Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6 of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.

A comparison of monkey and human motion processing mechanisms

Single-cell recording studies have provided vision scientists with a detailed understanding of motion processing at the neuronal level in non-human primates. However, despite the development of brain imaging techniques, it is not known to what extent the response characteristics of motion-sensitive neurons in monkey brain mirror those of human motion sensitive neurons. Using a motion adaptation paradigm, the direction aftereffect, we recently provided evidence of a strong resemblance in the response functions of motion-sensitive neurons in monkey and human to moving dot patterns differing in dot density. Here we describe a series of experiments in which measurements of the direction aftereffect are used to infer the response characteristics of human motion-sensitive neurons when viewing transparent motion and moving patterns that differ in their signal-to-noise ratio (motion coherence). In the case of transparent motion stimuli, our data suggest suppressed activity of motion-sensitive neurons similar to that reported for macaque monkey. In the case of motion coherence, our results are indicative of a linear relationship between signal intensity (coherence) and neural activity; a pattern of activity which also bears a striking similarity to macaque neural activity. These findings strongly suggest that monkey and human motionsensitive neurons exhibit similar response and inhibitory characteristics.

A plasma membrane Ca2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

Prognostic and therapeutic relevance of c-FLIP in acute myeloid leukaemia

Chemoresistance is a major contributor to the aggressiveness of AML and is often due to insufficient apoptosis. The CFLAR gene is expressed as long and short splice forms encoding the anti-apoptotic proteins c-FLIP(L) and c-FLIP(S) (CFLAR(L) and CFLAR(S) , respectively) that play important roles in drug resistance. In univariate analyses of CFLAR mRNA expression in adult AML patients, those individuals with higher than median mRNA expression of the long splice form CFLAR(L) (but not the short splice form) had significantly lower 3 year overall survival (P = 0·04) compared to those with low expression. In cell line studies, simultaneous down-regulation of c-FLIP(L) and c-FLIP(S) proteins using siRNA induced apoptosis in U937 and NB-4 AML cells, but not K562 or OCI-AML3 cells. However, dual c-FLIP(L/S) downregulation sensitized all four cell lines to apoptosis induced by recombinant tumour necrosis factor-related apoptosis-inducing ligand (rTRAIL). Moreover, specific downregulation of c-FLIP(L) was found to recapitulate the phenotypic effects of dual c-FLIP(L/S) downregulation. The histone deacetylase (HDAC)1/2/3/6 inhibitor Vorinostat was found to potently down-regulate c-FLIP(L) expression by transcriptional and post-transcriptional mechanisms and to sensitize AML cells to rTRAIL. Further analyses using more selective HDAC inhibitors revealed that HDAC6 inhibition was not required for c-FLIP(L) down-regulation. These results suggest that c-FLIP(L) may have clinical relevance both as a prognostic biomarker and potential therapeutic target for HDAC inhibitors in AML although this requires further study.

Intraretinal Leakage and Oxidation of LDL in Diabetic Retinopathy

Purpose: The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory.

Methods: Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modi?ed LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary
pericytes (HRCPs) was assessed.

Results: Intraretinal immuno?uorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in
HRCPs.

Conclusions: These ?ndings suggest a potentially important role for extravasated, modi?ed LDL in promoting DR by promoting apoptotic pericyte loss.

Glycation, carbonyl stress, EAGLEs, and the vascular complications of diabetes

The association between poor metabolic control and the microvascular complications of diabetes is now well established, but the relationship between long-term metabolic control and the accelerated atherosclerosis of diabetes is as yet poorly defined. Hyperglycemia is the standard benchmark by which metabolic control is assessed. One mechanism by which elevated glucose levels may mediate vascular injury is through early and advanced glycation reactions affecting a wide variety of target molecules. The "glycation hypothesis'' has developed over the past 30 years, evolving gradually into a "carbonyl stress hypothesis'' and taking into account not only the modification of proteins by glucose, but also the roles of oxidative stress, a wide range of reactive carbonyl-containing intermediates (derived not only from glucose but also from lipids), and a variety of extra- and intracellular target molecules. The final products of these reactions may now be termed "Either Advanced Glycation or Lipoxidation End-Products'' or "EAGLEs.'' The ubiquity of carbonyl stress within the body, the complexity of the reactions involved, the variety of potential carbonyl intermediates and target molecules and their differing half-lives, and the slow development of the complications of diabetes all pose major challenges in dissecting the significance of these processes. The extent of the reactions tends to correlate with overall metabolic control, creating pitfalls in the interpretation of associative data. Many animal and cell culture studies, while supporting the hypothesis, must be viewed with caution in terms of relevance to human diabetes. In this article, the development of the carbonyl stress hypothesis is reviewed, and implications for present and future treatments to prevent complications are discussed.

Self-assembly of hybrid organic–inorganic polyoxovanadates: functionalised mixed-valent clusters and molecular cages’

Herein we report the intra- and inter-molecular assembly of a {V5O9} subunit. This mixed-valent structural motif can be stabilised as [V5O9(L1–3)4]5−/9− (1–3) by a range of organoarsonate ligands (L1–L3) whose secondary functionalities influence its packing arrangement within the crystal structures. Variation of the reaction conditions results in the dodecanuclear cage structure [V12O14(OH)4(L1)10]4− (4) where two modified convex building units are linked via two dimeric {O4VIV(OH)2VIVO4} moieties. Bi-functional phosphonate ligands, L4–L6 allow the intramolecular connectivity of the {V5O9} subunit to give hybrid capsules [V10O18(L4–6)4]10− (5–7). The dimensions of the electrophilic cavities of the capsular entities are determined by the incorporated ligand type. Mass spectrometry experiments confirm the stability of the complexes in solution. We investigate and model the temperature-dependent magnetic properties of representative complexes 1, 4, 6 and 7 and provide preliminary cell-viability studies of three different cancer cell lines with respect to Na8H2[6]·36H2O and Na8H2[7]·2DMF·29H2O.

Electrospun fibrous mats for skin and abdominal wall repair

Esta tese centra-se no desenvolvimento de materiais biodegradáveis e nãodegradáveis produzidos por eletrofiação com aplicação na área biomédica. O poli(3-hidroxibutirato-co-3-hidroxivalerato) (PHBV), um poliéster biodegradável, foi selecionado como base dos materiais biodegradáveis, enquanto o poli(tereftalato de etileno) (PET), um polímero sintético, estável e biocompatível, foi selecionado para a produção das matrizes não degradáveis. Adicionou-se quitosana aos sistemas com o objetivo de melhorar o processo de eletrofiação e as propriedades morfológicas, físico-químicas e biológicas dos materiais resultantes. A composição química, bem como as características morfológicas e físicoquímicas dos materiais em estudo, foram manipuladas de modo a otimizar a sua performance como suportes celulares para engenharia de tecidos. Foram realizados estudos in vitro com cultura de fibroblastos L929 para avaliar o comportamento das células, i.e. viabilidade, adesão, proliferação e morte, quando cultivadas nas matrizes produzidas por eletrofiação. Adicionalmente foram realizados ensaios in vivo para investigar o potencial dos materiais em estudo na regeneração cutânea e como tela abdominal. Os principais resultados encontrados incluem: o desenvolvimento de novas matrizes híbridas (PHBV/quitosana) adequadas ao crescimento de fibroblastos e ao tratamento de lesões de pele; o desenvolvimento de um sistema de eletrofiação com duas seringas para a incorporação de compostos bioativos; diversas estratégias para manipulação das características morfológicas dos materiais de PHBV/quitosana e PET/quitosana produzidos por eletrofiação; uma melhoria do conhecimento das interações fibroblastos-suporte polimérico; a verificação de uma resposta inflamatória desencadeada pelos materiais nãodegradáveis quando utilizados no tratamento de defeitos da parede abdominal, o que sugere a necessidade de novos estudos para avaliar a segurança do uso de biomateriais produzidos por eletrofiação.

Nanocompósitos de PMMA/HA/grafeno para aplicações biomédicas

Os cimentos ósseos à base de PMMA para aplicações em artroplastia da anca apresentam como grande limitação o facto do seu constituinte principal ser um elemento bioinerte o que leva à falta de integração entre as interfaces cimento ósseo/tecido ósseo, comprometendo assim o desempenho mecânico da prótese ortopédica ao longo do tempo. Esta dissertação tem como objetivo principal a preparação de novas formulações de cimentos ósseos com a capacidade de estabelecer interações com os tecidos vivos circundantes. De modo a melhorar a bioatividade do sistema e facilitar a sua osseointegração, os cimentos ósseos comerciais foram reforçados com cargas significativas de HA. No entanto o recurso a elevadas cargas de HA (~60% m/m) no cimento ósseo promove debilidades do ponto de vista estrutural, levando a uma baixa resistência mecânica do material final. No sentido de ultrapassar esta limitação, foram inseridas nanoestruturas de carbono (GO ou CNTs) em baixas percentagens na matriz polimérica por forma a maximizar a sua performance mecânica através da perfeita integração de todos os componentes. A primeira fase deste trabalho consistiu no desenvolvimento de metodologias que permitissem a síntese de GO através da exfoliação química da grafite em solução aquosa. Os resultados obtidos demonstraram a obtenção de folhas de GO em larga escala e com número de camadas uniforme. A funcionalização orgânica superficial via ATRP do GO obtido, com cadeias de PMMA possibilitou o desenvolvimento de novos materiais nanocompósitos, no entanto alguns fatores de natureza tecnológica inviabilizaram o seu uso como agente de reforço na matriz idealizada. O desenvolvimento de novas formulações de cimentos ósseos consistiu numa matriz de PMMA/HA (1:2 (m/m)) reforçada com pequenas percentagens de GO ou CNTs (0,01, 0,1, 0,5 e 1,0% m/m). A síntese destes materiais nanocompósitos resultou da combinação de diversas técnicas: ultrassons, granulação por congelamento e liofilização. A análise estrutural dos nanocompósitos obtidos demonstrou a eficácia da metodologia desenvolvida na homogeneização de todos os elementos do sistema. Os estudos desenvolvidos após a conformação e caracterização estrutural dos novos materiais nanocompósitos permitiram verificar que as nanoestruturas de carbono apresentavam efeitos adversos na polimerização via radicalar do PMMA. A análise da fração orgânica permitiu verificar a presença de espécies oligoméricas o que reduziu significativamente o comportamento mecânico dos nanocompósitos. Através do estudo do aumento da concentração das espécies radicalares iniciais foi possível suplantar este problema e tirar o máximo rendimento dos agentes de reforço, tendo-se destacado os nanocompósitos reforçados com GO. A validação do ponto de vista mecânico das novas formulações de cimentos ósseos recaiu sobre o procedimento descrito na norma europeia ISO 5833 de 2002 – Implantes para cirurgia – cimentos acrílicos, tendo sido realizados os testes de compressão e de flexão. A avaliação biológica do comportamento dos cimentos ósseos assentou em duas abordagens complementares: estudos de mineralização em SBF e estudos de biocompatibilidade em meios celulares. Após a incubação das amostras em SBF ficou demonstrada a excelente capacidade para promoverem a integração de uma camada apatítica. Através de estudos celulares com Fibroblastos L929 e Osteoblastos Saos-2, nos quais foram avaliados a proliferação celular, viabilidade celular, espécies reativas de oxigénio, apoptose e morfologia celular, foi possível verificar bons níveis de biocompatibilidade para os materiais devolvidos.

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

The Synthesis and Coordination Chemistry of Two Families of Polydentate Ligands - Exploring Their Potential for the Preparation of Molecule-Based Magnets

The synthesis and studies of two classes of poly dentate ligands are presented as two projects. In project 1, four new carboxamide ligands have been synthesised via the condensation of 2,2',6,6'-tetrachloroformyl-4,4'-bipyridine or 2,6-dichloroformyl pyridine together with heterocyclic amines containing pyridine or pyrazole substituents. The coordination chemistry of these ligands has been investigated and studies have shown that with a Cu(II) salt, two carboxamide ligands LJ and L2 afford large clusters with stoichiometries [Cu8(L1)4Cl16].CHCl3.5H2O.7CH3OH (I) and [Cu9(L2)6Cl6].CH3OH.5H2O.(C2H5)3N (II) respectively. [molecular diagram availabel in pdf]. X-ray diffraction studies of cluster (I) reveal that it has approximate S4 symmetry and is comprised of four ligands and eight copper (II) centers. Here, coordination takes place via amide 0 atoms, and pyrazole nitrogens. This complex is the first reported example of an octanuclear copper cluster with a saddle-shaped structure. The second cluster comprises nine copper ions that are arranged in a cyclic array. Each ligand coordinates three copper centers and each copper ion shares two ligands to connect six ligands with nine copper ions. The amide nitrogens are completely deprotonated and both amide Nand 0 atoms coordinate the metal centres. The cluster has three-fold symmetry. There are six chloride ions, three of which are bridging two neighbouring Cu(II) centres. Magnetic studies of (I) and (II) reveal that both clusters display weak antiferromagnetic interactions between neighbouring Cu(II) centers at low temperature. In the second project, three complexes with stoichiometries [Fe[N302](SCN)2]2 (III), R,R-[Fe[N3O2](SCN)2 (IV) and R,R-]Fe[N3O2](CN)2] (V) were prepared and characterized, where [N302] is a pentadentate macrocycle. Complex (III) was prepared via the metal templated Schiff-base condensation of 2,2',6,6'-tetraacetyl-4,4'-bipyridine together with 3,6-dioxaoctane-I,8-diamine and comprises of a dimeric macro cycle where the two Fe(II) centres are in a pentagonal-bipyramidal environment with the [N302] ligands occupying the equatorial plane and two axial NCS ligands. Complexes (IV) and (V) were prepared via the condensation of 2,6-diacetylpyridine together with a chiral diamine in the presence of FeCh. The synthetic strategy for the preparation of the chiral diamine (4R,5R)-4,5-diphenyl-3,6-dioxa-I,8-octane-diamine was elucidated. The chirality of both macrocycles (IV) and (V) was probed by circular dichroism spectroscopy. The crystal structure of (IV) at 200 K contains two independent molecules in the unit cell, both of which contain a hepta-coordinated Fe(II) and axial NCS ligands. Variable temperature magnetic susceptibility and structural studies are consistent with a high spin Fe(II) complex and show no evidence of any spin crossover behaviour. In contrast, the bis cyanide derivative (V) crystallizes with two independent molecules in the unit cell, both of which have different coordination geometries consistent with different spin states for the two Fe(II) centres. At 250 K, the molecular structure of (V) shows the presence of both 7- and a 6-coordinate Fe(II) complexes in the crystal lattice. As the temperature is lowered, the molecules undergo a structural change and at 100 K the structural data is consistent with a 6- and 5-coordinate Fe(II) complex in the unit cell. Magnetic studies confirm that this complex undergoes a gradual, thermal, spin crossover transition in the solid state. Photomagnetic measurements indicate this is the first chiral Fe (II) sea complex to exhibit a LIESST.

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Evaluaton of a genetic marker within the organ of Corti of transgenic mice

This paper reviews a study to evaluate a genetic marker within the organ of Corti in transgenic mice for potential use in cell lineage studies.

PEGylated polyethyleneimine-entrapped gold nanoparticles for enhanced and targeted gene delivery applications

Gene therapy, which involves the transfer of nucleic acid into target cells in patients, has become one of the most important and widely explored strategies to treat a variety of diseases, such as cancer, infectious diseases and genetic disorders. Relative to viral vectors that have high immunogenicity, toxicity and oncogenicity, non-viral vectors have gained a lot of interest in recent years. This is largely due to their ability to mimic viral vector features including the capacity to overcome extra- and intra-cellular barriers and to enhance transfection efficiency. Polyethyleneimine (PEI) has been extensively investigated as a non-viral vector. This cationic polymer, which is able to compact nucleic acid through electrostatic interactions and to transport it across the negatively charged cell membranes, has been shown to effectively transfect nucleic acid into different cell lines. Moreover, entrapment of gold nanoparticles (Au NPs) into such an amine-terminated polymer template has been shown to significantly enhance gene transfection efficiency. In this work, a novel non-viral nucleic acid vector system for enhanced and targeted nucleic acid delivery applications was developed. The system was based on the functionalization of PEI with folic acid (FA; for targeted delivery to cancer cells overexpressing FA receptors on their surface) using polyethylene glycol (PEG) as a linker molecule. This was followed by the preparation of PEI-entrapped Au NPs (Au PENPs; for enhancement of transfection efficiency). In the synthesis process, the primary amines of PEI were first partially modified with fluorescein isothiocyanate (FI) using a molar ratio of 1:7. The formed PEI-FI conjugate was then further modified with either PEG or PEGylated FA using a molar ratio of 1:1. This process was finally followed by entrapment of Au NPs into the modified polymers. The resulting conjugates and Au PENPs were characterized by several techniques, namely Nuclear Magnetic Resonance, Dynamic Light Scattering and Ultraviolet-Visible Spectroscopy, to assess their physicochemical properties. In the cell biology studies, the synthesized conjugates and their respective Au PENPs were shown to be non-toxic towards A2780 human ovarian carcinoma cells. The role of these materials as gene delivery agents was lastly evaluated. In the gene delivery studies, the A2780 cells were successfully transfected with plasmid DNA using the different vector systems. However, FA-modification and Au NPs entrapment were not determinant factors for improved transfection efficiency. In the gene silencing studies, on the other hand, the Au PENPs were shown to effectively deliver small interfering RNA, thereby reducing the expression of the B-cell lymphoma 2 protein. Based on these results, we can say that the systems synthesized in this work show potential for enhanced and targeted gene therapy applications.

Prodrugs for the treatment of neglected diseases

Recently, World Health Organization ( WHO) and Medicins San Frontieres (MSF) proposed a classification of diseases as global, neglected and extremely neglected. Global diseases, such as cancer, cardiovascular and mental (CNS) diseases represent the targets of the majority of the R&D efforts of pharmaceutical companies. Neglected diseases affect millions of people in the world yet existing drug therapy is limited and often inappropriate. Furthermore, extremely neglected diseases affect people living under miserable conditions who barely have access to the bare necessities for survival. Most of these diseases are excluded from the goals of the R&D programs in the pharmaceutical industry and therefore fall outside the pharmaceutical market. About 14 million people, mainly in developing countries, die each year from infectious diseases. From 1975 to 1999, 1393 new drugs were approved yet only 1% were for the treatment of neglected diseases [ 3]. These numbers have not changed until now, so in those countries there is an urgent need for the design and synthesis of new drugs and in this area the prodrug approach is a very interesting field. It provides, among other effects, activity improvements and toxicity decreases for current and new drugs, improving market availability. It is worth noting that it is essential in drug design to save time and money, and prodrug approaches can be considered of high interest in this respect. The present review covers 20 years of research on the design of prodrugs for the treatment of neglected and extremely neglected diseases such as Chagas' disease ( American trypanosomiasis), sleeping sickness ( African trypanosomiasis), malaria, sickle cell disease, tuberculosis, leishmaniasis and schistosomiasis.