Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em Bioengenharia

TUBERCULOSIS INFECTION MIGHT INCREASE THE RISK OF INVASIVE CANDIDIASIS IN AN IMMUNOCOMPETENT PATIENT

Deep Candida infections commonly occur in immunosuppressed patients. A rare case of a multiple deep organ infection with Candida albicansand spinal tuberculosis was reported in a healthy young man. The 19-year-old man complained of month-long fever and lower back pain. He also had a history of scalded mouth syndrome. Coinfection with Mycobacterium tuberculosis and Candida albicans was diagnosed using the culture of aspirates from different regions. Symptoms improved considerably after antifungal and antituberculous therapy. This case illustrates that infection with tuberculosis might impair the host's immune system and increase the risk of invasive candidiasis in an immunocompetent patient.

The avirulence of the cultivated PF strain of Trypanosoma cruzi: V - the evaluation of parasitologic tests after vaccination of different animal species

After vaccination with the live PF strain of Trypanosoma cruzi, 194 blood cultures were performed in 143 mice, 9 dogs, 5 Cebus monkeys and 7 human subjects. Some of these blood cultures were simultaneously done with xenodiagnosis, subinoculation in baby mice and/or culture of viscerae. The trypanosomes isolated from the few positive cases (6,1%) were incapable of infecting baby mice were considered as cases of immunotolerance. All the other tests were negative.

Establishment of environmental and biological bases to optimise the production of the European clam Ruditapes decussatus (Linnaeus, 1758)

Dissertação para obtenção do Grau de Doutor em Ambiente

The hemorrhagic syndrome of leptospirosis: an experimental study in guinea pigs

The hemorrhagic syndrome of leptospirosis was studied in guinea pigs. The study correlates hematological, histopathological and immunohistochemical alterations in sixty animals inoculated by the intraperitoneal route with lml of the culture of virulent strain of Leptospira interrogans serovar copenhageni. Leptospirae antigens were detected by immunoperoxidase, chiefly in liver, kidney and heart muscle capillaries. Possible pathogenic mechanisms responsible for hemorrhagic syndrome are discussed with emphasis on toxic and anoxic attacks causing damage to endothelia, platelet depletion and alterations to hemostasia rates: prothrombin time [FT], partial thromboplastin time [PIT] and fibrinogen concentrations. Tide clinical-laboratoiy picture is compatible with the histopathological observation of disseminated intravascular coagulation [D1C] in most of the guinea pigs from day 4 of infection.

Severe coinfection of melioidosis and dengue fever in northeastern Brazil: first case report

This report focuses on a fatality involving severe dengue fever and melioidosis in a 28-year-old truck driver residing in Pacoti in northeastern Brazil. He exhibited long-term respiratory symptoms (48 days) and went through a wide-ranging clinical investigation at three hospitals, after initial clinical diagnoses of pneumonia, visceral leishmaniasis, tuberculosis, and fungal sepsis. After death, Burkholderia pseudomallei was isolated in a culture of ascitic fluid. Dengue virus type 1 was detected by polymerase chain reaction in cerebrospinal fluid (CSF); this infection was the cause of death. This description reinforces the need to consider melioidosis among the reported differential diagnoses of community-acquired infections where both melioidosis and dengue fever are endemic.

Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.

A qualitative approach to define the prototype of a Portuguese expatriate leader in Angola

This qualitative research analyzes the individual life experiences of Portuguese expatriate leaders who left their home country to work at organizations in Angola, a place that offers better job opportunities. Through interviews with those professionals and their followers, a prototype of a Portuguese expatriate leader in Angola has been developed. Even though Angola is a former Portuguese colony, it was at war for many years and so the economic situation and culture of the country are distant from what Portuguese workers are used too, which requires them to adapt and be open-minded to change.

Influence of the wood mechanical properties in the dovetail joint behavior

The force distribution inside a dovetail joint is complex. Wood is simultaneously loaded in different directions in the several connected surfaces. The analytical solutions available for the analysis of the behavior of those carpentry joints rely on the mechanical properties of wood. In particular, the stiffness properties of wood under compression are crucial for the forces equilibrium. Simulations showed that the stiffness values considered in each of the springs normally assumed in the analytical models, have great influence in the bearing capacity and stiffness of the dovetail joints, with important consequence on the stress distribution over the overall structure. In a wide experimental campaign, the properties under compression of the most common wood species of existing timber structures have been determined. Then, a solved example of a dovetail joint is presented assuming different wood species and the corresponding strength and stiffness properties values obtained in the tests.

O ensino da flauta de bisel em grupo como inovação pedagógica: Uma investigação-ação no 2.º Ciclo do Ensino Básico

Tese de Doutoramento em Estudos da Criança (Especialidade em Educação Musical)

As universidades e a participação pública em ciência. Perceções e práticas de cientistas, profissionais de comunicação e cidadãos em Portugal e Espanha

Tese de doutoramento em Ciência da Comunicação.

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)

Semipermeable capsules wrapping a multifunctional and self-regulated co-culture microenvironment for osteogenic differentiation

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the â stem cell nicheâ , the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

A participação dos cidadãos na televisão: estudo de caso de programas informativos da SIC e SIC Notícias

Dissertação de mestrado em Ciências da Comunicação (área de especialização em Informação e Jornalismo)

Anaerobic oxidation of methane associated with sulfate reduction in a natural freshwater gas source

The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with 13C-labeled CH4 (13CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13C-labeled CO2 (13CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with 13CO2 production and no methanogenesis occurred, excluding TMO as a possible source for 13CO2 production from 13CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.