Interactions between mitochondria and inflammatory factors during cellular senescence

PhD ThesisCellular senescence, the irreversible loss of proliferating capacity of somatic cells, is an important tumour suppressor mechanism but also driver of ageing. These somehow contradictory functions are dependent on the development of the so-called senescent phenotype, which involves over-production of pro-inflammatory and pro-oxidant signals, however the exact mechanisms underlying its induction remain incompletely understood. In this thesis we aimed to understand how mitochondria and pro-inflammatory factors interact during senescence and how they contribute to the senescent phenotype. Firstly, we show that mitochondria are critical for the establishment and maintenance of cell senescence. Elimination of mitochondria rejuvenated senescent human fibroblasts, abrogating the pro-inflammatory phenotype, heterochromatin foci and expression of cyclin-dependent kinase inhibitors p21 and p16. Importantly, a considerable percentage of these cells were able to resume proliferation. Mechanistically, we show that mTORC1 integrates signals from the DNA damage response towards PGC-1β-dependent mitochondrial biogenesis, playing a causal role in the development of senescence. Secondly we show that inhibition of IL-8, a prominent proinflammatory cytokine of the SASP, partially abrogated the senescent phenotype by reducing mTOR-dependent mitochondrial mass and ROS production during senescence. Finally, we demonstrate that inhibition of mitochondrial content in vivo by either rapamycin or PGC-1β deletion prevents age-dependent increase in senescence in mouse liver. Our results suggest mitochondria as an important target for interventions aiming to reduce the load of senescent cells in ageing tissues.FCT

Multi-period capacitated facility location under delayed demand satisfaction

We address an extension of the classical multi-period facility location problem in which customers are sensitive to delivery lead times. Accordingly, two customer segments are considered. The first segment comprises customers that require timely demand satisfaction, whereas customers accepting delayed deliveries make up the second segment. Each customer belonging to the latter segment specifies a maximum delivery time. A tardiness penalty is incurred to each unit of demand that is not satisfied on time. In the problem that we study, a network is already in place with a number of facilities being operated at fixed locations. The network can be expanded by establishing new facilities at a finite set of potential sites and selecting their capacity levels from a set of available discrete sizes. In addition, existing facilities may be closed over the time horizon. Two mixed-integer linear programming formulations are proposed to re-design the network at minimum cost and a theoretical comparison of their linear relaxations is provided. We also extend the mathematical models to the case in which each customer accepting delayed demand satisfaction requires late shipments to occur at most once over the delivery lead time. To gain insight into the complexity of the problems at hand, a computational study is performed with randomly generated instances that are solved with a general-purpose solver. Useful insights are derived from analyzing the impact of different delivery lead time restrictions on the network structure and cost

A computational comparison of formulations for a multi-period facility location problem with modular capacity adjustments and flexible demand fulfillment

We consider a multi-period facility location problem that takes into account changing trends in customer demands and costs. To this end, new facilities can be established at pre-specified potential locations and initially existing facilities can be closed over a planning horizon. Furthermore, facilities operate with modular capacities that can be expanded or contracted over multiple periods. A distinctive feature of our problem is that two customer segments are considered with different sensitivity to delivery lead times. Customers in the first segment require timely demand satisfaction, whereas customers in the second segment tolerate late deliveries. A tardiness penalty cost is incurred to each unit of demand that is satisfied with delay. We propose two alternative mixed-integer linear formulations to redesign the facility network over the time horizon at minimum cost. Additional inequalities are developed to enhance the original formulations. A computational study is performed with randomly generated instances and using a general-purpose solver. Useful insights are derived from analyzing the impact of several parameters on network redesign decisions and on the overall cost, such as different demand patterns and varying values for the maximum delivery delay tolerated by individual customers

Minorias e defesa nacional

- A dificuldade em tratar de um tema tão complexo como é o das Minorias de per si, a qual agravada ainda pela necessidade de, dada a natureza do curso, o articicular com a defesa nacional dos países onde as mesmas se situam. - A obrigatoriedade de, ao tratá-lo, falar de memória colectiva dos povos, preservadora das respectivas identidades culturais, que os unem tanto na união temporal efectiva quanto em episódicas e sempre indesejadas interrupções relacionais. bem como do não menos complexo lema da etnicidade, principal gerador de grupos minoritários. - A impossibilidade de, com um mínimo de credibilidade, se falar da actual situação interna do nosso país sem que se proceda a uima verdadeira pesquisa antropológica e sociológica no terreno acerca das minorias territorial mente existentes. - A discriminação a que todas elas estão sujeitas face ao grupo socialmente dominante e aos principais métodos desta prática (aniquilamento, expulsão e segregação). - As reacções minoritáris mais comuns (aculturação, auto-segregaçao, separatismo e criação de organizações). - A também possível integração das mesmas como membros de pleno direito na sociedade maior e as respectivas práticas (assimilação, amalgamação, pluralismo cultural e integração). - A génese das minorias e o verdadeiro significado do termo. - Alguns exemplos de grupos minoritários formados (ou ressurgidos) nos últimos tempos, tanto na Áfriça "negra" quanto na "brança" Europa e a sua dinâmica no terren

Depletion of mitochondria in mammalian cells through enforced mitophagy

Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, stimulates widespread mitophagy and effectively eliminates mitochondria. In this protocol, we explain how to generate Parkin-expressing, mitochondria-depleted cells from scratch in 23 d, as well as offer a variety of methods for confirming mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells for up to 30 d with minimal loss of viability, for longitudinal studies. This method should prove useful for investigating the importance of mitochondria in a variety of biological processes

Presbycusis: social impact

Trabalho final de mestrado integrado em Medicina, apresentado à Faculdade de Medicina da Universidade de Coimbra.Introdução: A presbiacusia é uma condição cada vez mais prevalente nos dias-de-hoje devido ao envelhecimento progressivo da população, constituindo a causa mais frequente de hipoacusia no idoso. Trata-se de uma patologia que afeta a comunicação e o ajuste social, levando a repercussões significativas a nível emocional, com rebate importante na qualidade de vida do doente. Na ausência de tratamento, as consequências podem ser graves, destacando-se a maior incidência de depressão e demência. Contudo, apenas uma minoria destes indivíduos adere às soluções de reabilitação auditiva atualmente disponíveis, por diversos fatores. Objetivos: Precisar o número de doentes seguidos no Centro Hospitalar e Universitário de Coimbra (CHUC) por presbiacusia e determinar que percentagem destes utiliza prótese auditiva. Apreciar o impacto emocional da presbiacusia na qualidade de vida destes doentes. Métodos: Analisámos todos os processos clínicos do serviço de Otorrinolaringologia (ORL) do CHUC desde 2010. Paralelamente, aplicámos a versão portuguesa do questionário Hearing Handicap Inventory for the Elderly (HHIE) a 50 destes doentes, contabilizando apenas as 13 questões referentes à vertente emocional. Resultados: Contabilizámos um total de 2643 indivíduos com diagnóstico de presbiacusia corroborado por audiograma tonal típico. Apenas 3,14% utilizam prótese auditiva, dos quais 67,45% são do sexo feminino. A análise dos questionários revelou que 68% dos indivíduos apresenta rebate emocional na sua qualidade de vida, com 47% dos mesmos a admitir um impacto emocional grave. Conclusões: Apesar da elevada prevalência de indivíduos com presbiacusia seguidos no CHUC, somente uma diminuta parte destes recorre ao uso de prótese auditiva, sendo a sua utilização substancialmente superior no sexo feminino. Cerca de dois terços dos indivíduos apresentaram repercussões emocionais na sua qualidade de vida. No que toca ao impacto 3 emocional, verificou-se que este surge em maior proporção nos indivíduos do sexo feminino, e nos indivíduos com idade superior a 75 anos. Apesar destes resultados, não foi atingida significância estatística para comparação de impacto entre sexos e faixas etárias.Introduction: Presbycusis is a condition more and more prevalent nowadays due to progressive aging of the population, being this, the most frequent cause of hypocusis in the elderly. It is a pathology that affects communication and social adjustment, leading to significant emotional consequences with an important impact in patients’ quality of life. When untreated the consequences may be severe, with special focus to the increased incidence of depression and dementia. However, only a minority of these patients seem to adhere to the solutions available for audiological reabilitation due to various factors. Objectives: To specify the number of patients followed at Centro Hospitalar e Universitário de Coimbra (CHUC) due to presbycusis and to determine what percentage of those uses hearing aids. Simultaneously, we aim to assess the emotional impact of presbycusis on the quality of life of those patients. Methods: We analyzed all clinical files present in the department of Otorhinolaryngology (ORL) dating from 2010 onwards. Simultaneously, we administered a questionnaire to 50 of these patients. The questions composing this questionnaire were selected from the Hearing Handicap Inventory for the Elederly (HHIE) Portuguese short version, containing only the 13 questions that concerned the emotional impact. Results: We obtained a total of 2643 individuals diagnosed with presbycusis corroborated by a typical tonal audiogram. Only 3.14% use hearing aids, of which 67.45% are female. Analysis of the questionnaires showed that 68% of the individuals present emotional consequences in their quality of life, with 47% of these admiting a severe emotional impact. Conclusions: Despite the high prevalence of individuals with presbycusis in CHUC, only a small number of these use hearing aids, being this use substantially higher in females. Two thirds of the patients reported emotional repercussions in their quality of life. There were no statistically significant diferences found between sexes or age groups, although there seemed 5 to exist a greater proportion of patients who reported emotional impact amongst women and patients who were 75 or more years old

Fungal stains on paper: Melanins produced by fungi

Books, prints, drawings, watercolours, engravings as well as all other works of art based on paper comprise a great portion of our cultural heritage. Therefore, its preservation is a matter of great concern. Paper can be deteriorated due to physical, chemical and biological agents. Fungi are among the most common biodeteriogens affecting paper-based collections, causing severe material and information losses. This work focused on fungal stains on paper which are often coloured. These interfere with the readability of the artefacts diminishing their artistic and monetary value. Up to now, there is still no definitive answer for this problem. The successful cleaning of fungal stains from paper is a mandatory conservation task, considered a priority by paper conservators. However, most of the authors refer the stain colour and patterns but they do not indicate the colourant or colourants (or chemical compound) responsible for the stain. Black stains on paper are of major concern because, not only they are very frequent, as well as its dark colour leads to a great loss of visibility. Therefore, this work focused primarily in the extraction and characterization of fungal melanins from three different species: Aspergillus niger, Chaetomium globosum and Cladosporium cladosporioides, known to be responsible for black staining on paper and melanin production. UV-Vis, μ-FTIR and μ-Raman analyses were carried out for all three fungi melanin extracts. UV-Vis, μ-FTIR and μ-Raman results show that, after extraction and purification, purified melanin samples were obtained from the three-fungal species. Moreover, SSNMR allowed to characterize A. niger’s melanin as a L-DOPA type melanin, and Cl. cladosporioides as a DHN type melanin, by comparison with the synthetic L-DOPA melanin (Sigma-Aldrich) and the literature. This will allow for a colourant-specific testing of newly developed cleaning methods, considering the base structure of the polymer (melanin) to be removed from the paper

Integrated facility location and capacity planning under uncertainty

We address a multi-period facility location problem with two customer segments having distinct service requirements. While customers in one segment receive preferred service, customers in the other segment accept delayed deliveries as long as lateness does not exceed a pre-specified threshold. The objective is to define a schedule for facility deployment and capacity scalability that satisfies all customer demands at minimum cost. Facilities can have their capacities adjusted over the planning horizon through incrementally increasing or reducing the number of modular units they hold. These two features, capacity expansion and capacity contraction, can help substantially improve the flexibility in responding to demand changes. Future customer demands are assumed to be unknown. We propose two different frameworks for planning capacity decisions and present a two-stage stochastic model for each one of them. While in the first model decisions related to capacity scalability are modeled as first-stage decisions, in the second model, capacity adjustments are deferred to the second stage. We develop the extensive forms of the associated stochastic programs for the case of demand uncertainty being captured by a finite set of scenarios. Additional inequalities are proposed to enhance the original formulations. An extensive computational study with randomly generated instances shows that the proposed enhancements are very useful. Specifically, 97.5% of the instances can be solved to optimality in much shorter computing times. Important insights are also provided into the impact of the two different frameworks for planning capacity adjustments on the facility network configuration and its total cost.publishersversionpublishe

Equity report on caterpillar Inc

This paper is presented in the form of an equity report, aiming to evaluate Caterpillar Inc. per share value, comparing its market value with its fair value. This manuscript presents investors with two valuation methodologies – Market Multiples and the Adjusted Present Value – that define a target price range for Caterpillar’s stocks. The first model compares Caterpillar accountings with its major competitors’ data and in the second model a 10-year forecast valuation was built. Based on the referred methods and supported on the analysis of the firm’s performance and future outlook a sell investment recommendation was made by the author