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.(...)

Semantic relations extraction from unstructured information for domain ontologies enrichment

Based in internet growth, through semantic web, together with communication speed improvement and fast development of storage device sizes, data and information volume rises considerably every day. Because of this, in the last few years there has been a growing interest in structures for formal representation with suitable characteristics, such as the possibility to organize data and information, as well as the reuse of its contents aimed for the generation of new knowledge. Controlled Vocabulary, specifically Ontologies, present themselves in the lead as one of such structures of representation with high potential. Not only allow for data representation, as well as the reuse of such data for knowledge extraction, coupled with its subsequent storage through not so complex formalisms. However, for the purpose of assuring that ontology knowledge is always up to date, they need maintenance. Ontology Learning is an area which studies the details of update and maintenance of ontologies. It is worth noting that relevant literature already presents first results on automatic maintenance of ontologies, but still in a very early stage. Human-based processes are still the current way to update and maintain an ontology, which turns this into a cumbersome task. The generation of new knowledge aimed for ontology growth can be done based in Data Mining techniques, which is an area that studies techniques for data processing, pattern discovery and knowledge extraction in IT systems. This work aims at proposing a novel semi-automatic method for knowledge extraction from unstructured data sources, using Data Mining techniques, namely through pattern discovery, focused in improving the precision of concept and its semantic relations present in an ontology. In order to verify the applicability of the proposed method, a proof of concept was developed, presenting its results, which were applied in building and construction sector.

Musées et casinos dans une ville "patrimoine mondial": authenticité et hyperréalité, deux formes culturelles de l'espace urbain de Macao

Texto submetido para publicação em 2011.

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.

Studies on the eucalyptus leaf disease complex in Portugal

Native from south eastern Australia, Eucalyptus globulus is the main species in eucalypts plantations in Portugal. The most serious foliar disease in eucalypt plantations is linked to Mycosphaerella senso lato, which affects young trees in the juvenile phase foliage causing leaf necrosis. This disease results in reduced growth rate of the host and lower wood volume, thus causing significant productivity losses. The most common name for this disease was Mycosphaerella Leaf Disease that became inappropriate when most of the pathogens on eucalypts were re-distributed into several genera. The term "Eucalyptus Leaf Disease Complex" is now more appropriate. The overall aim of this thesis was to investigate the Eucalyptus Leaf Disease Complex in Portugal, focusing on species diversity, taxonomy and the role played by each species in the disease complex on Eucalyptus globulus. Literature on the Eucalyptus Leaf Disease Complex was reviewed and the species were distributed into several genera. A survey based on symptomatic leaves collected from several Eucalyptus globulus plantations and characterized by morphological and molecular tools provided an overview of species incidence and of the most frequent species in the disease complex. The present work reveals additional species of Mycosphaerella senso lato associated with eucalypt plantations in Portugal. Thus, five new records of Teratosphaeria and phylogenetically related species were added to the Iberian Peninsula, namely, Neodevriesia hilliana, for the first time on Myrtaceae; Quasiteratosphaeria mexicana, Teratosphaericola pseudoafricana, Teratosphaeria pluritubularis and Teratosphaeria lusitanica, a new species. Furthermore, new anamorphic structures were found and two new combinations were made. Regarding other genera, some species were observed for the first time, such as Cladosporium cladosporioides, Fusicladium eucalypti, Mycosphaerella madeirae, in the mainland. In addition to leave diseases, Teratosphaeria gauchensis was found causing a severe stem and trunk canker on Eucalyptus globulus. The aggressiveness of several species was compared to evaluate each species individually in the complex, permitting to distinguish different behaviours, from primary to secondary pathogens. Cladosporium cladosporioides, M. communis and M. lateralis, appeared to be more aggressive than Teratosphaeria nubilosa. In fact, contrary to the prevailing views on this disease complex, Teratosphaeria nubilosa is not the only species responsible for the disease, which clearly involves a complex of species acting together.

Assessment of a novel Cu (II) complex as a potential anticancer agent

Widely used in cancer treatment, chemotherapy still faces hindering challenges, ranging from severe induced toxicity to drug resistance acquisition. As means to overcome these setbacks, newly synthetized compounds have recently come into play with the basis of improved pharmacokinetic/pharmacodynamic properties. With this mind-set, this project aimed towards the antiproliferative potential characterization of a group of metallic compounds. Additionally the incorporation of the compounds within a nanoformulation and within new combination strategies with commercial chemotherapeutic drugs was also envisaged. Cell viability assays presented copper (II) compound (K4) as the most promising, presenting an IC50 of 6.10 μM and 19.09 μM for HCT116 and A549 cell line respectively. Exposure in fibroblasts revealed a 9.18 μM IC50. Hoechst staining assays further revealed the compound’s predisposition to induce chromatin condensation and nuclear fragmentation in HCT116 upon exposure to K4 which was later demonstrated by flow cytometry and annexin V-FITC/propidium iodide double staining analysis (under 50 % cell death induction). The compound further revealed the ability to interact with major macromolecules such as DNA (Kb = 2.17x105 M-1), inducing structural brakes and retardation, and further affecting cell cycle progression revealing delay in S-phase. Moreover BSA interactions were also visible however not conclusive. Proteome profiling revealed overexpression of proteins involved in metabolic activity and underexpression of proteins involved in apoptosis thus corroborating Hoechst and apoptosis flow cytometry data. K4 nanoformulation suffered from several hindrances and was ill succeeded in part due to K4’s poor solubility in aqueous buffers. Other approaches were considered in this regard. Combined chemotherapy assays revealed high cytotoxicity for afatinib and lapatinib strategies. Lapatinib and K4 proteome profiling further revealed high apoptosis rates, high metabolic activity and activation of redundant proteins as part of compensatory mechanisms.