Synthesis of Polymeric Nanoparticles for biomedical delivery applications

Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behaviors provided by their small size. Such materials could be used in a wide range of applications such as diagnostics and drug delivery. Advantages of PNPs include controlled release, protection of drug molecules and its specific targeting, with concomitant increasing of the therapeutic index. In this work, novel sucrose and cholic acid based PNPs were prepared from different polymers, namely polyethylene glycol (PEG), poly(D,L-lactic-co-glycolic acid) (PLGA) and PLGA-co-PEG copolymer. In these PNP carriers, cholic acid will act as a drug incorporation site and the carbohydrate as targeting moiety. The uptake of nanoparticles into cells usually involves endocytotic processes, which depend primarily on their size and surface characteristics. These properties can be tuned by the nanoparticle preparation method. Therefore, the nanoprecipitation and the emulsion-solvent evaporation method were applied to prepare the PNPs. The influence of various parameters, such as concentration of the starting solution, evaporation method and solvent properties on the nanoparticle size, size distribution and morphology were studied. The PNPs were characterized by using atomic force microscopy (AFM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) to assess their size distribution and morphology. The PNPs obtained by nanoprecipitation ranged in size between 90 nm and 130 nm with a very low polydispersity index (PDI < 0.3). On the other hand, the PNPs produced by the emulsion-solvent evaporation method revealed particle sizes around 300 nm with a high PDI value. More detailed information was found in AFM and SEM images, which demonstrated that all these PNPs were regularly spherical. ζ-potential measurements were satisfactory and evidenced the importance of sucrose moiety on the polymeric system, which was responsible for the obtained negative surface charge, providing colloidal stability. The results of this study show that sucrose and cholic acid based polymeric conjugates can be successfully used to prepare PNPs with tunable physicochemical characteristics. In addition, it provides novel information about the materials used and the methods applied. It is hoped that this work will be useful for the development of novel carbohydrate based nanoparticles for biomedical applications, specifically for targeted drug delivery.

Methicillin-resistant Staphylococcus aureus bloodstream infection: risk factors and clinical outcome in non-intensive-care units

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is spread out in hospitals across different regions of the world and is regarded as the major agent of nosocomial infections, causing infections such as skin and soft tissue pneumonia and sepsis. The aim of this study was to identify risk factors for methicillin-resistance in Staphylococcus aureus bloodstream infection (BSI) and the predictive factors for death. METHODS: A retrospective cohort of fifty-one patients presenting bacteraemia due to S. aureus between September 2006 and September 2008 was analysed. Staphylococcu aureus samples were obtained from blood cultures performed by clinical hospital microbiology laboratory from the Uberlândia Federal University. Methicillinresistance was determined by growth on oxacillin screen agar and antimicrobial susceptibility by means of the disk diffusion method. RESULTS: We found similar numbers of MRSA (56.8%) and methicillin-susceptible Staphylococcus aureus (MSSA) (43.2%) infections, and the overall hospital mortality ratio was 47%, predominantly in MRSA group (70.8% vs. 29.2%) (p=0.05). Age (p=0.02) was significantly higher in MRSA patients as also was the use of central venous catheter (p=0.02). The use of two or more antimicrobial agents (p=0.03) and the length of hospital stay prior to bacteraemia superior to seven days (p=0.006) were associated with mortality. High odds ratio value was observed in cardiopathy as comorbidity. CONCLUSIONS: Despite several risk factors associated with MRSA and MSSA infection, the use of two or more antimicrobial agents was the unique independent variable associated with mortality.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

INTRODUCTION: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. METHODS: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5′-3′-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. RESULTS: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95%I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. CONCLUSIONS: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

O emprego de uma força de segurança de natureza militar nas missões de paz

The conflicts currently taking place around the world demand that the international intervention fits the intensity and extent of the threat. This is particularly important in post-conflict scenarios, leading to a greater participation of the Security Forces in those scenarios, in order to foster lasting peace, enforce the order and improve law enforcement services in those regions. The transition from armed conflict to peacekeeping may entail high risk situations and greater instability periods, so-called “intermediate situations”. Accordingly, in the face of persisting high volatility, a robust response is still required post-conflict. Therefore, it is appropriate to deploy Security Forces with military nature and status, the gendarmeries, which have training and response capabilities similar to Armed Forces in peacekeeping operations. Their double facet as police and military forces enables them to perform police duties in high risk and unsafe environments. In light of these features, the Portuguese gendarmerie, Guarda Nacional Republicana (GNR), is able to carry out tasks in these scenarios, which it has been doing through individual operatives or larger units. This dissertation focuses on the use of Security Forces of military nature in peacekeeping missions, in particular the Portuguese GNR, relying mostly on the inductive approach and using literature research, document analysis, interviews and statistics. After a brief description of international peacekeeping missions, we describe the contribution of Security Forces of a military nature in such operations. Then we introduce and analyse the GNR, focusing on its deployment in different kinds of peacekeeping operations, from its first participation in 1995 until today. We also report some reactions to the performance of GNR. Finally, we discuss whether there is indeed a unique role for this type of forces in international peacekeeping missions.

Relatório de estágio na Caixa Geral de Depósitos

This report will describe the activities undertaken during my internship at the Personnel Department (DPE-UPE4.1) in Caixa Geral de Depósitos (CGD), Lisbon, between September 22, 2014, and February 28, 2015. I consider that it is important to note from the outset i) that the subject of my training was suggested by my supervisor in the DPE and accepted by me; and ii) that the internship consisted essentially of carrying out research and information gathering into the different social systems that coexist within the bank and the application of each legal system in solving concrete situations of the CGD employees. The research and analysis of information was important not only for my study but for the CGD itself, as it enables the department to have such an important matter, full of specific characteristics, condensed into a single document, i.e. this report. This is a complex reality. The various welfare systems differ according to the contractual agreement linking the employee to the employer at the date when the labour contract is signed, and also the unique/singular characteristics of the CGD. In the early stage I started by trying to understand the financial institution and its organization and role and the department where I worked. So I analyzed the CGD Statutes and the legal measures that crystallized the scheme for its employees and I also researched its domestic and international operations. The first month was devoted to the research and analysis of such legislation to understand the creation of the CGD and its path to date. In the second and third months I studied the legal social systems that are applied to different groups of CGD workers. This period was quite important to identify and understand the differences between those regimes of CGD employees as well as the procedure inherent in each case. I highlighted the non-implementation of “the social protection regime of convergence” to the workers of this institution; the differences regarding the allocation of sickness subsidies paid to workers who belong to Social Security and CGA contributors, as well as the enforcement of internal rules to all the workers when a work-related accident happens. Then I focused on to assessing and examining external legislation and several internal regulations in order to obtain solutions to questions raised and situations involving by the workers, in order to understand how the DPE solves these situations. Over the last three months of internship, after this more theoretical work, I began the analysis of concrete situations involving employees carrying out their duties in Portugal and abroad. Some of these situations had been received by the department before the beginning of my internship and others over this period. When I was “working” in the DPE I analyzed “cases” that had been solved and some others without a final solution because they were still in courts. As for the last ones (new cases) I was able to follow their assessment and sometimes their outcome. Some of them became study cases for me. Over these five months of my internship, several cases were analyzed and discussed by legal experts of DPE in which I could participate. I always worked hard. I know that this action contributed to elucidate me about the treatment of the issues, and allowed me to have a direct contact with some workers and be part of a dynamic work team. For these reasons, my internship report is not merely descriptive of activities. It consists of an analysis of rules (legislation) and a regulatory framework of activities and it is also a description of several specific situations solved or in a solution process. Through this work I intend to make known the particular reality of a modern Portuguese financial institution not only because of its importance in our country but also such a large number of employees work here (in Portugal and abroad). I should add that throughout my internship I was allowed to attend conferences, within the scope of the bank in order to get a broader view of some issues related to the daily life of the DPE and the CGD. So, I participated in I Jornadas Bancárias and the Conferência Internacional do Contrato a Termo, given that the CGD is a bank and the DPE deals with legal and labour relations.

Paracoccidioidomycosis case series with and without central nervous system involvement

INTRODUCTION: Paracoccidioidomycosis (PCM) is the most important systemic mycosis in South America. Central nervous system involvement is potentially fatal and can occur in 12.5% of cases. This paper aims to contribute to the literature describing eight cases of neuroparacoccidioidomycosis (NPMC) and compare their characteristics with patients without neurological involvement, to identify unique characteristics of NPCM. METHODS: A cohort of 213 PCM cases was evaluated at the Infectious Diseases Clinic of the University Hospital, Federal University of Minas Gerais, Brazil, from October 1976 to August 2008. Epidemiological, clinical, laboratory, therapeutic and follow-up data were registered. RESULTS: Eight patients presented NPCM. The observed NPCM prevalence was 3.8%. One patient presented the subacute form of PCM and the other seven presented the chronic form of the disease. The parenchymatous form of NPCM occurred in all patients. 60% of the patients who proceeded from the north/ northeast region of Minas Gerais State developed NPCM. The neurological involvement of a mother and her son was observed. NPCM patients exhibited demographical and clinical profiles similar to what is described in the literature. When NPCM cases were compared to PCM patients, there were differences in relation to origin and positive PCM family history. CONCLUSIONS: The results corroborate the clinical view that the neurological findings are extremely important in the evaluation of PCM patients. Despite the limitations of this study, the differences in relation to patient's origins and family history point to the need of further studies to determine the susceptibility factors involved in the neurological compromise.

DYNAMOD – A dynamic agent based modelling framework for digital businesses

Digital Businesses have become a major driver for economic growth and have seen an explosion of new startups. At the same time, it also includes mature enterprises that have become global giants in a relatively short period of time. Digital Businesses have unique characteristics that make the running and management of a Digital Business much different from traditional offline businesses. Digital businesses respond to online users who are highly interconnected and networked. This enables a rapid flow of word of mouth, at a pace far greater than ever envisioned when dealing with traditional products and services. The relatively low cost of incremental user addition has led to a variety of innovation in pricing of digital products, including various forms of free and freemium pricing models. This thesis explores the unique characteristics and complexities of Digital Businesses and its implications on the design of Digital Business Models and Revenue Models. The thesis proposes an Agent Based Modeling Framework that can be used to develop Simulation Models that simulate the complex dynamics of Digital Businesses and the user interactions between users of a digital product. Such Simulation models can be used for a variety of purposes such as simple forecasting, analysing the impact of market disturbances, analysing the impact of changes in pricing models and optimising the pricing for maximum revenue generation or a balance between growth in usage and revenue generation. These models can be developed for a mature enterprise with a large historical record of user growth rate as well as for early stage enterprises without much historical data. Through three case studies, the thesis demonstrates the applicability of the Framework and its potential applications.

Board independence and innovation

Legislation introduced in the U.S. in 2002/2003 significantly changed board composition of public firms by imposing a 50% independent directors’ ratio. Research on the effect of independent directors is not consensual, implying that this exogenous shock is a unique opportunity to study their importance. This study answers the question of whether or not independent directors can effectively mitigate agency conflicts between shareholders and the management, having a positive impact on the choice of successful R&D projects. We find that an increase of board independence has a positive impact on patent counts. Hence, the results support that independent directors truly spur innovation and risk taking.

Human immunodeficiency virus/Leishmania infantum in the first foci of urban American visceral leishmaniasis: clinical presentation from 1994 to 2010

INTRODUCTION:Human immunodeficiency virus (HIV) coinfection with Leishmania infantum or Leishmania donovani, the agents of visceral leishmaniasis (or kala-azar), has become a fatal public health problem in the tropics where kala-azar is endemic.METHODS:The clinical presentation of patients with HIV and L. infantum coinfection is described using two unique databases that together produce the largest case series of patients with kala-azar infected with HIV in South America. First, a retrospective study paired the list of all patients with kala-azar from 1994 to 2004 with another of all patients with HIV/AIDS from the reference hospital for both diseases in the City of Teresina, State of Piauí, Brazil. Beginning in 2005 through to 2010 this information was prospectively collected at the moment of hospitalization.RESULTS:During the study, 256 admissions related to 224 patients with HIV/L. infantum coinfection were registered and most of them were males between 20-40 years of age. Most of the 224 patients were males between 20-40 years of age. HIV contraction was principally sexual. The most common symptoms and signs were pallor, fever, asthenia and hepatosplenomegaly. 16.8% of the cohort died. The primary risk factors associated to death were kidney or respiratory failure, somnolence, hemorrhagic manifestations and a syndrome of systemic inflammation. The diagnosis of HIV and kala-azar was made simultaneously in 124 patients.CONCLUSIONS:The urban association between HIV and kala-azar coinfection in South America is worrisome due to difficulty in establishing the diagnosis and higher mortality among the coinfected then those with either disease independently. HIV/L. infantum coinfection exhibits some singular characteristics and due to its higher mortality it requires immediate assistance to patients and greater research on appropriate combination therapy.

Integrated communications strategy for mylabel

Integrated Communication Strategy for MyLabel Following paper presents Integrated Communication Strategy for Continente’s private label brand of cosmetics MyLabel. The main purpose of the project is to position MyLabel as venture brand which will gain strong market position in order to compete with the manufacturer brands. Therefore, based on the created brand equity model for the venture cosmetic brand, MyLabel will be approached from the branding perspective in order to improve perceived quality and consecutively, build brand recognition and credibility. In this respect, integrated communication strategy includes some of the branding tactics and marketing communication mix. Thereafter, MyLabel will be transformed into the sub-brand MyBeauty, which can exploit opportunities given by the new market image of retailers’ brands and gain special, unique position in the market.

The born global phenomenon- The Portuguese case

The growing significance of companies that conduct international business at or near their funding has been critically challenging previous incremental models of international expansion. This thesis aims to cross-compare, by means of a multiple-case study, eight Portuguese start-ups among themselves and with the theoretical concept of born global firms versus traditional ones. This work project finds that: (1) active entrepreneurs with global vision from inception are essential for the implementation of international strategies; (2) only formal network plays a key role for successful internationalization and (3) inimitable sources of value creation, niche-focused strategies and unique intangible assets are also crucial.

Does direct cash flow presentation help in predicting future operating cash flow?

Research literature and regulators are unconditional in pointing the disclosure of operating cash flow through direct method a section of unique information. Besides the intuitive facet, it is also consistent in forecasting future operating cash flows and a cohesive piece to financial statement puzzle. Bearing this in mind, I produce an analysis on the usefulness and predictive ability on the disclosure of gross cash receipts and payments over the disclosure of reconciliation between net income and accruals for two markets with special features, Portugal and Spain. Results validate the usefulness of direct method format in predicting future operating cash flow. Key

Understanding how dentritic cell glycans affect antitumor immune responses

RESUMO: As células dendríticas (DCs) têm a capacidade única de induzir respostas imunitárias contra as células tumorais, fagocitando antigénios tumorais e apresentando-os às células T, provocando respostas imunitárias específicas que conduzem à eliminação de células de tumorais. Por induzirem memória imunológica de longa duração, as DCs são uma estratégia atrativa para o tratamento e/ou prevenção do cancro. No entanto, os resultados terapêuticos obtidos em ensaios clínicos com DCs são escassos e pouco eficientes. O nosso grupo demonstrou que ácidos siálicos que contêm glicanos desempenham um papel funcional importante em DCs geradas ex vivo. Com o objetivo de estabelecer um modelo in vitro para avaliar a resposta anti-tumoral específica realizou-se um tratamento enzimático a DCs derivadas de monócitos (moDCs) com sialidase, enzima que cliva ácidos siálicos na superfície celular. O perfil de maturação de moDCs foi caracterizado por citometria de fluxo e expressão de citocinas. Os resultados mostram que a sialidase pode regular positivamente a expressão de moléculas co-estimuladoras na superfície de moDCs estimuladas com agonistas de Toll like receptors (TLRs). Para percebermos se o tratamento com sialidase afeta a sinalização dos TLRs foram usadas células HEK transfectadas de forma estável com TLRs 2, 4 and 7/8. Os dados mostraram que a desialilação não afeta a sinalização através estes recetores. Para investigar o impacto funcional da sialidase na capacidade de moDCs em apresentar um antigénio e ativar células T, moDCs foram tratadas, ou não, com sialidase e cultivadas com clones de células T CD8+ específicas para os péptidos derivados do antigénio tumoral gp100. Os resultados mostram que DCs HLA*02:01+ desialiladas exibem maior cross-presentation do péptido gp100280-288 às células T CD8+ específicas. Além disso o tratamento com sialidase também aumenta a capacidade de DCs de induzir a proliferação de células T CD4+. Em conjunto, os resultados indicam que moDCs com menos ácidos siálicos na superfície, têm melhor potencial imuno-estimulador, com maior capacidade de induzir respostas imunes anti-tumorais.--------------------- ABSTRACT: Dendritic cells (DCs) have a unique capacity to induce immune responses against tumor cells. They can phagocyte tumor antigens, maturate and present them to T cells, triggering antigen-specific immune responses that may lead to the elimination of tumor cells. Since they induce long-lasting immunological memory, DCs become an attractive strategy as cellular targets for vaccines in the treatment and/or prevention of cancer. However, the therapeutic results obtained in clinical trials with DCs are scarce and only few patients effectively respond to the DC vaccines. Our group has shown that sialic acid containing glycans play an important functional role in ex vivo generated DC. Here we aimed to establish an in vitro model to assess specific antitumor responses. To achieve this, an enzymatic treatment of monocyte-derived DCs (moDCs) was performed using sialidase to cleave surface sialic acids. The maturation profile of the moDCs was characterized by flow cytometry and cytokine expression. The results show that sialidase treatment can upregulate co-stimulatory molecules on surface of moDCs stimulated with Toll like receptor (TLR) agonists. To understand whether sialidase treatment affected the TLR signaling, we have used HEK cells stably transfected with TLRs 2, 4 and 7/8. The data showed that desialylation of moDCs does not affect the signaling via these receptors. To investigate the functional impact of sialidase treatment in the capacity of moDCs to present antigen and to activate antigen specific T cells, sialidase treated and untreated moDCs were co-cultured with CD8+ T cell clones specific for peptides derived from the gp100 tumor antigen. Our results show that desialylated HLA02:01+ DCs are superior in cross-presentation of the peptide to gp100280–288 specific CD8+ T cells. In addition, sialidase treatment also increased the DC capacity to induce CD4+ T cells proliferation. Together, these data indicate that moDCs with altered cell surface sialic acids, through a sialidase treatment, have a better immunostimulatory potential which could improve anti-tumor immune responses.

Epstein-Barr virus-positive gastric cancer: a distinct molecular subtype of the disease?

Abstract: Approximately 90% of the world population is infected by Epstein-Barr virus (EBV). Usually, it infects B lymphocytes, predisposing them to malignant transformation. Infection of epithelial cells occurs rarely, and it is estimated that about to 10% of gastric cancer patients harbor EBV in their malignant cells. Given that gastric cancer is the third leading cause of cancer-related mortality worldwide, with a global annual incidence of over 950,000 cases, EBV-positive gastric cancer is the largest group of EBV-associated malignancies. Based on gene expression profile studies, gastric cancer was recently categorized into four subtypes; EBV-positive, microsatellite unstable, genomically stable and chromosomal instability. Together with previous studies, this report provided a more detailed molecular characterization of gastric cancer, demonstrating that EBV-positive gastric cancer is a distinct molecular subtype of the disease, with unique genetic and epigenetic abnormalities, reflected in a specific phenotype. The recognition of characteristic molecular alterations in gastric cancer allows the identification of molecular pathways involved in cell proliferation and survival, with the potential to identify therapeutic targets. These findings highlight the enormous heterogeneity of gastric cancer, and the complex interplay between genetic and epigenetic alterations in the disease, and provide a roadmap to implementation of genome-guided personalized therapy in gastric cancer. The present review discusses the initial studies describing EBV-positive gastric cancer as a distinct clinical entity, presents recently described genetic and epigenetic alterations, and considers potential therapeutic insights derived from the recognition of this new molecular subtype of gastric adenocarcinoma.

Pain evaluation of patients with fibromyalgia, osteoarthritis, and low back pain

The purpose of this study was to evaluate and compare pain as reported by outpatients with fibromyalgia, osteoarthritis, and low back pain, in view of designing more adequate physical therapy treatment. PATIENTS AND METHODS: A Portuguese version of the McGill Pain Questionnaire - where subjects are asked to choose, from lists of pre-categorized words, one or none that best describes what they feel - was used to assess pain intensity and quality of 64 patients, of which 24 had fibromyalgia, 22 had osteoarthritis, and 18 had low back pain. The pre-categorized words were organized into 4 major classes -- sensory, affective, evaluative, and miscellaneous. RESULTS: Patients with fibromyalgia reported, comparatively, more intense pain through their choice of pain descriptors, both sensory and affective; they also chose a higher number of words from these classes than patients in the other groups and were the only ones to choose specific affective descriptors such as "vicious", "wretched", "exhausting", "blinding". CONCLUSION: Assuming that each disease presents unique qualities of pain experience, and that these can be pointed out by means of this questionnaire by patients' choice of specific groups of words, the findings suggest that fibromyalgia include not only a physical component, but also a psycho-emotional component, indicating that they require both emotional/affective and physical care.