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.

Experimental and modeling studies on reverse electrodialysis for sustainable power generation

A potentially renewable and sustainable source of energy is the chemical energy associated with solvation of salts. Mixing of two aqueous streams with different saline concentrations is spontaneous and releases energy. The global theoretically obtainable power from salinity gradient energy due to World’s rivers discharge into the oceans has been estimated to be within the range of 1.4-2.6 TW. Reverse electrodialysis (RED) is one of the emerging, membrane-based, technologies for harvesting the salinity gradient energy. A common RED stack is composed by alternately-arranged cation- and anion-exchange membranes, stacked between two electrodes. The compartments between the membranes are alternately fed with concentrated (e.g., sea water) and dilute (e.g., river water) saline solutions. Migration of the respective counter-ions through the membranes leads to ionic current between the electrodes, where an appropriate redox pair converts the chemical salinity gradient energy into electrical energy. Given the importance of the need for new sources of energy for power generation, the present study aims at better understanding and solving current challenges, associated with the RED stack design, fluid dynamics, ionic mass transfer and long-term RED stack performance with natural saline solutions as feedwaters. Chronopotentiometry was used to determinate diffusion boundary layer (DBL) thickness from diffusion relaxation data and the flow entrance effects on mass transfer were found to avail a power generation increase in RED stacks. Increasing the linear flow velocity also leads to a decrease of DBL thickness but on the cost of a higher pressure drop. Pressure drop inside RED stacks was successfully simulated by the developed mathematical model, in which contribution of several pressure drops, that until now have not been considered, was included. The effect of each pressure drop on the RED stack performance was identified and rationalized and guidelines for planning and/or optimization of RED stacks were derived. The design of new profiled membranes, with a chevron corrugation structure, was proposed using computational fluid dynamics (CFD) modeling. The performance of the suggested corrugation geometry was compared with the already existing ones, as well as with the use of conductive and non-conductive spacers. According to the estimations, use of chevron structures grants the highest net power density values, at the best compromise between the mass transfer coefficient and the pressure drop values. Finally, long-term experiments with natural waters were performed, during which fouling was experienced. For the first time, 2D fluorescence spectroscopy was used to monitor RED stack performance, with a dedicated focus on following fouling on ion-exchange membrane surfaces. To extract relevant information from fluorescence spectra, parallel factor analysis (PARAFAC) was performed. Moreover, the information obtained was then used to predict net power density, stack electric resistance and pressure drop by multivariate statistical models based on projection to latent structures (PLS) modeling. The use in such models of 2D fluorescence data, containing hidden, but extractable by PARAFAC, information about fouling on membrane surfaces, considerably improved the models fitting to the experimental data.

Archaeological Remains Accounting for the Presence and Exploitation of the North Atlantic Right Whale Eubalaena glacialis on the Portuguese Coast (Peniche, West Iberia), 16th to 17th Century

The former occurrence of the North Atlantic right whale Eubalaena glacialis on the Portuguese coast may be inferred from the historical range of that species in Europe and in NW Africa. It is generally accepted that it was the main prey of coastal whaling in the Middle Ages and in the pre-modern period, but this assumption still needs firming up based on biological and archaeological evidence. We describe the skeletal remains of right whales excavated at Peniche in 2001-2002, in association with archaeological artefacts. The whale bones were covered by sandy sediments on the old seashore and they have been tentatively dated around the 16th to 17th centuries. This study contributes material evidence to the former occurrence of E. glacialis in Portugal (West Iberia). Some whale bones show unequivocal man-made scars. These are associated to wounds from instruments with a sharp-cutting blade. This evidence for past human interaction may suggest that whaling for that species was active at Peniche around the early 17th century.

Development of an experimental setup for metal cutting research

Analytical, numerical and experimental models have been developed over time to try to characterize and understand the metal cutting process by chip removal. A true knowledge of the cutting process by chip removal is required by the increasing production, by the quality requirements of the product and by the reduced production time, in the industries in which it is employed. In this thesis an experimental setup is developed to evaluate the forces and the temperature distribution in the tool according to the orthogonal cutting model conditions, in order to evaluate its performance and its possible adoption in future works. The experimental setup is developed in a CNC lathe and uses an orthogonal cutting configuration, in which thin discs fixed onto a mandrel are cut by the cutting insert. In this experimental setup, the forces are measured by a piezoelectric dynamometer while temperatures are measured by thermocouples placed juxtaposed to the side face of the cutting insert. Three different solutions are implemented and evaluated for the thermocouples attachment in the cutting insert: thermocouples embedded in thermal paste, thermocouples embedded in copper plate and thermocouples brazed in the cutting insert. From the tests performed in the experimental setup it is concluded that the adopted forces measurement technique shows a good performance. Regarding to the adopted temperatures measurement techniques, only the thermocouples brazed in the cutting insert solution shows a good performance for temperature measurement. The remaining solutions show contact problems between the thermocouple and the side face of the cutting insert, especially when the vibration phenomenon intensifies during the cut. It is concluded that the experimental setup does not present a sufficiently robust and reliable performance, and that it can only be used in future work after making improvements in the assembly of the thermocouples.

Acute renal failure due to abdominal compartment syndrome: report on four cases and literature review

We report on 4 cases of abdominal compartment syndrome complicated by acute renal failure that were promptly reversed by different abdominal decompression methods. Case 1: A 57-year-old obese woman in the post-operative period after giant incisional hernia correction with an intra-abdominal pressure of 24 mm Hg. She was sedated and curarized, and the intra-abdominal pressure fell to 15 mm Hg. Case 2: A 73-year-old woman with acute inflammatory abdomen was undergoing exploratory laparotomy when a hypertensive pneumoperitoneum was noticed. During the surgery, enhancement of urinary output was observed. Case 3: An 18-year-old man who underwent hepatectomy and developed coagulopathy and hepatic bleeding that required abdominal packing, developed oliguria with a transvesical intra-abdominal pressure of 22 mm Hg. During reoperation, the compresses were removed with a prompt improvement in urinary flow. Case 4: A 46-year-old man with hepatic cirrhosis was admitted after incisional hernia repair with intra-abdominal pressure of 16 mm Hg. After paracentesis, the intra-abdominal pressure fell to 11 mm Hg.

Computed tomography-guided cutting needle biopsy of pulmonary lesions

PURPOSE: To report the experience of a radiology department in the use of computed tomography - guided cutting needle biopsy of pulmonary nodules, by evaluating diagnostic yield and incidence of complications. METHODS: This is a retrospective analysis of 52 consecutive patients who underwent lung lesion biopsy guided by computed tomography, performed between May 1997 and May 2000. Thirty-five patients were male and 17 were female, with ages ranging from 5 to 85 years (median, 62 years). The size of the lesions ranged from 1.8 to 15 cm (median, 5.4 cm). RESULTS: In a total of 52 biopsies of lung lesions, 51 biopsies (98.1%) supplied appropriate material for histopathological diagnosis, with 9 diagnosed (17.3%) as benign and 42 (80.8%) as malignant lesions. Specific diagnosis was obtained in 44 (84.6%) biopsies: 4 benign (9.1%) and 40 (90.9%) malignant lesions. The sensitivity, specificity, and accuracy of the cutting needle biopsies for determining presence of malignancy were 96.8%, 100%, and 97.2%, respectively. Complications occurred in 9 cases (17.3%), including 6 cases (11.5%) of small pneumothorax, 1 (1.9%) of hemoptysis, 1 (1.9%) of pulmonary hematoma, and 1 (1.9%) of thoracic wall hematoma. All had spontaneous resolution. There were no complications requiring subsequent intervention. CONCLUSION: The high sensitivity and specificity of the method and the low rate of complications have established cutting needle biopsy as an efficient and safe tool for the diagnosis of lung lesions. In our hospital, cutting needle biopsy is considered a reliable procedure for the evaluation of indeterminate pulmonary nodules.

Computed tomography-guided biopsy of mediastinal lesions: fine versus cutting needles

PURPOSE: To report the experience of a radiology department in the use of computed tomography guided biopsies of mediastinal lesions with fine and cutting needles, describing the differences between them. The results of adequacy of the sample and histologic diagnoses are presented according to the type of needle used. METHODS: We present a retrospective study of mediastinal biopsies guided by computed tomography performed from January 1993 to December 1999. Eighty-six patients underwent mediastinal biopsy in this period, 37 with cutting needles, 38 with fine needles, and 11 with both types (total of 97 biopsies). RESULTS: In most cases, it was possible to obtain an adequate sample (82.5%) and specific diagnosis (67.0%). Cutting-needle biopsy produced a higher percentage of adequate samples (89.6% versus 75.5%, P = 0.068) and of specific diagnosis (81.3% versus 53.1%, P = 0.003) than fine-needle biopsy. There were no complications that required intervention in either group. CONCLUSION: Because they are practical, safe, and can provide accurate diagnoses, image-guided biopsies should be considered the procedure of choice in the initial exploration of patients with mediastinal masses. In our experience, cutting needles gave higher quality samples and diagnostic rates. We recommend the use of cutting needles as the preferred procedure.

Distribution of Atta (Hymenoptera - Formicidae) in "terra-firme" rain forest of Central Amazonia: density, species composition and preliminary results on effects of forest fragmentation

One hundred and fourteen hectares of a "terra-fiirme" rain forest 70 km north of Manaus, Amazonas, Brazil, were surveyed for leaf-cutting ant colonies (Atta spp). One half of this area was in isolated forest fragments (surrounded by pastures or second growth) of two sizes: 1 and 10 ha. The other half was in non-isolated fragments (connected to a large parch of forest) of the same sizes. Only two species occured in this forest: Atta sexdens sexdens L. and A. cepfhalotes L. The first was the most abundant species with a mean density of 0.35 colonies per ha. The mean density of A. cephalotes colonies was 0.03 per ha. The density of colonies was not significantly different between the isolated fragments and the continuous forest. Furthermore, the species composition did not change with isolation. However, pre-isolation data and long term monitoring are necessary to conclude that the isolation of a forest fragment has no effect upon Atta colonies. The non-uniform spatial distribution of Atta colonics within the "terra-firme" forest must be taken into account when selecting conservation areas in the Amazon, in order to preserve this important group of ants together with their native habitat.

Synthesis and characterization of C3-symmetric 1,3,5 - Benzenetricarboxamide derived supramolecular systems

The aim of this work was to study the self-assembly process of C3-symmetric molecules. To accomplish this objective 1,3,5 – benzentricarboxamides (BTA) derivatives were obtained. Five C3-symmetric molecules were synthesized in moderate to good yields (39-72%) using azo-benzene, aniline, benzylamine, tryptophan and tyrosine. The aggregation behavior of the BTA derivatives was probed with 1H-NMR spectroscopy, 1H-1H 2D Nuclear Overhauser Effect Spectroscopy (NOESY) and Diffusion Ordered Spectroscopy (DOSY). These experiments allowed to study the influence of H-bonding groups, aromatic rings, unsaturated bonds and the overall geometry in the molecular self-assembly associated with the different structural patterns present on these molecules. The stacking and large molecule behavior where observed in BTA 1, aniline derivative, BTA 4, tyrosine derivative or BTA 5, tryptophan derivative, with several of those discussed functional groups such as unsaturated bonds and H-bonding groups. BTA 5 was used in a few preliminary interaction studies with glucose and ammonium chloride showing interaction with the ammonium ion.

The relationship between stem vessel parameters and the development of strata in the early stages of secondary forest succession in Amazonia

In order to compare the development of strata in the early stages of secondary forest succession with vessel parameters of the tree species, a forest inventory was carried out in 4-year (Q1: 48 m2), 11-year (Q2: 400 m2) and 20-year (Q3: 400 m2) forests and vessel parameters were investigated from stem cross sections of 18 species obtained in Q2. Thirty three species (21 families), 77 species (35 families), 39 species (20 families) were found in Ql, Q2, Q3, respectively. The percentage of dead individuals, dead stems and the percentage of individuals with multiple stems increased with time after clear cutting. Also, the total D2H of Q3 was 26.1 times that of Q1, and the development of strata started in Q2 and Q3. The image analysis of vessel size, area and number of vessels revealed that species which reach the forest canopy had a large D2H value, vessel diameter and area, while species which remain near the forest floor had smaller ones. Poecilanthe effusa (Huber) Ducke is an example of the latter case, with a large number of individuals and abundant sprouting of new stems from stumps, but with high mortality.

Proteins on microbial identification: past achievements, present state-of-the-art, and future challenges

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

Towards high performance and multi-functional structural membranes using advanced fibrous and textile materials

Scientific and technological advancements in the area of fibrous and textile materials have greatly enhanced their application potential in several high-end technical and industrial sectors including construction, transportation, medical, sports, aerospace engineering, electronics and so on. Excellent performance accompanied by light-weight, mechanical flexibility, tailor-ability, design flexibility, easy fabrication and relatively lower cost are the driving forces towards wide applications of these materials. Cost-effective fabrication of various advanced and functional materials for structural parts, medical devices, sensors, energy harvesting devices, capacitors, batteries, and many others has been possible using fibrous and textile materials. Structural membranes are one of the innovative applications of textile structures and these novel building skins are becoming very popular due to flexible design aesthetics, durability, lightweight and cost benefits. Current demand on high performance and multi-functional materials in structural applications has motivated to go beyond the basic textile structures used for structural membranes and to use innovative textile materials. Structural membranes with self-cleaning, thermoregulation and energy harvesting capability (using solar cells) are examples of such recently developed multi-functional membranes. Besides these, there exist enormous opportunities to develop wide varieties of multi-functional membranes using functional textile materials. Additionally, it is also possible to further enhance the performance and functionalities of structural membranes using advanced fibrous architectures such as 2D, 3D, hybrid, multi-layer and so on. In this context, the present paper gives an overview of various advanced and functional fibrous and textile materials which have enormous application potential in structural membranes.