Genetic and functional analysis of the bacillus subtilis BSP1 gam cluster

Mannans (linear mannan, glucomannan, galactomannan and galactoglucomannan) are the major constituents of the hemicellulose fraction in softwoods and show great importance as a renewable resource for fuel or feedstock applications. As complex polysaccharides, mannans can only be degraded through a synergistic action of different mannan-degrading enzymes, mannanases. Microbial mannanases are mainly extracellular enzymes that can act in wide range of pH and temperature, contributing to pulp and paper, pharmaceutical, food and feed, oil and textile successful industrial applications. Knowing and controlling these microbial mannan-degrading enzymes are essential to take advantage of their great biotechnological potential. The genome of the laboratory 168 strain of Bacillus subtilis carries genes gmuA-G dedicated to the degradation and utilization of glucomannan, including an extracellular -mannanase. Recently, the genome sequence of an undomesticated strain of B. subtilis, BSP1, was determined. In BSP1, the gmuA-G operon is maintained, interestingly, however, a second cluster of genes was found (gam cluster), which comprise a second putative extracellular β-mannanase, and most likely specify a system for the degradation and utilization of a different mannan polymer, galactoglucomannan. The genetic organization and function of the gam cluster, and whether its presence in BSP1 strain results in new hemicellulolytic capabilities, compared to those of the laboratory strain, was address in this work. In silico and in vivo mRNA analyses performed in this study revealed that the gam cluster, comprising nine genes, is organized and expressed in at least six different transcriptional units. Furthermore, cloning, expression, and production of Bbsp2923 in Escherichia coli was achieved and preliminary characterization shows that the enzyme is indeed a β-mannanase. Finally, the high hemicellulolytic capacity of the undomesticated B. subtilis BSP1, demonstrated in this work by qualitative analyses, suggests potential to be used in the food and feed industries.

Complex networks and data mining: toward a new perspective for the understanding of complex systems

Complex systems, i.e. systems composed of a large set of elements interacting in a non-linear way, are constantly found all around us. In the last decades, different approaches have been proposed toward their understanding, one of the most interesting being the Complex Network perspective. This legacy of the 18th century mathematical concepts proposed by Leonhard Euler is still current, and more and more relevant in real-world problems. In recent years, it has been demonstrated that network-based representations can yield relevant knowledge about complex systems. In spite of that, several problems have been detected, mainly related to the degree of subjectivity involved in the creation and evaluation of such network structures. In this Thesis, we propose addressing these problems by means of different data mining techniques, thus obtaining a novel hybrid approximation intermingling complex networks and data mining. Results indicate that such techniques can be effectively used to i) enable the creation of novel network representations, ii) reduce the dimensionality of analyzed systems by pre-selecting the most important elements, iii) describe complex networks, and iv) assist in the analysis of different network topologies. The soundness of such approach is validated through different validation cases drawn from actual biomedical problems, e.g. the diagnosis of cancer from tissue analysis, or the study of the dynamics of the brain under different neurological disorders.

La Réforme des soins en santé mentale en Belgique: analyse d'un modéle

RESUMO: O relatório anual de 2001 da Organização Mundial de Saúde (OMS), anunciou o interesse social dos problemas crescentes em matéria de saúde mental e relativo à necessidade urgente de uma extensão, como também, para uma melhor organização da oferta de tratamento. Sob proposta da Bélgica, esta observação foi traduzida em uma resolução « Mental health :responding to the call for action » (déclaração de intenção do 18 de maio de 2002). Nós notamos que certos países, como os Estados Unidos ou a Grã Bretanha transformam sistematicamente o tratamento residencial em um tratamento mais dirigido para a comunidade. Eles também se concentram na descoberta prematura dos problemas psiquiátricos. Este fenômeno de désinstitutionalisação obteve resultados concretos e traz um ganho certo, tanto para os atores do campo da saúde mental, como também para os candidatos a um tratamento. O tratamento das pessoas dentro do seu meio de vida é muito claramente reconhecido. As evoluções que marcaram os tratamentos em saúde mental na Bélgica durante estes utimos 40 anos mostram que vários paços importantes já foram feitos. A reforma tem como objetivo converter a oferta hospitalar e montar tratamentos dirigidos para e dentro da comunidade pela criação de equipas móveis que se inscrevem no dispositivo comunitário, historicamente muito activo bem que sub-financiado. A reorganização dos sistemas de tratamento está baseada na criação de redes, para construir um dispositivo flexível e contínuo que considera as necessidades dos pacientes. Esta reforma é, ao mesmo tempo, ambiciosa e complexa,. É uma visão nova, uma mudança de cultura, não só, para todos os atores da saúde mental, mas também para os pacientes e suas famílias. A reforma está baseada numa visão global e integrada que associa no mesmo movimento todas as autoridades competentes relativamente a saúde que elas sejam federais, regionais ou comunitárias.-----------ABSTRACT: The World Health Organization’s (WHO) annual report of 2001 identified a social interest for the increasing problems related to mental health and the urgent need of an extension and a better organization of mental health care. On a proposal of Belgium this statement was transformed into a resolution « Mental health: responding to the call for action» (Declaration of Intent, May 18th, 2002). Some countries such as the USA or the UK systematically dismantled in-patient residential care in favor of more community-based care and a focus on early detection of psychiatric problems. This de -institutionalization has clearly and concretely paid off and the value of bringing mental health care to claimants and treating them in their own residence was acknowledged. In Belgium, the evolutions which have shaped mental health care in the last forty years indicate that a number of important steps are already taken. The Reform aims to convert the supply of hospital care into community-based mental health care services through the creation of mobile teams which offer services in everyday life of the user. These teams take place in the community, historically highly active howbeit underfunded. The reorganization of health care system relies upon the creation of networks in order to build a flexible and continuous device which take into account user’s needs. This Reform can be seen as both ambitious and complex. It is a completely new vision, a major cultural shift for all mental health care stakeholders, but also for users and their relatives. The Reform is based on a global and integrated approach which links I, a same movement all relevant health authorities whether they be federal, regional or community. de-institutionalization – community mental health – recovery - users and families involvement - networking.

Epidemiological pattern of leprosy in an endemic area of North-East Brazil, 1996-2005: the supporting role of a Nongovernmental Organization

In an endemic area of North-East Brazil (the town of Picos, State of Piauí), a nongovernmental organization (NGO) supported the activity against leprosy in connection with governmental health organizations and local agents. The indicators of leprosy elimination were compared over time (within Picos) and across space (Picos versus Piauí). The case detection rate, above 8 per 10,000 people in the last two years of observation, increased over time in Picos (p=0.010). This finding could be due to active detection activities rather than expanding endemicity, as suggested by the reduction in leprosy in children (p=0.053) and the decrease in the proportion of new cases with grade 2 disability (p<0.001). These indicators showed a more favorable time trend in the city than in the State, suggesting that NGO activity was supportive in the battle towards leprosy control.

Integrated care approaches and skills mix policies in Europe

ABSTRACT - Background: Integration of health care services is emerging as a central challenge of health care delivery, particularly for patients with elderly and complex chronic conditions. In 2003, the World Health Organization (WHO) already began to identify it as one of the key pathways to improve primary care. In 2005, the European Commission declared integrated care as vital for the sustainability of social protection systems in Europe. Nowadays, it is recognized as a core component of health and social care reforms across European countries. Implementing integrated care requires coordination between settings, organizations, providers and professionals. In order to address the challenge of integration in such complex scenario, an effective workforce is required capable of working across interdependent settings. The World Health Report 2006 noted that governments should prepare their workforce and explore what tasks the different levels of health workers are trained to do and are capable of performing (skills mix). Comparatively to other European countries, Portugal is at an early stage in what integrated care is concerned facing a growing elderly population and the subsequent increase in the pressure on institutions and professionals to provide social and medical care in the most cost-effective way. In 2006 the Portuguese government created the Portuguese Network for Integrated Care Development (PNICD) to solve the existing long-term gap in social support and healthcare. On what concerns health workforce, the Portuguese government already recognized the importance of redefine careers keeping professional motivation and satisfaction. Aim of the study: This study aims to contribute new evidence to the debate surrounding integrated care and skills mix policies in Europe. It also seeks to provide the first evidence that incorporates both the current dynamics of implementing integrated care in Portugal and the developments of international literature. The first ambition of our study is to contribute to the growing interest in integrated care and to the ongoing research in this area by identifying its different approaches and retrieve a number of experiences in some European countries. Our second goal of this research is to produce an update on the knowledge developed on skills mix to the international healthcare management community and to policy makers involved in reforming healthcare systems and organizations. To better inform Portuguese health policies makers in a third stage we explore the current dynamics of implementing integrated care in Portugal and contextualize them with the developments reported in the international literature. Methodology: This is essentially an exploratory and descriptive study using qualitative methodology. In order to identify integrated care approaches in Europe, a systematic literature review was undertaken which resulted in a paper published in the Journal of Management and Marketing in Health care titled: Approaches to developing integrated care in Europe: a systematic literature review. This article was recommended and included into a list of references identified by The King's Fund Library. A second systematic literature review was undertaken which resulted in a paper published in the International Journal of Healthcare Management titled: Skills mix in healthcare: An international update for the management debate. Semi-structured interviews were performed on experts representing the regional coordination teams of the Portuguese Network for Integrated Care Development. In a last stage a questionnaire survey was developed based on the findings of both systematic literature reviews and semi-structured interviews. Conclusions: Even though integrated care is a worldwide trend in health care reforms, there is no unique definition. Definitions can be grouped according to their sectorial focus: community-based care, combined health and social care, combined acute and primary care, the integration of providers, and in a more comprehensive approach the whole health system. Indeed, models that seek to apply the principles of integrated care have a similar background and are continually evolving and depend on the different initiatives taken at national level. . Despite the fact that we cannot argue that there is one single set typology of models for integrated care, it is possible to identify and categorize some of the basic approaches that have been taken in attempts to implement integrated care according to: changes in organizational structure, workforce reconfiguring, and changes in the financing system. The systematic literature review on skills mix showed that despite the widely acknowledged interest on skills mix initiatives there is a lack of evidence on skills mix implications, constraints, outcomes, and quality impact that would allow policy makers to take sustained and evidence-based decisions. Within the Portuguese health system, the integrated care approach is rather organizational and financial, whereas little attention is given to workforce integration. On what concerns workforce planning Portugal it is still in the stage of analyzing the acceptability of health workforce skills mix. In line with the international approaches, integration of health and social services and bridging primary and acute care are the main goals of the national government strategy. The findings from our interviews clarify perceptions which show no discrepancy with the related literature but are rather scarce comparing to international experience. Informants hold a realistic but narrow view of integrated care related issues. They seem to be limited to the regional context, requiring a more comprehensive perspective. The questionnaire developed in this thesis is an instrument which, when applied, will allow policy makers to understand the basic set of concepts and managerial motivations behind national and regional integrated care programs. The instrument developed can foster evidence on the three essential components of integrated care policies: organizational, financial, and human resources development, and can give additional input on the context in which integrated care is being developed, the type of providers and organizations involved, barriers and constraints, and the workforce skills mix planning related strategies. The thesis was successful in recognizing differences between countries and interventions and the instrument developed will allow a better comprehension of the international options available and how to address the vital components of integrated care programs.

Pedestrian Priority Zones in the Context of Urban Mobility and Valorization of Public Space

The second half of the XX century was marked by a great increase in the number of people living in cities. Urban agglomerations became poles of attraction for migration flows and these phenomena, coupled with growing car-ownership rates, resulted in the fact that modern transport systems are characterized by large number of users and traffic modes. The necessity to organize these complex systems and to provide space for different traffic modes changed the way cities look. Urban areas had to cope with traffic flows, and as a result nowadays typical street pattern consists of a road for motorized vehicles, a cycle lane (in some cases), pavement for pedestrians, parking and a range of crucial signage to facilitate navigation and make mobility more secure. However, this type of street organization may not be desirable in certain areas, more specifically, in the city centers. Downtown areas have always been places where economic, leisure, social and other types of facilities are concentrated, not surprisingly, they often attract large number of people and this frequently results in traffic jams, air and noise pollution, thus creating unpleasant environment. Besides, excessive traffic signage in central locations can harm the image and perception of a place, this relates in particular to historical centers with architectural heritage.

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.

Epidemiology of the viral hepatitis B and C in female prisoners of Metropolitan Regional Prison Complex in the State of Goiás, Central Brazil

INTRODUCTION: Little information regarding hepatitis B virus (HBV) and hepatitis C virus (HCV) infections among Brazilian female prisoners exists. This study investigated the prevalence and risk factors associated with HBV and HCV infections and identified viral genotypes among female prisoners in Goiás, Central Brazil. METHODS: Women incarcerated in the largest prison in the State of Goiás were invited to participate in the study. All female prisoners were interviewed and tested for the detection of hepatitis B surface antigen (HBsAg), antibodies against HBsAg (anti-HBs), against hepatitis B core antigen (anti-HBc), and antibody against HCV (anti-HCV) by ELISA. HBsAg and anti-HCV positive samples were tested for HBV DNA and HCV RNA and genotyped, respectively. RESULTS: Participants (n=148; 98.6%) completed the study with an overall HBV prevalence of 18.9%. Age >30 years, a low education level, sex with a sexually transmitted diseases carrier, and a male sexual partner serving in the same penitentiary were associated with HBV infections. Only 24% of the women were anti-HBs positive suggesting previous HBV vaccination. Nine female prisoners (6.1%) were anti-HCV positive. Age >40 years, injecting drug use and length of incarceration were statistically associated with anti-HCV antibodies. Five samples were HCV RNA positive and classified as genotypes 1 (subtypes 1a; n=3 and 1b; n=1) and 3 (subtype 3a; n=1). The HBsAg-reactive sample was HBV DNA positive and genotype A. CONCLUSIONS: These findings highlight the necessity of public policies to control hepatitis B and C infections and emphasize the importance of hepatitis B vaccination in prison environments.

As 24 horas – Cooperação e performance em dois eventos formativos

Dissertação de Mestrado apresentada ao ISPA - Instituto Universitário

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Evaluation of the World Health Organization 2009 classification of dengue severity in autopsied individuals, during the epidemics of 2011 and 2012 in Brazil

Abstract: INTRODUCTION: The dengue classification proposed by the World Health Organization (WHO) in 2009 is considered more sensitive than the classification proposed by the WHO in 1997. However, no study has assessed the ability of the WHO 2009 classification to identify dengue deaths among autopsied individuals suspected of having dengue. In the present study, we evaluated the ability of the WHO 2009 classification to identify dengue deaths among autopsied individuals suspected of having dengue in Northeast Brazil, where the disease is endemic. METHODS: This retrospective study included 121 autopsied individuals suspected of having dengue in Northeast Brazil during the epidemics of 2011 and 2012. All the autopsied individuals included in this study were confirmed to have dengue based on the findings of laboratory examinations. RESULTS: The median age of the autopsied individuals was 34 years (range, 1 month to 93 years), and 54.5% of the individuals were males. According to the WHO 1997 classification, 9.1% (11/121) of the cases were classified as dengue hemorrhagic fever (DHF) and 3.3% (4/121) as dengue shock syndrome. The remaining 87.6% (106/121) of the cases were classified as dengue with complications. According to the 2009 classification, 100% (121/121) of the cases were classified as severe dengue. The absence of plasma leakage (58.5%) and platelet counts <100,000/mm3 (47.2%) were the most frequent reasons for the inability to classify cases as DHF. CONCLUSIONS: The WHO 2009 classification is more sensitive than the WHO 1997 classification for identifying dengue deaths among autopsied individuals suspected of having dengue.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

The World Health Organization Multi-Professional Patient Safety Curriculum : implementation of key modules and its impact on patient safety knowledge, skills, and attitudes of medical students at the University of Algarve

ABSTRACT - Objectives: We attempted to show how the implementation of the key elements of the World Health Organization Patient Safety Curriculum Guide Multi-professional Edition in an undergraduate curriculum affected the knowledge, skills, and attitudes towards patient safety in a graduate entry Portuguese Medical School. Methods: After receiving formal recognition by the WHO as a Complementary Test Site and approval of the organizational ethics committee , the validated pre-course questionnaires measuring the knowledge, skills, and attitudes to patient safety were administered to the 2nd and3rd year students pursuing a four-year course (N = 46). The key modules of the curriculum were implemented over the academic year by employing a variety of learning strategies including expert lecturers, small group problem-based teaching sessions, and Simulation Laboratory sessions. The identical questionnaires were then administered and the impact was measured. The Curriculum Guide was evaluated as a health education tool in this context. Results: A significant number of the respondents, 47 % (n = 22), reported having received some form of prior patient safety training. The effect on Patient Safety Knowledge was assessed by using the percentage of correct pre- and post-course answers to construct 2 × 2 contingency tables and by applying Fishers’ test (two-tailed). No significant differences were detected (p < 0.05). To assess the effect of the intervention on Patient Safety skills and attitudes, the mean and standard deviation were calculated for the pre and post-course responses, and independent samples were subjected to Mann-Whitney’s test. The attitudinal survey indicated a very high baseline incidence of desirable attitudes and skills toward patient safety. Significant changes were detected (p < 0.05) regarding what should happen if an error is made (p = 0.016), the role of healthcare organizations in error reporting (p = 0.006), and the extent of medical error (p = 0.005). Conclusions: The implementation of selected modules of the WHO Patient Safety Curriculum was associated with a number of positive changes regarding patient safety skills and attitudes, with a baseline incidence of highly desirable patient safety attitudes, but no measureable change on the patient safety knowledge, at the University of Algarve Medical School. The significance of these results is discussed along with implications and suggestions for future research.

Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

On the function of the floccular complex of the vertebrate cerebellum: implications in paleoneuroanatomy

The cerebellum floccular complex lobes (FCLs) are housed in the FCL fossa of the periotic complex. There is experimental evidence indicating that the FCLs integrate visual and vestibular information, responsible for the vestibulo-ocular reflex, vestibulo-collic reflex, smooth pursuit and gaze holding. Thus, the behavior of extinct animals has been correlated with FCLs dimension in multiple paleoneuroanatomy studies. Here I analyzed braincase endocasts of a representative sample of Mammalia (48 species) and Aves (59 species) rendered using tomography and image segmentation and tested statistical correlations between the floccular complex volume, ecological and behavioral traits to assess various previously formulated paleobiological speculations. My results demonstrate: 1) there is no significant correlation between relative FCL volume and body mass; 2) there is no significant correlation between relative FCL and optic lobes size in birds; 3) average relative FCL size is larger in diurnal than in nocturnal birds but there is no statistically significant difference in mammals; 4) feeding strategies are related with different FCL size patterns in birds, but not in mammals; 5) locomotion type is not related with relative FCL size in mammals; 6) agility is not significantly correlated with FCL size in mammals. I conclude that, despite the apparent relation between FCL size and ecology in birds, the cerebellum of tetrapods is a highly plastic structure and may be adapted to control different functions across different taxonomic levels. For example, the european mole (Talpa europaea) which is fossorial and practically blind, has a FCL fossae relative size larger than those of bats, which are highly maneuverable. Therefore, variation in FCL size may be better explained by a combination of multiple factors with relation to anatomical and phylogenetic evolutionary constraints.