Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Structural and mechanistic studies of iron containing proteins

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica, ramo de Bioquímica-Física, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Periplasmic nitrate reductases: structural and spectroscopic studies

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica, especialidade Bioquímica-Física, pela Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa

Low-loss austempered ductile iron gears: experimental evaluation comparing materials and lubricants

An experimental study to evaluate the power dissipation of gears was performed. Three low-loss gear models were manufactured using standard 20° pressure angle tools. Austempered ductile iron (ADI) and 20MnCr5 carburized steel gears were tested in an FZG gear test machine using mineral, ester and polyalphaolephine (PAO)-based oils. The results compare power dissipation, the influence of different tooth flank geometries, materials and lubricants. This work concludes that conventional power-transmission gears can be replaced by these improved and more efficient low–loss models, which can be produced using common tools and that steel gears can be successfully replaced by austempered ductile iron gears.

Low torque loss gears: austempered ductile iron versus carburized steel

Low-loss power transmission gears operate at lower temperature than conventional ones because their teeth geometry is optimized to reduce friction. The main objective of this work is to compare the operating stabilization temperature and efficiency of low-loss austempered ductile iron (ADI) and carburized steel gears. Three different low-loss tooth geometries were adopted (types 311, 411 and 611, all produced using standard 20° pressure angle tools) and corresponding steel and ADI gears were tested in a FZG machine. The results obtained showed that low-loss geometries had a significant influence on power loss, gears 611 generating lower power loss than gears 311. At low speeds (500 and 1000 rpm) and high torque ADI gears generated lower power loss than steel gears. However, at high speed and high torque (high input power and high stabilization temperature) steel gears had better efficiency.

New iron(II) cyclopentadienyl derivative complexes: synthesis and antitumor activity against human leucemia cancer cells

A new family of "Fe-II(eta(5)-C5H5)" half sandwich compounds bearing a N-heteroaromatic ligand coordinated to the iron center by a nitrile functional group has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for complexes 1 and 3, which crystallized in the monoclinic P2(1)/c and monoclinic P2(1)/n space groups, respectively. Studies of interaction of these five new complexes with plasmid pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC50 values revealed excellent antiproliferative activity compared to cisplatin. (C) 2014 Elsevier B.V. All rights reserved.

Solvent-free microwave-assisted peroxidative oxidation of alcohols catalyzed by Iron(III)-TEMPO catalytic systems

The iron(III) complexes [H(EtOH)][FeCl2(L)(2)] (1), [H(2)bipy](1/2)[FeCl2(L)(2)].DMF (2) and [FeCl2(L)(2,2'-bipy)] (3) (L = 3-amino-2-pyrazinecarboxylate; H(2)bipy = doubly protonated 4,4'-bipyridine; 2,2'-bipy = 2,2'-bipyridine, DMF = dimethylformamide) have been synthesized and fully characterized by IR, elemental and single-crystal X-ray diffraction analyses, as well as by electrochemical methods. Complexes 1 and 2 have similar mononuclear structures containing different guest molecules (protonated ethanol for 1 and doubly protonated 4,4'-bipyridine for 2) in their lattices, whereas the complex 3 has one 3-amino-2-pyrazinecarboxylate and a 2,2'-bipyridine ligand. They show a high catalytic activity for the low power (10 W) solvent-free microwave assisted peroxidative oxidation of 1-phenylethanol, leading, in the presence of TEMPO, to quantitative yields of acetophenone [TOFs up to 8.1 x 10(3) h(-1), (3)] after 1 h. Moreover, the catalysts are of easy recovery and reused, at least for four consecutive cycles, maintaining 83 % of the initial activity and concomitant rather high selectivity. 3-Amino-2-pyrazinecarboxylic acid is used to synthesize three new iron(III) complexes which act as heterogeneous catalysts for the solvent-free microwave-assisted peroxidative oxidation of 1-phenylethanol.

Utilization of food industry wastes for the production of zero-valent iron nanoparticles

The proper disposal of the several types of wastes produced in industrial activities increases production costs. As a consequence, it is common to develop strategies to reuse these wastes in the same process and in different processes or to transform them for use in other processes. This work combines the needs for new synthesis methods of nanomaterials and the reduction of production cost using wastes from citrine juice (orange, lime, lemon and mandarin) to produce a new added value product, green zero-valent iron nanoparticles that can be used in several applications, including environmental remediation. The results indicate that extracts of the tested fruit wastes (peel, albedo and pulp fractions) can be used to produce zero-valent iron nanoparticles (nZVIs). This shows that these wastes can be an added value product. The resulting nZVIs had sizes ranging from 3 up to 300 nm and distinct reactivities (pulp > peel > albedo extracts). All the studied nanoparticles did not present a significant agglomeration/settling tendency when compared to similar nanoparticles, which indicates that they remain in suspension and retain their reactivity.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Transformation of Medicago truncatula with the arginine decarboxylase gene to modify polyamine metabolism toward water deficit resistance

Dissertation submitted to obtain a Ph.D. (Doutoramento) degree in Biology at the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Bridging environmental conflicts with social metabolism : forestry expansion and socioeconomic change

Dissertação apresentada para obtenção do Grau de Doutor em Ciências do Ambiente, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Identification of Cryptosporidium spp. oocysts in fecal smears stained with Heidenhain's iron hematoxylin

There is no paucity of methods for diagnosing Cryptosporidium spp. infection. The merits of immunoassays notwithstanding, microscopic identification of Cryptosporidium spp. oocysts in fecal samples remains an important diagnostic procedure. It owes the persistence of its use to such characteristics as dispensing with expensive equipment and kits, requiring only basic laboratory facilities, and having a low probability of false positive results when permanent slides are prepared, which can be re-examined in case of doubt. Cryptosporidium spp. oocysts can be readily identified in fecal smears prepared according to a regressive iron hematoxylin staining technique. The number of steps and their duration, as well as costs, were reduced to a minimum without loss of image quality and permanence of the preparations.

Metals in proteins from sulphate-reducing bacteria: adenylate kinase and ATP sulfurylase. Proteins containing cobalt, zinc, iron (II) ions

A Thesis submitted at the Faculty Science and Technology of the New University of Lisbon for a degree in Doctor of Philosophy in Biochemistry with specialization in Physical Biochemistry