Dissertations / Theses on the topic 'Malaria – Drug therapy'

With the increase in recent years in the prevalence of malaria, and in drug resistance of Plasmodium falciparum, there has been much interest in natural plant products for new antimalarials with novel modes of action against Plasmodium. Artemisinin or Qinghaosu is one such antimalarial isolated from a Chinese herb, Anemisia annua (Asteraceae) and it is currently undergoing phase I and II clinical trials. The Southern African species, Artemisia afra (African wormwood, wildeals, lengana) is commonly used by local traditional healers for symptoms of malaria, in particular fever. Thus it seemed appropriate to investigate this species for antimalarial activity. Crude petroleum ether soxhlet extracts of Anemisia afra had demonstrated antimalarial activity against Plasmodium falciparum, FCR-3, cultured in vitro. The IC₅₀ values ranged from 5-13μg/ml. The extract from leaves and flowers was then screened against D10 (chloroquine-sensitive) and FAC8 (chloroquineresistant) P. falciparum, in vitro, with IC₅₀ values of 1.03μg/ml and l.5μg/ml respectively. This extract was fractionated by column chromatography using silica gel-60 and the fractions obtained were screened for antimalarial activity. The most active fraction had an IC₅₀ of 0.5μg/ml against D10 and FAC8. Using TLC and HPLC-UV analysis with pure artemisinin as a standard, no artemisinin could be detected in this fraction. This result was confirmed by thermospray LC-MS analyses. Purification of this fraction yielded ultimately a single pure compound; a clear colourless oil identified by MS and NMR analyses as hydroxydavanone. The compound was screened against a variety of P. falciparum strains with varying degrees of sensitivity and resistance to both chloroquine and mefloquine. Their sensitivity against artemisinin was also established. IC₅₀ values obtained for the isolated pure compound against P. falciparum ranged from 0.87 to 2.54μg/ml. The IC₅₀ values obtained for general cytotoxicity of the crude extract and isolated pure compound against RAT-I fibroblast cells were 34.78 ± 8.23 and 6.29 ± 0.95 μg/ml (n=4) respectively. Thus the crude extract and isolated pure compound exhibited a greater antimalarial than cytotoxic effect. Hence, there are implications for A. afra to be used as a phytomedicine for the treatment of malaria. In vivo studies are recommended for hydroxydavanone in order to fully assess its potential for clinical use.

no
New antimalarial drugs are urgently needed to combat emerging multidrug resistant strains of malaria parasites. This Highlight focuses on plant-derived natural products that are of interest as potential leads towards new antimalarial drugs including synthetic analogues of natural compounds, with the exception of artemisinin derivatives, which are not included due to limited space. Since effective antimalarial treatment is often unavailable or unaffordable to many of those who need it, there is increasing interest in the development of locally produced herbal medicines; recent progress in this area will also be reviewed in this Highlight.

Malaria is a health, social and economic burden in Nigeria and consistently ranks amongst the four most common causes of childhood deaths. Treatment of malaria is usually started at home
care is only sought from the health facility when the treatment is ineffective (McCombie, 1996). Patent medicine vendors (PMVs) have been identified as a widely patronized source for drugs used in the home treatment of malaria (Breiger et al, 2001
Goodman, et al, 2007
Salako et al, 2001). Inadequate or poor knowledge and practices in the use of anti-malaria drugs (AMDs) increases morbidity and mortality, undermines therapeutic efficacy, and promotes the emergence and spread of drugresistant malaria. Aim: The aim of the study was to describe and quantify the knowledge and self-reported practices of PMVs in the use of antimalarials, particularly artemisinin-based combination therapies (ACTs), in a poor urban community in Lagos state, Nigeria.

Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.

Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery

The most severe form of malaria in humans is caused by the intracellular parasite Plasmodium falciparum. The African continent bears the greatest burden of malaria with 90% of all malaria deaths occurring in sub-Saharan Africa where the high risk populations include pregnant woman and children under the age of five. Fatal cases of malaria are often a result of the progression of the disease to a life threatening syndrome where intravenous quinine or artesunate are administered as an emergency treatment, however a 15-20% mortality rate is still observed among treated individuals. Pathogenesis of severe malaria is associated with the mature or late trophozoite stage of the parasite s intra-erythrocyte life cycle. At this stage the intracellular parasite expresses parasite derived proteins on the surface of the red blood cell (RBCs) that bind to host endothelial receptors. This cytoadhesion ultimately allows the parasite to multiply unhindered by the host resulting in high parasitaemia levels which is associated with the extent of the symptoms associated with severe malaria. There is currently no effective vaccine available for malaria and available antimalarial drugs are often compromised by rapidly developing drug resistance. Therefore there is an urgent need to identify novel drug targets for the treatment of severe malaria and to explore therapeutic agents that can be used as adjuncts to the currently available anti-malarial drugs. Statins, also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have been identified as a potential adjunctive therapy for severe malaria due to their pleiotropic effects which include anti-cytoadhesive activity. The aim of this study was to characterise cytoadhesive related parasite proteins that are expressed on the surface of parasitised RBCs and then to assess the effects of a classic statin, lovastatin, on these parasite proteins. This was done in order to provide improved insight and extend existing knowledge on the potential role statins may have in treating severe malaria. The P. falciparum 3D7 strain was efficiently synchronised and cultured according to standard culturing procedures and selective harvesting provided a 90% enrichment of narrowly synchronised cultures at the late trophozoite stage (35 -40 hours post invasion). Scanning electron microscopy (SEM) was used to successfully illustrate parasite induced morphological changes to the surface of parasitised RBCs which included parasitic knob structures, a characteristic feature of severe malaria. Isolation of membranes of equivalent RBCs and parasitised RBCs that were untreated and treated with 10 ?M of lovastatin was optimised and surface proteins were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Bands of interest that were visually apparent on Stain Free imaged gels and Coomassie brilliant blue stained gels were excised and in-gel trypsinised followed by peptide sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). Advanced proteomic software was then used to successfully confirm the identity of key RBC structural membrane proteins as well as several parasite surface proteins, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), P. falciparum erythrocyte membrane protein 3 (PfEMP3), P. falciparum knob associated histidine rich protein (PfKAHRP), P. falciparum mature infected erythrocyte surface antigen (PfMESA) and P. falciparum cytoadhesion linked asexual gene (PfCLAG) proteins. These cytoadhesive related proteins were identified on the membranes of parasitised RBCs that were treated with lovastatin thus suggesting that although lovastatin has the ability to retard cytoadhesion it is apparently not through the inhibition of expression of parasite derived adhesion proteins. The study also highlighted the need for stringent controls when using label-free proteomics to assess differential protein expression. The effects of lovastatin on protein abundance were inconclusive due to the low abundance of these proteins. This study demonstrated the potential of combining pharmacological studies with advanced proteomic techniques where the effects of physiological relevant concentrations of a drug were successfully assessed at the parasite protein expression level.
Dissertation (MSc)--University of Pretoria, 2015.
Pharmacology
MSc
Unrestricted

Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.

Thesis (MSc)--Stellenbosch University, 2013.
Aminoquinolines are important class of drugs that have been used for malaria chemotherapy for centuries. However, long-term exposure to these drugs leads to extensive spread of drug resistance. As such, modified chloroquinoline derivatives are being studied as alternative antimalarial agents with the possibility to overcome drug resistance associated with chloroquine analogues. In this study, 15 aminoquinoline derivatives that are linked by a 1,4-disubstituted 1,2,3-triazole ring to an ethyl and propyl carbon spacer with a distal amine motif were designed and synthesized as novel antimalarial agents using the Cu(I)-promoted Huisgen reaction. The compounds have been synthesized from the 7-chloro-N-(prop-2-yn-1-yl)quinolin-4-amine alkyne precursor and the azides of ethyl and propyl amino moieties using a 1,3-dipolar cycloadditioncoupling in the presence of CuI catalyst to obtain moderate to good yields (53 – 85%). These compounds have been characterized by the combination of NMR, ESI+ HRMS and IR spectroscopic methods. The antiplasmodial activity of the compounds was investigated in vitro against P. falciparum strain NF54 using chloroquine as a reference drug together with a standard antimalarial drug artesunate. Of the 15 novel chloroquinoline derivatives, 11 have demonstrated to possess promising potency by way of the inhibition concentrations less than 250 nM with the lowest being 28 nM. The observed activities have been ascribed to the overall modifications such as the introduction of a triazole linker and changing of carbon chain length as these were the variables. The compounds are accordingly under further biological investigations and only the chloroquine sensitive results are reported in this work.

For many centuries, malaria has remained the most common parasitic disease in sub-Saharan Africa potentially placing 32 million pregnancies at risk each year. Malaria in pregnancy (MiP) in malaria endemic Africa is mostly asymptomatic or paucisymptomatic, yet associated with maternal anaemia and intra-uterine growth retardation resulting in low birth weight (LBW) which is an important risk factor for infant mortality (chapter 1). In Mali, several observational studies have determined the risk and consequences of malaria in pregnancy. However, national estimates of the burden of MiP and its potential impact are lacking. This thesis describes the results of a series of surveys conducted in different malaria transmission settings countrywide from 2005 to 2010, to quantify the burden and consequences of MiP in Mali (chapter 2). Results demonstrate that the risk of malaria infection at delivery was generally high ([average prevalence 11.6%]) and showed marked diversity between regions and transmission settings. Coverage of intermittent preventive treatment (IPTp) with sulfadoxine-pyrimethamine (SP) and impregnated treated bednets (ITNs) was low (30.4% and 60.7%) and indicated important miss opportunities for the control of PAM. To prevent the disease and its consequences in pregnancy, the World Health Organization recommends IPTp using SP and use of an ITN. For IPTp, the recommended regimen consists of at least 2 doses of SP given during the second and third trimesters for HIV negative women and at least 3 doses for HIV-positive women not receiving cotrimoxazole. However, there are concerns that the 2-dose regimen, which provides at most 12 weeks of prophylaxis, leaves many women unprotected for as much as half of the 20-26 pregnancy weeks after quickening. Re-infection with the 2-dose regimen is common, especially during the transmission seasons and amongst women who complete their last dose early in the third trimester. A trial was therefore conducted to compare the standard 2-dose regimen versus 3 doses using SP, hypothesizing that the third dose may add significant benefit over the 2-dose regimen in preventing placental malaria and other birth outcomes (chapter 3). The study concluded that IPTp-SP with 3-doses was superior to the standard 2-dose regimen and resulted in better birth outcomes. The results of this trial were then combined with 6 similar trials as part of a meta-analysis assessing if 3 or more doses of IPTp-SP are more effective than the current standard 2-dose regimen. The pooled results suggested a marked benefit of adding extra SP doses over the standard 2-dose regimen in both regions of high and low SP resistance as measured by the prevalence of dihydropteroate synthase K540E mutations (chapter 4). Although studies from western Africa favour the use of IPTp-SP, SP resistance is now a serious threat to the longevity of IPTp with SP in parts of eastern and southern Africa where the quintuple dihydofolate reductase (N51I, C59L, S108N) /dhps (A437G, K540E) mutation is saturated in many places. In order to get a better understanding of the impact of SP resistance on IPTp effectiveness, this thesis also determined the in vivo response of parasites in asymptomatic parasitaemic pregnant women who received IPTp-SP and the effectiveness of IPTp-SP on birth parameters in West-Africa (chapter 5 & 6) Overall, the study concluded that SP resistance in Mali and Burkina Faso is low and that the IPT-SP is associated with clinically relevant improvements in birth outcomes in Mali.

Malaria is a disease of major public health importance, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women in many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the parasite Plasmodium falciparum which is responsible for 80% of infections and 90% of deaths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementation of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to the available drugs has emerged, rendering the majority of them ineffective. Furthermore, the high cost of those drugs represents a major obstacle to their dispensation in areas of the world where the affected people are often the less fortunate. The enzyme Cytochrome b5 Reductase (cb5r) and its electron acceptor Cytochrome b5 (cb5) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, including Plasmodium falciparum. It is important to note that plant cb5r has been identified as a novel herbicidal target. Considering the close phylogenetic relationship between plant cb5r and Plasmodium falciparum cb5r, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs against the parasite. In this project, we want to obtain the biochemical and enzymatic characterization of cb5r and cb5 in order to establish whether these two proteins represent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of novel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.

Magister Public Health - MPH
The introduction of antiretroviral therapy (ART) brought new hope to HIV patients as it has transformed a fatal disease to a chronic manageable condition. In 2009 there were over 920,000 Malawians infected with HIV and 110,000 new infections. Malawi like other countries in the sub-Saharan Africa has made great strides in ensuring access to ART. The government of Malawi introduced free antiretroviral therapy (ART) in June 2004. By 2010, a total of 250,987 patients in the country were receiving ART. The success of ART requires, amongst others, a sustained adherence rate to medication of more than 95% to prevent viral replication and the development of drug resistant HIV strains. Identifying the factors that influence adherence is essential for the long-term success of public ART programmes. This study explored patient, socio-economic, cultural, and religious and health systems factors that influence adherence to ART in Zomba district in Malawi. An explorative qualitative study was conducted amongst ART patients and health workers in four health facilities in Zomba district of the Southern Region of Malawi. Data collection was through individual in-depth interviews with 25 ART patients and semi-structured key informant interviews with 13 health workers that were actively involved in the ART programme. Data was audio-recorded and transcribed verbatim. Thematic and content analysis of transcribed data was done. The study found high individual commitment, having social support from family and friends and continuous good counselling to be facilitators to adherence to ART. HIV-related stigma and discrimination, none disclosure of HIV status, lack of partner support, travelling to attend funerals and religious beliefs were noted barriers to adherence. Health system factors such as congestion in the clinic, negative staff attitudes and a lack of privacy at the pharmacy were also identified as barriers to clinic attendance and keeping appointments. Although pill burden was not mentioned, patients reported drug reactions as a barrier to adherence. Although there is good road network in the district, transport cost was still mentioned as a hindrance to treatment adherence. Treatment success was reported to be both a facilitator and a barrier to adherence. HIV-related stigma and discrimination among people need to be addressed to increase support to PLWHIV and encourage disclosure of HIV status. The improvement of the socio-economic status of ART patients needs to be addressed to reduce dependence on support from other people and provide money to make follow-up appointments. The health systems need to reduce clinic congestion and waiting times so that patients are not deterred from accessing ART.

Brugia malayi is one of the causative agents of the disabling and disfiguring disease known as Lymphatic Filariasis (LF). This infection is a well-established ailment in tropical and subtropical countries and recently the drug ivermectin has been introduced for the LF control programs. Ivermectin (IVM) is an excellent microfilaricide, but is not markedly macrofilaricidal. However, it causes a long-lasting reduction in the production of new larvae by female worms, suggesting that adult stages are affected. However, the mechanism by which IVM produces such effect in the adult worm is not well understood. One major reason is our incomplete understanding about the biological effect of IVM on adult stages. The present study was carried out to examine the in vitro effects of IVM on B. malayi adult worms using Brugia-gerbil animal model. And also to have some leads in understanding the drug-uptake and location of probable targets in the worm body by using fluorescent labeled IVM and confocal microscopy.
The antifilarial effects of IVM were examined using three parameters: mf release by female worms, and motility, and viability in both male and female worms. The results reported in this study demonstrate that although IVM did not kill the adult worm, but showed significant antifilarial effects on B. malayi adult stages when examined in an in vitro system. Confocal microscopy images of the worms incubated in bodipy FITC-IVM showed strong specific localization signal in the anterior cephalic region of both male and female worms. These observations suggest the early/initial interactions of the drug with its probable receptors that could be located specifically in the head region.

Les symptômes psychologiques et comportementaux de la démence (SPCD) sont fréquents et peuvent concerner jusqu’à 90 % des patients atteints de la maladie d’Alzheimer. Considérant l’efficacité limitée et l’ampleur des effets secondaires observés avec les traitements psychotropes en France, la majorité des directives existantes soulignent l’importance de la recherche clinique sur la maladie d’Alzheimer et une amélioration de l’évaluation des approches non pharmacologiques (ANP). En 2016, Le recours à la médiation animale, comme prise en soins, en établissements d’hébergement pour personnes âgées dépendantes (EHPAD) est de plus en plus fréquent. Nous avions montré les bienfaits de cette ANP sur l'apathie dans la maladie d'Alzheimer et avons souhaité en démontrer son efficacité. Notre étude évalue et mesure, principalement à l’aide de l’Inventaire Neuropsychiatrique version Equipe soignante (NPI-ES), comment la présence du chien dans la psychothérapie des malades Alzheimer est associée à des niveaux de SPCD chez des femmes et des hommes, âgés en moyenne de 85 ans qui vivent en institution. Nous nous sommes concentrés sur le bien-être et la construction d’émotions positives de la personne âgée démente, en particulier sur la revalorisation de l’estime de soi, la stimulation, la remobilisation et le maintien des capacités cognitives préservées, comme base thérapeutique possible dans l’association de la présence du chien avec la diminution significative des SPCD
Today, the use of animal mediation as a form of treatment in nursing homes has become more and more common. Our study assesses and measures, largely through the use of the Neuropsychiatric Inventory for Nursing Home (NPI-NH), how the presence of a dog in Alzheimer’s patient psychotherapy affects psycho-behavioral disorder (BPSD) levels in French men and women with a mean age of 85 years living in an institution. Our study focuses on well-being and on building positive emotion in the elderly with dementia, particularly through regaining self-esteem, stimulation, remobilization and maintenance of preserved cognitive abilities. This therapeutic basis is made possible through exposure to a dog with the goal of decreasing BPSD

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

Pang, Lap-yin.
"42" in title is subscript.
"July 2000."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (p. 163-173).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

A DISSERTATION SUBMITTED TO THE FACULTY OF MEDICINE, UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG, FR THE DEGREE OF MASTER OF SCIENCE IN MEDICINE.
The influence of iron on the outcome of various infections have been extensively reviewed. Clinical observations suggests that iron deficiency may be protective against malaria. Various researchers have shown that certain iron chelators blocked the proliferation of plasmodium falciparum in vitro and in vivo. (Abbreviation abstract)
Andrew Chakane 2018

In the second part of the project, the immunology of PfMSP-133 was studied. During the invasion of merozoites, PfMSP--142 is processed into two fragments with molecular weight of 33kDa and 19kDa. The 19kDa fragment (PfMSP-119) originating from the carboxyl--terminal of PfMSP--142 is relatively more immuno-dominant in different malarial species such as P. falciparum, P. vivax and P. yoelii. In the past, only limited researches about PfMSP-1 33 were performed. Apart from its difficulty in expression, PfMSP-1 33 was also believed to be incapable of inducing protection.
Nevertheless, following the breakthrough of expressing recombinant PfMSP-1 33 in our laboratory, we have demonstrated in this study that recombinant MSP-133 can elicit antibodies with a titer up to a million. Also, we observed that MSP-133 can help MSP-119 to induce protective immunity and such effect is independent from the covalent linkage between these two proteins. Most importantly, our results show that recombinant PfMSP-133 can elicit the production of antibodies that can potentiate the inhibitory effect of anti-MSP-142 serum at high serum dilution. Results of this study give new insights in malarial vaccine development in terms of optimizing the use of adjuvant and immunization regimens.
The 42kDa carboxyl terminal fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP--142) is one of the most promising candidate antigens in the development of malarial vaccine. In vivo experiments in the 1990's showed that Aotus monkeys immunized with PfMSP--142 were protected from malarial challenge. Later on, other experiments also demonstrated the possibility of using recombinant PfMSP-142 as candidate antigen for malarial vaccine. Previously, recombinant PfMSP-142 (Bvp42) was expressed with the baculovirus expression system and characterized in our laboratory.
The aim of the first part of this project is to improve the production of Bvp42. Experimental results have shown that the expression level of Bvp42 was increased under a BMN compatible baculovirus expression vector---pVL1393. Besides, a codon optimized MSP-142 nucleotide is constructed for the construction of a baculovirus carrying codon optimized MSP-142 gene and aimed for higher expression level. Unfortunately, no Bvp42 expression is observed in the transfection samples and the reason of this observation is unclear. Meanwhile, the purification of Bvp42 was also improved. Pretreatment of the hemolymph with Q--sepharose before affinity chromatography could enhance the purity of the final product.
Yuen, Sai-hang Don.
"July 2007."
Adviser: Walter K. K. Ho.
Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0220.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 183-195).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Chan Ping Kei.
Thesis submitted in: December 2001.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 135-143).
Abstracts in English and Chinese.
ACKNOWLEGEMENTS --- p.i
ABSTRACT --- p.ii
TABLE OF CONTECTS --- p.v
LIST OF FIGURE --- p.viii
LIST OF ABBREVIATIONS --- p.xii
CHAPTER
Chapter 1 --- INTRODUCTION
Chapter 1.1 --- Epidemilogy --- p.1
Chapter 1.2 --- Malaria disease --- p.1
Chapter 1.3 --- Life cycle of Malaria --- p.1
Chapter 1.4 --- Current measure to control Malaria --- p.6
Chapter 1.5 --- Anti-malaria vaccine candidate --- p.7
Chapter 1.6 --- Anti-erythrocytic malaria vaccine MSP-1 --- p.10
Chapter 1.7 --- Baculovirus Expression System --- p.20
Chapter 1.8 --- hexa-histidine tagged fusion protein --- p.25
Chapter 1.9 --- IMAC --- p.26
Chapter 1.10 --- Aim of study --- p.26
Chapter 2 --- MATERIALS AND METHODS
Chapter 2.1 --- Materials --- p.29
Chapter 2.2 --- Methods --- p.40
Chapter 3 --- CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BmNPV CARRYING PfMSP-l42
Chapter 3.1 --- Cloning of C-HisPfMSP-l42 into pBM030 --- p.71
Chapter 3.2 --- Construction of Recombinant BmNPV Carrying PfMSP-l42 --- p.72
Chapter 3.3 --- Purification of Recombinant BmNPVs --- p.78
Chapter 3.4 --- In vitro expression of Recombinant --- p.80
Chapter 3.5 --- In Vivo Expression of Recombinant PfMSP-l42 Protein --- p.80
Chapter 4 --- PURIFICATION OF BmNPV-EXPRESSED RECOMBINANT C- TERMIAL HEXA-HIS-TAGGED PfMSP-l42 PROTEIN
Chapter 4.1 --- Nickel ion charged Chelating Sepharose Fast Flow (immobilized metal affinity chromatography) --- p.88
Chapter 4.2 --- POROS HS/M (Strong Cation Exchanger) --- p.105
Chapter 4.3 --- Combination of chromatographic separations --- p.107
Chapter 5 --- CHARACTERIZATION OF RECOMBINANT C-HISPfMSP-l42 PROTEIN
Chapter 5.1 --- Proper formation of disulphide bridges in epidermal growth factor (EGF) like domains --- p.115
Chapter 5.2 --- Characterization of the integrity of hexa-histidines residue on recombinant PfMSP-142 protein --- p.117
Chapter 5.3 --- Immunogenicity of Recombinant C-HisPfMSP-l42 Protein --- p.117
Chapter 6 --- DISCUSSION
Chapter 6.1 --- Construction of recombinant BmNPV carrying HisPfMSP-l42 --- p.122
Chapter 6.2 --- Expression of recombinant HisPfMSP-l42 proteins --- p.123
Chapter 6.3 --- Purification of recombinant C-HisPfMSP-l42 protein --- p.125
Chapter 6.4 --- Characterization of recombinant C-HisPfMSP-l42 protein --- p.128
Chapter 6.5 --- Future prospects --- p.130
REFERENCE --- p.135
APPENDICES
Chapter 1. --- Appearance of Mulberry leaves
Chapter 2. --- Biomark 2000 (Beckman) program for sandwich ELISA protocol
Chapter 3. --- Nucleotide Sequence of PfMSP-l42 3D7 Isolate
Chapter 4. --- Nucleotide sequence of PfMSP-l42 FVO isolate
Chapter 5. --- Efficiency of the mAb5.2 immunoaffinity column in purifying the recombinant PfMSP-l42 protein

Leung Wai-hang.
"November 2002."
On t.p. "33" and "42" are subscripts following the word "MSP-1" in the title.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (p. 162-171).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

by Ng Wang Kit.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 131-139).
Abstracts in English and Chinese.
Acknowledgements --- p.iii
Abstract --- p.v
List of Tables --- p.ix
List of Figures --- p.x
List of Abbreviations --- p.xiii
Table of Contents --- p.xv
Chapter Chapter 1: --- General Introduction --- p.1
Chapter Chapter 2: --- Literature Review --- p.3
Chapter 2.1 --- Malaria --- p.3
Chapter 2.1.1 --- Global picture --- p.3
Chapter 2.1.2 --- Malaria mechanics --- p.4
Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4
Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5
Chapter 2.2.1 --- Antimalarial drugs --- p.5
Chapter 2.2.2 --- Drug resistance --- p.6
Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7
Chapter 2.3.1 --- Malarial vaccine developments --- p.7
Chapter 2.3.2 --- Transmission blocking vaccines --- p.7
Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9
Chapter 2.3.4 --- Blood stage vaccines --- p.10
Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11
Chapter 2.5 --- Plants as bioreactors --- p.12
Chapter 2.5.1 --- Products of transgenic plants --- p.13
Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14
Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15
Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15
Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16
Chapter 2.7.1 --- Arabidopsis --- p.16
Chapter 2.7.2 --- Tobacco --- p.17
Chapter 2.8 --- Transformation methods --- p.17
Chapter 2.8.1 --- Direct DNA uptake --- p.17
Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17
Chapter 2.8.1.2 --- Biolistic transformation --- p.18
Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18
Chapter 2.8.2.1 --- Leaf-disc technique --- p.18
Chapter 2.8.2.2 --- In planta transformation --- p.19
Chapter 2.9 --- Phaseolin --- p.20
Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21
Chapter 2.11 --- Aims of study and hypotheses --- p.22
Chapter Chapter 3: --- Materials and Methods --- p.24
Chapter 3.1 --- Introduction --- p.24
Chapter 3.2 --- Chemicals --- p.24
Chapter 3.3 --- Expression in tobacco system --- p.24
Chapter 3.3.1 --- Plant materials --- p.24
Chapter 3.3.2 --- Bacterial strains --- p.25
Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25
Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26
Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30
Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30
Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33
Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34
Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35
Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36
Chapter 3.3.8 --- GUS assay --- p.37
Chapter 3.3.9 --- Genomic DNA isolation --- p.37
Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38
Chapter 3.3.11 --- Southern blot analysis --- p.38
Chapter 3.3.12 --- Total seeds RNA isolation --- p.39
Chapter 3.3.13 --- RT-PCR --- p.39
Chapter 3.3.14 --- Northern blot analysis --- p.40
Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40
Chapter 3.3.16 --- Western blot analysis --- p.41
Chapter 3.4 --- Expression in Arabidopsis system --- p.42
Chapter 3.4.1 --- Plant materials --- p.42
Chapter 3.4.2 --- Bacterial strains --- p.42
Chapter 3.4.3 --- Chimeric gene construction --- p.42
Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43
Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45
Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45
Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45
Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49
Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49
Chapter 3.4.7 --- Arabidopsis Transformation --- p.49
Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50
Chapter 3.4.9 --- Selection of successful transformants --- p.51
Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51
Chapter 3.4.11 --- GUS assay --- p.52
Chapter 3.4.12 --- Genomic DNA isolation --- p.52
Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52
Chapter 3.4.14 --- Southern Blot analysis --- p.52
Chapter 3.4.15 --- Total siliques RNA isolation --- p.53
Chapter 3.4.16 --- RT-PCR --- p.53
Chapter 3.4.17 --- Northern blot analysis --- p.53
Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54
Chapter 3.4.18 --- Western blot analysis --- p.54
Chapter 3.5 --- In vitro transcription and translation --- p.54
Chapter 3.5.1 --- In vitro transcription --- p.54
Chapter 3.5.2 --- In vitro translation --- p.55
Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56
Chapter 3.6.1 --- Chimeric gene constructs --- p.56
Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61
Chapter Chapter 4: --- Results --- p.63
Chapter 4.1 --- Tobacco system --- p.63
Chapter 4.1.1 --- Chimeric gene constructs --- p.63
Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65
Chapter 4.1.3 --- GUS activity assay --- p.67
Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68
Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68
Chapter 4.1.4.2 --- Southern blot analysis --- p.70
Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72
Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72
Chapter 4.1.5.2 --- Northern blot analysis --- p.75
Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76
Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78
Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78
Chapter 4.1.7.2 --- Western blot analysis --- p.78
Chapter 4.2 --- Arabidopsis system --- p.83
Chapter 4.2.1 --- Chimeric gene constructs --- p.83
Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85
Chapter 4.2.3 --- Selection of transgenic plants --- p.87
Chapter 4.2.4 --- Assay of GUS activity --- p.91
Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92
Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92
Chapter 4.2.5.2 --- Southern blot analysis --- p.96
Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99
Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99
Chapter 4.2.6.2 --- Northern blot analysis --- p.106
Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107
Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108
Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108
Chapter 4.2.8.2 --- Western blot analysis --- p.108
Chapter 4.3 --- In vitro transcription and translation --- p.112
Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115
Chapter Chapter 5: --- Discussion and Future perspectives --- p.117
Chapter 5.1 --- Failure in detecting transgene expression --- p.117
Chapter 5.2 --- Poor transgene expression --- p.120
Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120
Chapter 5.2.2 --- AT-richness --- p.121
Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123
Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125
Chapter 5.2.5 --- Codon usage --- p.126
Chapter 5.3 --- Future perspectives --- p.127
Chapter Chapter 6: --- Conclusion --- p.129
References --- p.131

Malaria affects about forty percent of the world’s population. Annually more than 1.5 million fatalities due to malaria occur and parasite resistance to existing antimalarial drugs such as mefloquine has already reached disturbingly high levels in South-East Asia and on the African continent. Consequently, there is a dire need for new drugs or formulations in the prophylaxis and treatment of malaria. Artesunate, an artemisinin derivative, represents a new category of antimalarials that is effective against drug-resistant Plasmodium falciparum strains and is of significance in the current antimalarial campaign. As formulating an ACT double fixed-dose combination is technically difficult, it is essential that fixed-dose combinations are shown to have satisfactory ingredient compatibility, stability, and dissolution rates similar to the separate oral dosage forms. Since the general deployment of a combination of artesunate and mefloquine in 1994, the cure rate increased again to almost 100% from 1998 onwards, and there has been a sustained decline in the incidence of Plasmodium falciparum malaria in the experimental studies (Nosten et al., 2000:297; WHO, 2010:17). However, the successful formulation of a solid oral dosage form and fixed dosage combination of artesunate and mefloquine remains both a market opportunity and a challenge. Artesunate and mefloquine both exhibited poor flow properties. Furthermore, different elimination half-lives, treatment dosages as well as solubility properties of artesunate and mefloquine required different formulation approaches. To substantiate the FDA’s pharmaceutical quality by design concept, the double fixed-dose combination of artesunate and mefloquine required strict preliminary formulation considerations regarding compatibility between excipients and between the APIs. Materials and process methods were only considered if theoretically and experimentally proved safe. Infrared absorption spectroscopy (IR) and X-ray powder diffraction (XRPD) data proved compatibility between ingredients and stability during the complete manufacturing process by a peak by peak correlation. Scanning Electron Micrographs (SEM) provided explanations for the inferior flow properties exhibited by the investigated APIs. Particle size analysis and SEM micrographs confirmed that the larger, rounder and more consistently sized particles of the granulated APIs contributed to improved flow under the specified testing conditions. A compressible mixture containing 615 mg of the APIs in accordance with the WHO recommendation of 25 mg/kg of mefloquine taken in two or three divided dosages, and 4 mg/kg/day for 3 days of artesunate for uncomplicated falciparum malaria was developed. Mini-tablets of artesunate and mefloquine were compressed separately and successfully with the required therapeutic dosages and complied with pharmacopoeial standards. Preformulation studies eventually led to a formula for a double fixed-dose combination and with the specific aim of delaying the release of artesunate due to its short half-life. A factorial design revealed the predominant factors contributing to the successful wet granulation of artesunate and mefloquine. A fractional factorial design identified the optimum factors and factor levels. The application of the granulation fluid (20% w/w) proved to be sufficient by a spraying method for both artesunate and mefloquine. A compatible acrylic polymer and coating agent for artesunate, Eudragit® L100 was employed to delay the release of approximately half of the artesunate dose from the double fixed-dose combination tablet until a pH of 6.8. A compressible mixture was identified and formulated to contain 200 mg of artesunate and 415 mg of mefloquine per tablet. The physical properties of the tablets complied with BP standards. An HPLC method from available literature was adapted and validated for analytical procedures. Dissolution studies according to a USP method were conducted to verify and quantify the release of the APIs in the double fixed-dose combination. The initial dissolution rate (DRi) of artesunate and mefloquine in the acidic dissolution medium was rapid as required. The enteric coated fraction of the artesunate exhibited no release in an acidic environment after 2 hours, but rapid release in a medium with a pH of 6.8. The structure of the granulated particles of mefloquine may have contributed to its first order release profile in the dissolution mediums. A linear correlation was present between the rate of mefloquine release and the percentage of mefloquine dissolved (R2 = 0.9484). Additionally, a linear relationship was found between the logarithm of the percentage mefloquine remaining against time (R2 = 0.9908). First order drug release is the dominant release profile found in the pharmaceutical industry today and is coherent with the kinetics of release obtained for mefloquine. A concept pre-clinical phase, double fixed-dose combination solid oral dosage form for artesunate and mefloquine was developed. The double fixed-dose combination was designed in accordance with the WHO’s recommendation for an oral dosage regimen of artesunate and mefloquine for the treatment of uncomplicated falciparum malaria. The specifications of the double fixed-dose combination were developed in close accordance with the FDA’s quality by design concept and WHO recommendations. An HPLC analytical procedure was developed to verify the presence of artesunate and mefloquine. The dissolution profiles of artesunate and mefloquine were investigated during the dissolution studies.
PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

While the molecular defect that cause sickle cell disease has well been established, the cause of vaso-occlusive crisis remains elusive and largely debated upon. Majority of studies have linked the painful episodes to polymerization of sickle hemoglobin following its deoxygenation. The variability of the disease symptoms among patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator of Hb F induction and a widely used treatment, has ameliorated the complication of SCD but it is only effective in 50% of the patients. Expression of Hb F lowers the content of Hb S in blood and hence reduces oxidative stress caused by Hb S denaturation. Sickle cell disease severity depends on several factors. Most importantly, the ability of red cell to sickle dominates all other determinants. While deoxygenation of sickle hemoglobin may be inevitable, the duration with which the red cell remains in the deoxygenated state can be manipulated. Deoxygenation is a transient process that when compared to the time taken to develop the long filaments of deoxyhemoglobin to causes severe sickling, the red cell would have been cycled back to the lungs and re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle cells would flow as fast as healthy erythrocytes, the detrimental impacts of sickling such as vaso-occlusive crisis, would not be a concern for this disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation through auto-oxidation, which imposes oxidative stress to the cells. The oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which subsequently impair their constitutive action against the tyrosine kinases. In the end, a net tyrosine phosphorylation state in the red cell membrane proteins, most notably the transmembrane protein band 3, succeeds. Band 3 tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage to the membrane thus engendering membrane destabilization. A destabilized erythrocyte sheds membrane fragments in form of microvesicles/microparticles and discharges free hemoglobin into the extra cellular matrix. In consequence, the microparticles power initiation of coagulation cascade through activation of thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is necessary for vasodilation and induces further oxidative stress within the microvasculature, and activates expression of adhesion receptors on the endothelium. Taken together, these events culminate in entrapment of red cells (not naming leucocytes and platelets) in the microvasculature, blockade of blood vessels and further damage of erythrocytes through prolonged deoxygenated state thus terminating in tissue injury, strokes, and organ damage, amid vaso-occlusive episodes which always require hospitalization and extensive medical care for survival. Band 3 tyrosine phosphorylation and membrane weakening is not unique just to SCD, but also a druggable target for malaria. Malaria, a disease that is touted as the evolutionary cause of sickle cell disease, surprisingly thrives through the same mechanism. Briefly, malaria parasite consumes hemoglobin for its DNA synthesis, and in the process generate reactive oxygen species from denatured hemoglobin that feeds into the oxidative stress which triggers band 3 tyrosine phosphorylation. In this case however, a destabilized membrane offers perfect conditions for merozoites’ (malaria daughter parasites) egress/exit out of the cell to begin infecting other red cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s death. Treatment of diseased cells with imatinib and other Syk inhibitors effectively reversed membrane weakening. A stabilized membrane not only survives longer in circulation to alleviate SCD symptoms but also traps and starves malaria parasite leading to termination of the parasitic infection. With band 3 tyrosine phosphorylation at center stage, this dissertation explores the above events in an effort to unveil a novel therapy for sickle cell and malaria diseases. First, the therapeutic strategy regarding SCD is discussed in detail beginning with non-transfused patients and ending in additional mechanistic study on inactivation of the principal erythrocyte’s protein tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial proof of concept on efficacy of imatinib in treatment of malaria as a monotherapy and its efficacy when used in a triple combination therapy with the standard of care treatment. Finally, I outline an alternative possible mechanism of action of quinine against malaria.