Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species

Fungi of the genus Aspergillus are widespread in the environment. Some Aspergillus species, most commonly Aspergillus fumigatus, may lead to a variety of allergic reactions and life-threatening systemic infections in humans. Invasive aspergillosis occurs primarily in patients with severe immunodeficiency, and has dramatically increased in recent years. There are several factors at play that contribute to aspergillosis, including both fungus and host-related factors such as strain virulence and host pulmonary structure/immune status, respectively. The environmental tenacity of Aspergilllus, its dominance in diverse microbial communities/habitats, and its ability to navigate the ecophysiological and biophysical challenges of host infection are attributable, in large part, to a robust stress-tolerance biology and exceptional capacity to generate cell-available energy. Aspects of its stress metabolism, ecology, interactions with diverse animal hosts, clinical presentations and treatment regimens have been well-studied over the past years. Here, we synthesize these findings in relation to the way in which some Aspergillus species have become successful opportunistic pathogens of human- and other animal hosts. We focus on the biophysical capabilities of Aspergillus pathogens, key aspects of their ecophysiology and the flexibility to undergo a sexual cycle or form cryptic species. Additionally, recent advances in diagnosis of the disease are discussed as well as implications in relation to questions that have yet to be resolved.

Developmental biology and cytogenetics of B. xylophilus

The pine wood nematode Bursaphelenchus xylophilus reproduces bisexually: a haploid sperm fertilizes a haploid oocyte, and the two pronuclei rearrange, move together, fuse, and begin diploid development. Early embryonic events taking place in the B. xylophilus embryo are similar to those of Caenorhabditis elegans, although the anterior-posterior axis appeares to be determined oppositely to that observed for C. elegans. Thai is, in the B. xylophilus embryo, the male pronucleus emerges at the future anterior end, whereas the female pronucleus appeares laterally. To understand the evolution of nematode developmental systems, we cloned the full length of Bx-tbb-1 (beta tubulin) from B. xylophilus cDNA and attempted to apply reverse genetics analysis to B. xylophilus. Several lengths of double stranded RNA (dsRNA) for the Bx-tbb-1 gene were synthesized by in vitro transcription, and both B. xylophilus and C. elegans were soaked in dsRNA for RNAi. Both nematodes could suck up the dsRNA, and we could detect the abnormal phenotypes caused by Bx-tbb-1 dsRNA in C. elegans, but not in B. xylophilus. We suspect that systemic RNAi might be suppressed in B. xylophilus and are attempting to establish other methods for functionally analyzing B. xylophilus genes.

Aß biology and its contribution to Alzheimer's disease

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva patologicamente caracterizada pela presença de placas de amilóide (placas senis) insolúveis e também pela presença de tranças neurofibrilhares,formadas pela proteína Tau hiperfosforiladada. O principal constituinte das placas senis é o peptídeo beta-amilóide (Ab), que deriva do processamento proteolítico da proteína precursora de amilóide de Alzheimer (APP). Embora Ab exista como um agregado pouco solúvel nas placas senis, ele é secretado pelas células como uma molécula solúvel. O Ab “per se” pode afectar o metabolismo da APP. Alguns autores sugerem que o Ab exerce o seu efeito alterando o processamento ou catabolismo da APP, outros sugerem que ele também induz a transcrição da APP, onde aumentando os níveis da APP pode estar a contribuir para a sua própria produção (mecanismo de “feedback” positivo). Assim sendo, torna-se difícil consolidar todas estas observações e identificar as potenciais funções fisiológicas do Ab “in vivo”, ou as consequências da sua produção. Neste trabalho caracterizaram-se os efeitos do Ab no metabolismo da APP. Os nossos estudos revelaram que um dos mecanismos induzidos pelo Ab é a acumulação intracelular do fragmento neuroprotector sAPP (isAPPa) em estruturas com características vesiculares associadas ao citosqueleto. Estudos adicionais em culturas primárias revelaram que o Ab estava a exercer o seu efeito ao nível da secreção vesicular, provavelmente interferindo com o transporte de APP/sAPP ao longo da rede do citosqueleto. Esta hipótese é sustentada pelo facto do Ab estar a afectar a estabilidade e a polimerização de proteínas envolvidas na dinâmica do citosqueleto. Contrariamente a publicações anteriores o Ab não induziu a transcrição da APP, na verdade em culturas primárias neuronais foi observado uma diminuição nos níveis de expressão da APP. Isto foi acompanhado por um aumento nos fragmentos C-terminais da APP (CTFs) e uma diminuição na localização nuclear do seu domínio intracelular (AICD), sugerindo alterações na sinalização nuclear da APP. O Ab pode afectar outras vias de sinalização, particularmente alterando o balanço entre as actividades das proteínas cinases e fosfatases, o que pode ter consequências para o desenvolvimento da doença. Os dados obtidos indicam que o Ab é capaz de inibir a actividade da proteína fosfatase1, a sua importância numa perspectiva de futuras terapias é discutida. Devido à relevância da agregação do Ab para a sua toxicidade, a formação de complexos com proteínas que promovem a sua desagregação/degradação e o seu efeito no processamento da APP foi avaliado. Na presença destes complexos observou-se uma reversão da acumulação isAPP, demonstrando o potencial terapêutico destas proteínas como moduladores do metabolismo da APP. Este trabalho permitiu compreender melhor os mecanismos envolvidos nos efeitos do Ab no processamento da APP e descobrir algumas moléculas que podem ser relevantes numa perspectiva de diagnóstico e terapia na DA.

Embryonic and larval ecology and biochemistry of offshore decapods

Understanding the biology of offshore species is hardened by the difficulties of sampling in the deep-sea environment. Additionally, due to the vastness of the open ocean, knowledge of early life histories of pelagic larvae is still relatively scarce. In decapod species with bentho-pelagic lifestyle, the transition from life in the seafloor to the water column not only is associated with drastic morphological metamorphosis, but also with changes in behavior and feeding ecology. The purpose of the present thesis was to investigate physiological, biochemical and behavioral adaptation occurring during early development of such species. The Norway lobster, Nephrops norvegicus, and the crab Monodaeus couchi were used as a model as these two species are encountered off the NE Atlantic shelf at depth greater than 300 m. Chapter 1 introduces the challenges faced by both adult and larvae inhabiting such remote habitats, including the effect of food availability on development and oceanographic processes on dispersal and recruitment. The thesis follows early life histories, starting with within-brood variability in the fatty acid (FA) profile displayed by developing N. norvegicus embryos. There were no differences in the FA composition of embryos sampled from both sides of the brooding chamber in most females. However, all females exhibited significant differences in the FA profiles of embryos sampled from different pleopods. Potential causes for the variations recorded may be differential female investment during oocyte production or shifts in FA catabolism during the incubation period promoted by embryo’s location within the brooding chamber. Next, feeding rates and digestive enzymes activity of the early stage larvae was investigated in N. norvegicus. Both stages were able to maximize food intake when larvae were scarce and showed increased feeding rate following periods of starvation. Amylase activity indicated that carbohydrates are not the primary energy reserve and that feeding may be required soon after hatching to trigger amylase activity. Protease activity indicated that protein reserves are catabolized under starvation. These results indicate that larvae may maximize prey ingestion in the presence of plankton patches with higher food abundance and minimize the deleterious effects induced by previous periods of intermittent starvation or unsuitable prey densities/types. Additionally, changes in enzymatic activity may allow newly hatched N. norvegicus larvae to metabolize protein reserves to overcome short-term starvation. Vertical migration behavior and the influence of oceanographic properties were studied next. All zoeal stages of M. couchi displayed reverse diel vertical migration. Abundance of early stages was correlated with chlorophyll a levels. An ontogenic shift in vertical distribution explained the results; earlier zoeal stages remain in the food-rich upper water column while later stages migrate to the bottom for settlement. This vertical migration behavior is likely to affect horizontal distribution of larvae. Indeed, global current patterns will result in low inter-annual variations in decapod larvae recruitment, but short term variations such as upwelling events will cause deviation from the expected dispersal pattern. Throughout development, from the embryo to metamorphosis into benthic juvenile, offshore decapods face many challenges. For the developing individual survivorship will depend heavily on food availability but also on the reserves passed on by the mother. Even though vertical migration behavior can allow the larvae to take advantage of depth varying currents for transport, the effect of general circulation pattern will superimpose local current and influence feeding conditions and affect dispersal and recruitment.

Biology, population dynamics, management and conservation of deep water lantern sharks, Etmopterus spinax and Etmopterus pusillus (Chondrichthyes: Etmopteridae) in southern Portugal (northeast Atlantic)

Tese dout., Ciências e Tecnologias das Pescas, Universidade do Algarve, 2007

Ecology of the early life-history stages of shad Alosa fallax fallax (Lacépède, 1803) in the River Mira, with a note on Alosa spp. larvae in the River Guadiana

Tese dout., Ecologia, Universidade do Algarve, 2006

Assessing the atmospheric oscillations effects on the biology of the bullet tuna (Auxis rochei) and its possible linkage with global warming

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve; Instituto Español de Oceanografia; 2015

Role of β-adrenergic signaling in ovarian cancer cells biology

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2014

Metagenomic systems biology: frameworks for modeling and characterizing the gut microbiome

Thesis (Ph.D.)--University of Washington, 2014

ß-lactamases in the biochemistry and molecular biology laboratory

β-lactamases are hydrolytic enzymes that inactivate the β-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of β-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative enterobacteria, amongst others. However, only some studies refer to the detection and development of β-lactamases carriers in healthy humans, sick animals, or even in strains isolated from environmental stocks such as food, water, or soils. Considering this, we proposed a 10-week laboratory programme for the Biochemistry and Molecular Biology laboratory for majors in the health, environmental, and agronomical sciences. During those weeks, students would be dealing with some basic techniques such as DNA extraction, bacterial transformation, polymerase chain reaction (PCR), gel electrophoresis, and the use of several bioinformatics tools. These laboratory exercises would be conducted as a mini research project in which all the classes would be connected with the previous ones. This curriculum was compared in an experiment involving two groups of students from two different majors. The new curriculum, with classes linked together as a mini research project, was taught to a major in Pharmacy and an old curriculum was taught to students from environmental health. The results showed that students who were enrolled in the new curriculum obtained better results in the final exam than the students who were enrolled in the former curriculum. Likewise, these students were found to be more enthusiastic during the laboratory classes than those from the former curriculum.

Effect of temperature and prey in the biology of Scymnus subvillosus (Goeze) (Coleoptera: Coccinellidae)

Dissertação de Mestrado, Biotecnologia em Controlo Biológico, 18 de Dezembro de 2013, Universidade dos Açores.

Sei whale (Balaenoptera borealis) ecology and management in the North Atlantic

Tese de Doutoramento em Ciências do Mar, especialidade em Ecologia Marinha.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.

Molecular estimation of dispersal for ecology and population genetics

The dispersal process, by which individuals or other dispersing agents such as gametes or seeds move from birthplace to a new settlement locality, has important consequences for the dynamics of genes, individuals, and species. Many of the questions addressed by ecology and evolutionary biology require a good understanding of species' dispersal patterns. Much effort has thus been devoted to overcoming the difficulties associated with dispersal measurement. In this context, genetic tools have long been the focus of intensive research, providing a great variety of potential solutions to measuring dispersal. This methodological diversity is reviewed here to help (molecular) ecologists find their way toward dispersal inference and interpretation and to stimulate further developments.