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Plasmodium falciparum is a protozoan parasite responsible for the most severe form of human malaria, with infection often resulting in death. Efforts to control malaria have been hindered by an increased spread of parasite resistance to previously effective antimalarial drugs, leading to an intensified search for novel antimalarial drug targets. A group of proteins suggested as potentially effective targets are the integral membrane transport proteins, since they play key roles in Plasmodium parasite growth and replication. One such membrane protein recently characterised was the P. falciparum copper efflux transporter. Treatment of cultured P. falciparum parasites with the intracellular copper chelator neocuproine inhibited parasite growth, suggesting that additional mechanisms for malaria parasite copper homoeostasis are likely to be present. Copper is an essential trace element involved in enzymatic processes requiring redox-chemistry. In higher eukaryotes copper is transported across the plasma membrane via the copper transport protein, Ctr1, and distributed intracellularly by copper metallochaperones. The mechanisms for copper acquisition and distribution in the Plasmodium parasite are, however, yet to be characterised. An in silico Basic Local Alignment Search Tool for protein (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified sequences corresponding to a putative copper transporter, and associated copper metallochaperones, in eight species of the Plasmodium parasite. Each of the Plasmodium copper transport protein sequences was found to contain features common to the well characterised copper transporters. These features included predicted copper-binding motifs in the protein's amino terminus, three membrane spanning domains and the characteristic MxxxM and GxxxG motifs located in the second and third transmembrane domains, respectively. Affinity purified anti-peptide antibodies, generated against an immunogenic peptide (CSDKQSGDDECKPILD) in the amino terminus of a putative malaria parasite copper transporter (PY00413), detected the target protein in murine malaria parasites in association with a parasite membrane. The open reading frames corresponding to the amino terminal domains of one P. berghei [PBANKA_130290 (447 bp)] and two P. falciparum [PF14_0211 (132 bp) and PF14_0369 (282 bp)] putative copper transport proteins were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with maltose binding protein (MBP). The resulting sizes for the recombinant proteins were 61kDa for MBP-PbCtrNt, 48kDa for MBP-PfCtr211Ntᵀᴰ and 55kDa for MBP-PfCtr369Ntᵀᴰ, with each protein being recognised by a corresponding anti-peptide antibody. All three recombinant proteins bound copper in vitro and in vivo, with each having a binding preference for the reduced cuprous ion. This preference has been similarly established for the characterised copper transporters. Although the results supported the expression and copper binding ability of a Plasmodium parasite copper transport protein, the directional transport of copper, by this protein, requires experimental confirmation as does its specific location. The identification of a P. falciparum copper transporter, and other copper dependent proteins, implies a parasite metabolic requirement for copper. Mammalian and yeast cells require a Cox17 metallochaperone for copper delivery to cytochrome-c oxidase. Identification of P. falciparum orthologs for Cox17 (PF10_0252) and a number of cytochrome-c oxidase subunits (PF13_0327; PF14_0288; mal_mito_1; mal_mito_2; PFI1365w; PFI1375w), suggests the existence of similar parasite mechanisms for copper delivery. Analysis of the Plasmodium Cox17-like sequences identified essential amino acids conserved in the well characterised yeast and mammalian Cox17. This included the identification of six cysteine residues essential for Cox17 function. A homology model of P. falciparum Cox17, with human Cox17 as the template [PDB ID: 2RN9 (apoCox17); 2RN8 (Cu⁺-Cox17)], suggested that Plasmodium Cox17 orthologs would adopt a similar structural conformation. The open reading frames for full-length P. yoelii [PY03823 (192 bp)] and P. falciparum [PF10_0252 (195 bp)] Cox17 were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with either a His₆-tag or glutathione S-transferase (GST)-tag, respectively. The resulting sizes for the recombinant proteins were 11.6kDa for His₆-PyCox17 and 33.5kDa for GST-PfCox17, with each protein being recognised by a corresponding anti-peptide antibody. Both recombinant Cox17 proteins bound the cuprous ion in vitro and in vivo, similar to mammalian and yeast Cox17. This supported the likely existence of a mitochondrial copper metallochaperone pathway within the malaria parasite; however, this requires further experimental confirmation. Identification of a parasite copper transport protein, and associated metallochaperones, could provide novel targets for drug-based inhibition of parasite growth. Alternatively, the copper transporter may provide a novel mechanism for drug delivery into the Plasmodium parasite. The potential of these malaria parasite proteins being effective drug targets does, however, remain to be confirmed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties, and proximal limb weakness. The autosomal dominant form of this disease is caused by the expansion of a polyalanine stretch from 10 to 12--17 alanines in the N-terminus of PABPN1. Mutated PABPN1 (mPABPN1) is able to induce the formation of filamentous intranuclear inclusions that are the pathological hallmark of OPMD. PABPN1 is predominantly localized to the nucleus, binds RNA poly(A) tail, forms oliogmers, and is involved in polyadenylation. In this study we first demonstrated that oligomerization of PABPN1 is mediated by two potential oligomerization domains (OD), while inactivating oligomerization of mPABPN1 by deletions of 6--8 residues in either of the ODs prevents intranuclear protein aggregation. Expression of mPABPN1 in COS-7 cells is associated with cell death, whereas preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. We then identified two PABPN1 interacting proteins, hnRNP A1 and A/B, using a yeast two-hybrid library screen. The interaction between PABPN1 and hnRNP A1 or A/B was confirmed by GST pull-down and co-immunoprecipitation assays. When coexpressed with mPABPN1 in COS-7 cells, predominantly nuclear localized hnRNP A1 and A/B co-localize with mPABPN1 to the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. We finally found a nuclear localization signal (NLS) in PABPN1 that is not homologous to any known NLSs. The 18 amino acids 289RGRVYRGRARATSWYSPY 306 in PABPN1 are necessary and sufficient for nuclear translocation. Attaching this sequence to cytoplasmic protein PKM2 completely re-localizes it to the nucleus. Alanine-scanning mutagenesis analysis showed that the last 9 residues 298RATSWYSPY306 are crucial to the function as an NLS. Our studies showed that mPABPN1 induced intran

The process of metastasis is initiated through the acquisition of inherent and autonomous motile and invasive properties by tumor cells. These phenomena are initiated through a balance between forward cancer cell membrane protrusion and tail retraction, and occur via cell cytoskeleton remodeling, actin reorganization, and coordinated focal adhesion assembly and disassembly events. Among the vast network of cytoskeletal proteins, the actin-bundling protein fascin plays a major function in cell cytoskeleton remodeling. It is a 55-kDa protein involved in the formation of filopodia and cell migration, and found to be upregulated in many cancers. We report herein key functions for fascin in the regulation of prostate and breast cancer progression. Fascin expression is upregulated in localized and hormone refractory prostate cancer, responsible for a more aggressive clinical course. In addition, functional dissection of fascin reveals a novel function in the regulation of focal adhesion turnover dynamics, by modulating the phosphorylation state of central focal adhesion proteins through a potential collaboration with the protein tyrosine phosphatase, PEST. Together, our data support the importance of fascin in cancer cell invasion and as a significant prognostic marker and a potential therapeutic target for aggressive cancers.

Conjugated polymers are promising candidates for applications in all kinds of organic optoelectronic devices: OLEDs, organic field-effect transistors (OFETs) and organic photovoltaic cells. The main goal of this work was to investigate transport features of photogenerated electrical charge in pi-conjugated polymers by means of novel technique based on time-resolved electric field-induced second harmonic generation (TREFISH). TREFISH measurement setup was implemented in the laboratory of Molecular compounds physics, and applicability of the method has been verified. Measurements were performed on three different model polymers: methyl substituted ladder-type poly(para-phenylene) (MeLPPP), poly(fluorene-co-benzothiadiazole) (F8BT) and poly(spirobifluorene-co-benzothiadiazole) (PSF-BT), having different morphological and chemical structure. It has been found that motion of photogenerated charge carriers in π-conjugated polymer films experiences rapid dynamics after excitation. Different time domains of charge transport were distinguished. Initial fast transport of photogenerated charge carriers corresponds to the carrier motion along the single polymer chain or conjugated segment of the polymer chain. Slowest carrier motion phase is well described by the stochastic drift, which is attributed to interchain jumps and determines the macroscopic equilibrium mobility. Thus, the equilibrium mobility value is not applicable to the transport on nanometer scale up to tens of nanometers... [to full text] / Konjuguotieji polimerai kaip funkcinės medžiagos gali būti panaudoti įvairiuose prietaisuose: organiniuose šviestukuose, organiniuose lauko tranzistoriuose, organiniuose saulės elementuose. Šio darbo tikslas - nustatyti fotogeneruotų krūvininkų pernašos dėsningumus π – konjuguotuose polimeruose panaudojant naują žadinimo-zondavimo metodą pagrįstą išoriniu elektriniu lauku indukuota antrosios optinės harmonikos generacija. Pagrindinis dėmesys buvo skiriamas pernašos dinamikai. Molekulinių darinių fizikos laboratorijoje buvo įrengta matavimų schema ir įvertintas metodo tinkamumas krūvio pernašos tyrimams. Buvo atlikti krūvio pernašos matavimai trijuose skirtinguose konjuguotuosiuose polimeruose. Nustatyta, kad fotogeneruotų krūvininkų judris tuoj po sužadinimo yra daug didesnis lyginant su stacionaria judrio verte, o krūvio pernašos dinamiką lemia konjuguoto polimero struktūrinė hierarchija, krūvininkų judėjimas yra daugialypis, susidedantis iš greito judėjimo viena polimero grandine ar konjuguotais polimero grandinės segmentais ir lėto šokavimo tarp atskirų polimero grandinių Pirmą kartą detaliai išnagrinėta šviesa sugeneruotų krūvininkų pernašos dinamika konjuguotuose polimeruose. Darbo rezultatai suteikia žinių apie fundamentalius krūvininkų pernašos mechanizmus konjuguotuose polimeruose, kurios gali būti panaudotos kuriant organinius elektronikos prietaisus.

An explosive growth in demand for broadband mobile wireless services is currently being fuelled by cellular telephone users who, encouraged by service providers, are no longer content with voice transmission only but are demanding real-time video services, including multi-user, interactive games and 'movie' programmes. As these applications develop, expectations mount in other mobile user markets, especially the public safety arena, for comparable user features but with greater emphasis on reliability and robustness of the equipment and supporting network in adverse propagation conditions, remote locations and emergencies. These applications all have in common the requirements for efficient use of wireless bandwidth and of battery power, as well as seamless operation when moving, sometimes at high vehicle speeds, from one type of environment to another in a multi-user scenario. Orthogonal frequency-division multiplexed (OFDM) signals have been found to compare favourably with other modulation systems in these applications, the multi-carrier format being more tolerant of delay spread. It has been used in both code-division (MC-CDMA) and frequency-division (OFDMA) multi-user schemes, the latter having the advantage of maintaining orthogonality among users in fading-signal environments, with consequent simplification of signal processing. The major drawback of OFDM has been the high peak-to-average power ratio (PAPR) that is characteristic of signals with multiple sub-carriers. A result of this is that the transmitter requires a linear power amplifier (PA) that generally has to be 'backed off' to accommodate the high PAPR. Additional back-off is required to achieve linearity, as well as sometimes-complex linearisation circuitry. The power usage and cost of such a transmitter is more acceptable in a base station, tending to limit the application of OFDM to downlinks. The potential application to hand-portable terminals has severe constraints of size, cost and battery life, exacerbated by the use of video-capable LCD displays, increasing motivation for the use of MIMO (multi-antenna) technology and the development of mobile ad-hoc networks, the latter being particularly applicable in the public safety arena. Previous efforts to ameliorate the PAPR problem have been principally directed at two areas, the reduction of signal PAPR, by block coding, clipping or other techniques, and methods of achieving PA linearisation with improved power efficiency. The first object of the present research was to establish, as far as practicable, the current state of the art in these areas, to set a performance baseline. The next step was to develop an improved transmitter modulation scheme that would not only be able to take advantage of any existing peak reduction methods but would transmit a signal that would be compatible with existing OFDM receivers. A novel modulation technique is now presented, termed Quadrature Phase-Time Modulation (QPTM), that has been found to meet the requirements for linearity, simplicity and low cost, whilst being able to take advantage of constant-envelope PA technology, with its attendant power efficiency. After final amplification, the signal is restored by a passive narrow-band filter to standard OFDM form, having both phase and amplitude modulation. The QPTM system of modulation relies on a dual baseband pulse-width modulation process, performed at a substantially-higher rate than the upper baseband frequency, followed by direct quadrature modulation of a carrier signal. The work undertaken has been in the nature of a feasibility study, commencing with the theoretical basis of the technique, from which a behavioural system model was designed and simulated. After the system was simulated successfully, in several forms, a model was designed for realisation with available high-frequency integrated circuits. From this design, prototypes were constructed and tested. The prototype circuit boards also included an experimental UHF Class-D PA circuit, excluding the output filter, to facilitate ongoing development of the PA and filter subsystem as a separate project. This type of PA was seen as a potential complement to the QPTM modulator, although the technology was at an early stage of development. The prototype PA has a novel push-pull arrangement of GaAs FETs that employs a broadside-coupled tapered-stripline balun instead of the usual transformer. Preliminary measurements were made on the PA using both a spectrum analyser and a newly-available 8GHz-bandwidth digital oscilloscope to confirm basic operating characteristics. The performance of the QPTM technique at frequencies needed for broadband operation is dependent on its practical implementation, which has therefore been a major focus. The inherent difficulties in realising a highly-linear 40MHz triangle-wave reference generator, with a precise ultra-high-speed comparator and modulator system, have been overcome with the chosen design techniques and attention to several critical aspects. The result has been the successful demonstration of QPTM as an efficient PA modulation technique that is equally applicable to either narrow-band, high-capacity UHF or broadband OFDM microwave systems.

According to IPCC reports, the greenhouse gas CO2 is responsible for global climate change. Studies show that CO2 concentration reached a level of 400 ppm in 2013, or 40 % above pre-industrial levels. The contribution of CO2 from industrial activity to increasing global CO2 concentrations is widely accepted and points to the need to reduce the emission of this greenhouse gas.One possible combustion technology that shows promise for reducing CO2 emissions is chemical looping combustion (CLC). It is an oxy-fuel technology, but has the advantages of in situ oxygen separation, low NOx emissions and low cost of CO2 emission abatement; it entails the use of an oxygen carrier (OC) to provide oxygen for combusting fuels. OC development is an important task in CLC. Iron based OCs have attracted most research attention in recent years, mainly due to their inexpensive and non-toxic nature. Bi-metal oxide OCs usually impart better CLC performance than mono-metal oxide OCs, one example of which is the introduction of CeO2 as a partially reducible material capable of generating oxygen vacancies that lead to oxygen storage and transfer. In this study, CeO2 was used as an additive to a Fe2O3-based OC and its effect on physical properties, such as morphology, surface area and mechanical strength, was analyzed in detail. The reactivity of OCs is studied using TGA-MS and a bench scale CLC setup. The results show that the reduction reaction at the surface is independent of whether CeO2 is present or not, but after the surface oxygen had been consumed, the OC with CeO2 provided faster oxygen transfer rates from the bulk to the surface to produce better average reaction rates. The OCs after reduction and oxidation were analyzed using XRD and Raman spectroscopy; based on these analytical data, a model for the promoting role of CeO2 is discussed. Furthermore, the reaction kinetics of the OCs were also studied using shrinking core model, the kinetics parameters were obtained and compared. Scale-up of laboratory-scale CLC reactors is another important task necessary to develop an understanding of the potential and efficiencies of CLC. In this study, scaling laws were used as a guide to design and then build two different-sized CLC reactors. Testing of the reactors involved a focus on chemical similarities. Comparisons of the performance of both reactors showed good consistency, thereby validating the scale modeling method and the scale laws for CLC reactors.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

The Drosophila melanogaster eIF4E binding protein (d4E-BP) inhibits translation initiation and is implicated in cell growth as a downstream effector of the Drosophila insulin signaling pathway. Since d4E-BP null flies show similar growth and development to control flies, the possibility of a conditional phenotype was explored through stress treatments. Adult d4E-BP null flies show sensitivity to oxidative stress, and d4E-BP null larvae die faster than controls under starvation and protein starvation. Expressing a mutant d4E-BP that doesn't bind to eIF4E in the d4E-BP null background does not rescue this stress sensitivity, which suggests that wild-type stress resistance requires binding of d4E-BP to eIF4E. / The Drosophila forkhead transcription factor dFOXO is a transcriptional activator of d4E-BP. There is a strong reduction of d4E-BP peptide in a dFOXO null background. dFOXO null flies are also sensitive to oxidative stress, and rescue of this sensitivity through ectopic expression of UAS-d4E-BP(wt) in a dFOXO null background suggests d4E-BP is a downstream mediator of dFOXO oxidative stress resistance.

Early Drosophila development is a coordinated series of temporal and spatial events leading to specific localized gene expression. The maternally expressed gene Bicaudal-C (Bic-C) encodes a KH-domain RNA binding protein required in the developing oocyte for anterior-posterior patterning and follicle cell migration. The dominant heterozygous phenotype results in the development of embryos with bicaudal and head defects. A two-hybrid screen using BIC-C as "bait" identified the novel protein ANTIMEROS (ATMS) and the SH3-domain containing protein MILE END (MILE). / ATMS is highly conserved between humans and mice, its expression is almost entirely female-specific, and is limited to certain developmental stages. Mutant alleles for atms are able to dominantly enhance the phenotype of Bic-C heterozygotes confirming the Bic-C-atms interaction. Here I show that NOS mislocalization causes the trans-heterozygous phenotype, as introduction of a nos mutation strongly suppresses the bicaudal phenotype. nos transcripts show a hyper-polyandenylation in atms mutant ovaries, an indicator of translational activation, suggesting that ATMS and BIC-C function as translational repressors of nos through changes in its poly(A) tail length. / MILE, contains two highly conserved SH3 domains at the C-terminus. Experiments involving the analysis of mutant alleles and overexpression mile transgenic lines show that MILE is a negative regulator of both Torso and Egfr RTK signaling. Its not clear what functional role BIC-C may have with RTK signaling, but recent evidence suggests that posterior group gene expression influence terminal pole RTK signaling.