Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Absorption and dispersion by a multiple driven two-level atom

We investigate the absorption and dispersion properties of a two-level atom driven by a polychromatic field. The driving field is composed of a strong resonant (carrier) frequency component and a large number of symmetrically detuned sideband fields (modulators). A rapid increase in the absorption at the central frequency and the collapse of the response of the system from multiple frequencies to a single frequency are predicted to occur when the Rabi frequency of the modulating fields is equal to the Rabi frequency of the carrier field. These are manifestations of the undressing or a disentanglement of the atomic and driving field states, that leads to a collapse of the atom to its ground state. Our calculation permits consideration of the question of the undressing of the driven atom by a multiple-modulated field and the predicted spectra offer a method of observing undressing. Moreover, we find that the absorption and dispersion spectra split into multiplets whose structures depend on the Rabi frequency of the modulating fields. The spectral features can jump between different resonance frequencies by changing the Rabi frequency of the modulating fields or their initial phases, which can have potential applications as a quantum frequency filter.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Estudo comparativo entre sistemas RoF com modulação directa e com modulação externa

O RoF (Radio over Fiber) é uma tecnologia que permite a transmissão de sinais rádio de elevada largura de banda, fornecida pela fibra óptica, e simultaneamente mantêm a característica de mobilidade das redes de comunicação móvel. Esta dissertação de mestrado tem como objectivo estudar, simular e comparar sistemas RoF com modulação directa e com modulação externa, utilizando um sinal WiMAX, por ser uma tecnologia recente e com potencial de utilização futura. Desta forma foram avaliados três tipos de moduladores externos, sendo que o modulador EA (Electro-Absorption) é o que permite obter melhores valores de EVM e de SNR devido ao facto deste ter menores perdas de inserção na fibra. Da comparação entre o esquema com modulação directa e o esquema com modulação externa, é possível concluir que para larguras de banda mais baixas a utilização de modulação directa é mais eficiente que a modulação externa, mas à medida que a largura de banda aumenta a modulação externa apresenta claramente melhor desempenho. Isto deve-se ao facto de a modulação directa produzir mais chirp que a modulação externa, sendo que o chirp limita a largura de banda e o comprimento da fibra. De forma a melhorar o desempenho do sistema com modulação directa foi introduzido uma fibra com compensação de dispersão. Foi possível concluir que a utilização de fibra com compensação de dispersão é uma boa solução quando se pretende transmitir sinais de elevada largura de banda em esquemas com modulação directa.

A marcha humana: circuitos neuronais entre estruturas corticais vs sub-corticais

As conexões entre os níveis corticais e sub-corticais na activação da marcha Humana carecem de discussão. As alterações da marcha por lesão no território da Artéria Cerebral Média (ACM) podem ser explicadas pela disfunção de circuitos neuronais dos Núcleos da Base ao córtex e Núcleos Pedúnculo-Pontino. Este trabalho tem como objectivo identificar os percursos anatómicos das principais conexões entre as estruturas encefálicas referidas no território da ACM. Com base na topografia das conexões neuronais, é aceitável que as alterações da marcha sejam explicadas por alterações na função dos Núcleos da Base, através das suas conexões, enquanto moduladores da actividade motora.

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.

Circuitos neuronais entre estruturas corticais vs sub-corticais marcha humana

As conexões entre os níveis corticais e os níveis sub-corticais na activação da marcha Humana carecem de discussão. As alterações da marcha por lesão no território da Artéria Cerebral Média podem ser explicadas pela disfunção de circuitos neuronais dos Núcleos da Base ao córtex e Núcleos Pedúnculo-Pontinos, sendo para isso necessário identificar os percursos anatómicos das principais conexões entre as estruturas encefálicas referidas. Com base na topografia das conexões neuronais, é aceitável que as alterações da marcha possam existir também devido a alterações na função dos Núcleos da Base, através das suas conexões, enquanto moduladores da actividade motora.

Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancer

Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patients

New water-soluble Ruthenium(II) cytotoxic complex: biological activity and celular distribution

A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=eta(5)-cyclopentadienyl, mTPPMS = diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy = 2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.

Marx-Type solid-state bipolar modulator topologies: Performance comparison

The operation of generalized Marx-type solid-state bipolar modulators is discussed and compared with simplified Marx-derived circuits, to evaluate their capability to deal with various load conditions. A comparative analysis on the number of switches per cell, fiber optic trigger count, losses, and switch hold-off voltages has been made. A circuit topology is obtained as a compromise in terms of operating performance, trigger simplicity, and switching losses. A five-stage laboratory prototype of this circuit has been assembled using 1200 V insulated gate bipolar transistors (IGBTs) and diodes, operating with 1000 V dc input voltage and 1 kHz frequency, giving 5 kV bipolar pulses, with 2.5 mu s pulse width and 5 mu s relaxation time into resistive, capacitive, and inductive loads.

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Hox genes control the specification of global vertebral domains

Dissertação apresentada para obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Design of switched-capacitor filters using low gain amplifiers

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.