21 research outputs found
Working Paper 35 - Privatization of Public Enterprises in Zambia: An Evaluation of the Policies, Procedures and Experiences
No full textThe term Privatization is often loosely used to mean a number of related activities, including any expansion of the scope of private sector activity in an economy and the adoption by the public sector of efficiency enhancing techniques commonly employed by the private sector. While acknowledging that no definition of privatization is water tight, we will define privatization, for the purpose of this paper, as the transfer of productive asset ownership and control from the public to the private sector.1 The transfer of assets can be total, partial or functionary, with the sale being implemented by methods such as private sales, leasing arrangements, employee buy outs and share issues. In Africa, many governments have embraced the idea of privatization, brought to the fore mainly as a part of the adjustment and stabilization programs of the mid-eighties and the nineties. Privatization now frequently features in government policy statements and in conditionalities from donors. The past decade has also seen the World Bank and other donors get increasingly involved in lending operations towards parastatal sector reforms that included privatization components. African countries share a number of common features in relation to the drive towards privatization. For most of these countries, the first twenty years of independence were characterized by rapid growth, driven by favorable terms of trade and high levels of public investments in infrastructure and services. The development of import substituting industries brought in the dramatic rise of parastatal corporations, which were also used as vehicles for increased local participation in the economies. Many governments moved to nationalize existing foreign interests in their countries and also to create new state enterprises to carry out the various production and trading functions. Parastatal corporations rapidly dominated the extractive industries, manufacturing and financial sectors of their economies, and acquired important economic and political status, becoming major sources of employment. The moderate growth experienced in the seventies, however, was quickly reversed by the financial crisis of the early eighties, and associated inefficiencies made parastatal sector reform a major element in the reform efforts implemented by the countries. Zambia was one of the earlier countries to embark on a major privatization exercise as part of its economic reform program started in 1992. Although progress was initially slow, mainly due to the inertia associated with start up activities and generally opposition from interested parties2, the program picked up momentum in the last two years, culminating in the rapid divestiture of public enterprises that many have compared only to privatization programs in eastern Europe. This paper reviews the privatization program in Zambia, highlighting the major tools and mechanisms employed, and the achievements and constraints faced by the authorities in privatizing one of the largest public sectors in Africa. The paper begins with a brief overview of the main economic issues surrounding moves towards privatization of public enterprises.
Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.
No full textWhile all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections
Chlorophyll biosynthesis under the control of arginine metabolism
No full textSummary: In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis
Structural basis of vilazodone dual binding mode to the serotonin transporter
No full textThe serotonin transporter (SERT) plays a pivotal role in regulating serotonin (5-HT) signaling and is a key target in the treatment of psychiatric disorders. SERT has a binding site (S1) for 5-HT that also serves as a high-affinity binding site for antidepressants. The antidepressant vilazodone has been shown to inhibit SERT by binding to an allosteric site. Here, we present the cryo-EM structure of SERT with vilazodone bound to the S1 site and extending towards the allosteric site. We systematically dissect the vilazodone molecule into fragments and find that the terminal indole ring is the key determinant of its high affinity to SERT. Further, unlike typical Na+-dependent SERT-selective antidepressants, vilazodone exhibits a dissociation constant (KD) for purified SERT in the nanomolar range both in the presence and absence of Na+. We substantiate this binding mode by exploring the conformational impact of vilazodone binding to SERT using site-specific insertion of the fluorescent non-canonical amino acid Anap. Our results offer molecular insight into the distinct pharmacological profile of a clinically used polymodal antidepressant.</p
The active site of magnesium chelatase
No full textThe insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons
Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of ade novo-designed heme protein
No full textProtein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the modelEscherichia coliTat system to recognize and translocatede novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no hemebcofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of theEcolitrimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of hemeb-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality control mechanism
Critical assessment of LC3/GABARAP ligands used for degrader development and ligandability of LC3/GABARAP binding pockets
No full textRecent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development.15
Dinâmica da guanilato ciclase solúvel na sepse: uma janela de oportunidade
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia.A alta produção de óxido nítrico (NO) é um dos principais responsáveis pela hipotensão e hiporeatividade a vasoconstritores, características clínicas do choque séptico. A ativação da enzima guanilato ciclase solúvel (sGC) responde pelos principais efeitos do NO durante esta patologia. Entretanto, há controvérsias a respeito se a utilização de inibidores desta enzima é uma opção efetiva e segura para o tratamento do choque séptico. A incubação in vitro de pulmões, retirados de animais controles ou injetados com LPS 24 h antes, com nitroprussiato de sódio (SNP), gerou um aumento de 20 vezes nos níveis de cGMP. Já em tecidos de animais injetados com LPS 8 h antes, o SNP falhou em aumentar os níveis de cGMP. Os níveis protéicos da sGC foram reduzidos no tempo de 8 h após a injeção de LPS e voltaram a valores normais em ratos injetados com LPS 24 h antes. A reposta vasoconstritora para fenilefrina foi reduzida em torno de 50 % em 8 e 24 h após a injeção de LPS. O azul de metileno (MeB), inibidor da sGC, restaurou a reatividade para fenilefrina em animais injetados com LPS 24 h antes, mas falhou em animais injetados 8 h antes. Para avaliar o efeito do azul de metileno sobre a mortalidade os animais foram submetidos ao procedimento de ligadura e perfuração do ceco (CLP). Quando os animais receberam MeB 8 h após o CLP, a mortalidade dos animais piorou. Entretanto, quando os animais receberam MeB no tempo de 20 h após o procedimento de CLP, ocorreu uma redução no índice de mortalidade. Portanto, o padrão de resposta da enzima sGC durante a sepse pode determinar o sucesso ou a falha do tratamento com seus inibidores. Assim, o azul de metileno pode ser uma estratégia terapêutica útil se administrado em uma janela correta de oportunidade
Analysis of the effects of a Constructivist-Based Mathematics Problem Solving Instructional Program on the achievement of Grade Five Students in Belize, Central America.
No full textThis thesis examined whether social constructivist activities can improve the mathematical competency of grade five students in Belize, Central America. The sample included 342 students and eight teachers from two rural and urban schools. A switching replication design was employed enabling students in the experimental groups to be taught using social constructivist activities for 12 weeks and the controls exposed to similar instructional practices from weeks 7 to 12. Students‘ performance was assessed using Pre-test, Post test 1 and 2 with an internal consistency of 0.89, 0.90 and 0.93 respectively. As revealed by the repeated measures ANOVA within subject analysis, there were significant differences among the pre-test and post test 1 and 2 results. That is, students in the control groups, who were instructed using a procedural approach from weeks 1 to 6, demonstrated higher gains than the experimental groups who were immersed in social constructivist activities. Furthermore, when the control groups became immersed in similar activities from weeks 7 to 12, they continued to outperform the experimental groups who were exposed to social constructivist activities alone. Hence, due to this unexpected result, the aim of this thesis became to explain why these results came about and what implications for teaching were highlighted by the consideration.
Besides the quantitative results highlighted above, qualitative data was also obtained as part of the study. For example, students were videoed within constructivist math groups and their performance analyzed using Pirie and Kieren‘s (1994) Model of Growth for Mathematical Understanding. The data from the video recording revealed that use of one step math problems did not enabled students to restructure their thinking to solve innovative problems. Data from semi-structured interviews also revealed that some students lacked basic math skills and were not exposed or guided to solve complex problems. Besides the need for careful examination of social constructivist activities on performance, this thesis underscores the importance of relevant teaching and learning activities, the important role of teachers during social constructivist activities and the need to identify suitable forms of assessment to measure performance
Characterising the Drosophila extracellular superoxide Dismutase gene
No full textThe indiscriminate action of reactive oxygen species (ROS), if left unregulated, haslong been considered contributory to a range of disease processes within the animalkingdom and is also a factor associated with ageing. Consequently modifying themolecular mechanisms that regulate ROS levels may prove therapeutic and could alsopositively affect longevity. One of the key components of this machinery is thesuperoxide dismutase (SOD) family of enzymes which regulate ROS levels byscavenging the ROS superoxide. Mammals have three distinct SOD enzymes eachresponsible for managing superoxide levels in different cellular compartments. InDrosophila homologues of two of the mammalian SODs, the intracellular (SOD1) andmitochondrial (SOD2) SODs, have been identified and studied extensivelydemonstrating a clear link between SOD and oxidative protection and survival.Recently the sequence of a third sod gene, homologous to both the relatively poorlycharacterised mammalian (sod3) and C. elegans (sod-4) extracellular sod, wasidentified in Drosophila and is also predicted to locate extracellularly (sod3). To date,no (published) work has been carried out to assess the role of sod3 within insects. Thisthesis reports the molecular and biochemical characteristics of sod3 in Drosophila.Detailed within are the steps taken to clone the sod3 gene which appears to beexpressed as two gene products formed by alternative splicing. Furthermore, acombination of gene expression, proteomic and functional analysis of a number of sodmutants was used to: i) reveal sex specific sod gene expression; ii) validate a sod3hypomorph mutant; iii) indicate a functional role for sod3 in protection against H2O2induced oxidative stress; iv) suggest a SOD1-SOD3 co-dependency for maintaining CuZn SOD activity; v) demonstrate the appearance of genetic modifiers in the sod3hypomorph. The findings of this report and further studies on the Drosophila sod3 geneshould encourage the re-evaluation of the previous work concerning SOD’s influenceon disease states and lifespan regulation
