were obtained at different times of day, respectively, in the afternoon and early morning. The brain areas were used to quantify the APP content and BACE and TACE activities. The circulating exosomes were isolated from serum and used to quantify CD63, reactive species and Aβ content, besides AChE and superoxide dismutase (SOD) activities.

Background and Aims: Morphometric features such as the Milan criteria serve as standard criteria for liver transplantation (LT) in patients with hepatocellular carcinoma (HCC). Since it has been recognized that these criteria are too restrictive and do not adequately display the tumor biology, additional selection parameters are emerging. Methods: Concise review of the current literature on patient selection for downstaging and LT for HCC outside the Milan criteria. Results: The major task in patients outside the Milan criteria is the need for higher granularity with patient selection, since the benefit through LT is not uniform. The recent literature clearly shows that beneath tumor size and number, additional selection parameters are useful in the process of patient selection for and during downstaging. For initial patient selection, the alpha fetoprotein (AFP) level adds additional information to the size and number of HCC nodules concerning the chance of successful downstaging and LT. This effect is quantifiable using newer selection tools like the WE (West-Eastern) downstaging criteria or the Metroticket 2.0 criteria. Also an initial PET-scan and/or tumor biopsy can be helpful, especially in the high risk group of patients outside the University of California San Francisco (UCSF) criteria. After this entry selection, the clinical course during downstaging procedures concerning the tumor and the AFP response is of paramount importance and serves as an additional final selection tool Conclusion: Selection criteria for liver transplantation in HCC patients are becoming more and more sophisticated, but are still imperfect. The implementation of molecular knowledge will hopefully support a more specific risk prediction for HCC patients in the future, but do not provide a profound basis for clinical decision-making at present.