Mechanism of Action Studies of Phenotypic Whole-cell Active Antimalarial Leads Through Target Identification

Chemotherapy has remained the backbone of malaria control and prevention. Over the past century, potent antimalarial drugs with different mechanisms of action have been successfully developed and used to treat malaria. However, the ability of the most virulent species, P. falciparum, to resist these available antimalarial chemotypes and compromise their potency has raised the importance of using combination therapies and developing new, safe, and efficacious molecules with novel modes of action for the treatment of malaria. Phenotypic whole-cell screening, followed by medicinal chemistry optimization efforts, identified the pyrido[1,2-a]benzimidazole compounds KP68 and KP124 and the benzimidazole compound DM253 as efficacious antimalarial leads. However, the essential details of their mechanism of action against P. falciparum remain unresolved. This thesis employs ‘omics-based techniques with support from fluorescence live-cell imaging, compound docking, and heme fractionation studies to generate insights into the action of these compounds against P. falciparum. The previous mechanism of action studies on these antimalarial chemotypes has focused mainly on the inhibition of hemozoin biocrystallization in the acidic digestive vacuole of the parasite. However, the intrinsic fluorescence properties of KP68 and KP124 were used to comprehensively study the subcellular accumulation of these compounds in an infected erythrocyte. Using the inherent fluorescence properties of these compounds is advantageous because accurate localization due to the compounds is observed with no KP68 PfNF54 IC50 = 0.03 µM PfK1 IC50 = 0.04 µM in vivo P. berghei (p.o) 4x50 mg/kg = 98.0% 3/3 malaria infected mice cured KP124 PfNF54 IC50 = 0.14 µM PfK1 IC50 = 0.13 µM DM253 PfNF54 IC50 = 0.012 µM PfK1 IC50 = 0.040 µm in vivo P. berghei (p.o) 4x50 mg/kg = 99.52 Mean survival days = 14 days influence from an external fluorophore. On the other hand, DM253 required the attachment of an external fluorophore for live-cell imaging. As such, a novel fluorescent derivative was designed and synthesized with guidance from extensive structure-activity relationship studies previously conducted in this series. 7-Nitrobenz-2-oxa-1,3-diazole (NBD) was identified as an appropriate external fluorophore and was attached to the compounds investigated. The spacer chain length between the compounds and the fluorophore was varied to find suitable fluorescent derivatives that would appropriately represent the parent compounds. The photophysical and physicochemical properties of all fluorescent compounds were evaluated. Although the fluorescent derivatives lost antiplasmodium potency relative to their parent compounds, all but NBD-labelled KP124 retained antiplasmodium activity in the chloroquine-sensitive strain of P. falciparum. Furthermore, a detergent-mediated assay indicated that all fluorescently labelled derivatives retained activity against βhematin formation compared to the parent molecules. These results suggest that except for KP124-NBD, all fluorescent compounds and the fluorescent analogues were suitable for live-cell fluorescence accumulation studies. Live-cell imaging showed selective accumulation of all fluorescent compounds within P. falciparum-infected red blood cells. Different accumulation patterns were observed when using the inherent fluorescence of the structurally related KP68 and KP124. KP124 was observed to colocalize in the parasite's digestive vacuole and associate with hemozoin crystals, whiles KP68 which differs from KP124 by the replacement of the imidazole[1,2- a:4,5-b′]dipyridine core with the benzimidazole core, as well as the presence of chloro substituents, showed no accumulation in the parasite's digestive vacuole. Quantitative colocalization studies of parasite cells co-stained with KP124, DM253-NBD, and LysoTracker Red demonstrated an excellent colocalization between these signals. This indicates a preference for these compounds in the parasite's acidic compartment. Furthermore, the quantitative analysis also revealed that none of the compounds localized in the nucleus, eliminating the nucleus as a site of action for these compounds. To mitigate the limitations of resolution, Airyscan and super-resolution structured-illumination microscopy (SR-SIM) were employed. Fluorescence imaging using the ER-Tracker Red revealed a broad colocalization between KP124 and DM253-NBD and the tracker dye, suggesting that both compounds accumulate in the endoplasmic reticulum (ER). However, no significant amounts of KP68 were localized in the ER. The mitochondrion was, however, implicated in the action of KP68. Although colocalization was not observed between the MitoTracker Deep Red and KP68, significant amounts of the compound localize around the mitochondrion membrane. Finally, all compounds were assessed in the cellular heme fractionation assay. Results from this assay indicate that inhibition of hemozoin formation is a mechanism of action for KP124 but not for KP68 and DM253. The recent growth in genomics and genetics has provided powerful tools for mode of action studies. In vitro resistance selections represent one of the genomics tools for target deconvolution of hit and lead molecules. The mechanism of resistance of the pyrido[1,2- a]benzimidazoles was investigated through resistance selection. Whole-genome sequencing of the mutant clones generated from KP68 under drug pressure showed a single nucleotide polymorphism in the mitochondrion carrier protein. It also revealed a few copy number variations, including the deamplification of the mitochondrialprocessing peptidase and the P. falciparum multidrug resistance transporter PfMDR1.This result, coupled with the significant amounts of KP68 observed to accumulate around the parasite's mitochondrion, confirms the mitochondrion as an organelle of interest in the compound's mode of action in P. falciparum. Furthermore, no cross-resistance was observed between KP68 and chloroquine, suggesting that both compounds may act through different resistance mechanisms and possibly different mechanisms of action. Similarly, no cross-resistance was observed between the mutant clones generated for KP68 and KP124, meaning that the parasite's mode of resistance and the action of both compounds may be mediated through different mechanisms. This also confirms the livecell imaging and heme fractionation assay results, which support hemozoin inhibition as a mode of action for KP124 but not KP68. Finally, chemical proteomics was employed to identify the protein binding partners of these antimalarial compounds in P. falciparum. Here, drug-labelled matrices were used to capture protein binding partners of KP68 and KP124 from P. falciparum cell lysates. Several protein binding partners specific to these compounds were detected from the parasite lysate prepared and identified by mass spectrometry and proteomic analysis. Out of the many proteins identified as protein binding partners for KP68, the high molecular weight EMP1-trafficking protein, PfEMP1 is of significant interest. This is because it is essential for the parasite's survival and has been implicated in the action of other antimalarials such as dihydroartemisinin. These results suggest that these compounds may impact different parasite pathways and processes. Besides hemozoin formation, KP124 has also been implicated in interfering with the parasite's protein synthesis. Overall, this work has developed new tools that have aided in understanding the mechanistic details of these compounds. The observations described here, and further studies using the techniques and approaches to target deconvolution discussed here may facilitate the identification of novel targets for treating malaria. Also, once chemically validated, the protein targets identified in this work can serve as suitable starting points for target-based antimalarial drug discovery efforts

Anti-Bribery Legislation in the United States and United Kingdom: A Comparative Analysis of Scope and Sentencing

Lawmakers and prosecutors continue to take aim at a major subset of global corruption - corporate bribery of foreign government officials. Specifically, while the enforcement of the Foreign Corrupt Practices Act in the United States has risen to new records, the United Kingdom has revolutionized its anti-bribery law following global criticism of its previously relaxed legal regime. Both U.S. and U.K. anti-bribery laws, furthermore, apply extraterritorially and have the capability to entangle even the largest multinational companies in their legal frameworks. These all-encompassing frameworks hold significant consequences for both corporations and their employees, but the increasing power of anti-bribery law raises important questions regarding the proper scope of legislation on the subject, as well as the sentencing approaches to these crimes

In the Voice of the Chief: The Role of the ‘Okyeame’ in Protecting the Gates of Traditional Royal Communication

The study set out to explore the rhetoric language of the Okyeame and the part it played as a gatekeeping strategy in Akan royal communication in the traditional court. As the art of using skill in language writing and speaking to persuade and influence, Rhetoric takes its source from a wide selection of human discourse. The research revealed that the Okyeame employs the use of certain linguistics arts such as proverbs, metaphors and symbols in communication which serve as a means of polishing and summarizing messages to and from the chief in the traditional court. In his desire to keep the royal gates of communication, the Okyeame exercises his skill in language use for the chief and audiences gathered at the traditional court.  It was revealed that the Okyeame’s use of rhetoric language enhances brevity and precision in Akan royal communication in the performance of his duty as the Okyeame. Keywords: Rhetoric; Okyeame; gatekeeping; proverbs; metaphors DOI: 10.7176/NMMC/91-03 Publication date:June 30th 202

Literary Analysis of ‘Kpaa’ Songs. A Didactic Legacy of Oral Performance. A Case Study of the People of Teshie, Accra, Ghana

The unwritten literature of Africa is usually viewed by many as unrefined. The study focuses on an oral form of literature of  Teshie among the Ga people in Ghana. The study dwelt on the literary analysis of ‘Kpaa[1]’ Songs which is performed during ‘Kpaashimo[2]’. It throws light on how it can be merged into the context of literature by revealing the use of poetic devices such as proverbs, rhetorical questions, metaphor, personification, imagery, repetitions, allusions, alliteration and assonance. The study further reveals the immeasurable wisdom and knowledge found in ‘Kpaa’ songs as well as its importance in terms of teaching the culture of the people of the Ga in Ghana. It further reveals the didactic qualities of the songs. The focus of the study is on the performance and the songs that are used. The songs were recorded and analysed as poetry. Keywords: Ga, Kpaa, Kpaashimo, Didactic, Performance [1] A type of song performed by the Ga in the Greater Accra Region of Ghana [2] The performance of Kpaa songs is termed as Kpaashim

Стартап-проект школи моди як ефективний напрям розвитку економіки Гани

У даній статі розглянуто розвиток ринку моди у Гані і сформульована пропозиція заснування стартап-проекту школи моди як ефективний напрямок економічного розвитку Гани. Показано, що економіка Гани пройшла скрізь період значних змін, викликаних різноманітними факторами, включаючи інноваційні технології і технічний розвиток, розвиток міжнародної торгівлі, глобальної конкуренції та конкурентною перевагою Гани, що інтегрується у глобальну економіку. Було висвітлено, у Гані дизайнери модного одягу усвідомлюють, що старі правила більше не працюють на ринку моди Гани так само, як це вже сталося у інших напрямках діяльності людей. Були продемонстровані окремі ринкові можливості та ринкові проблеми локальних дизайнерів модного одягу. З одного боку, у статті підкреслюється, що талановиті модні дизайнери не мають можливості просування власних дизайнерських робіт за кордоном, а також не мають відповідної освіти для цього. З іншого боку, у статті показується, що ринок модного одягу потребує нових маркетингових інструментів для впровадження, що обумовлює зростання попиту на спеціалізовану освіту з маркетингу, брендінгу модного одягу, а також ребрендінгу для завойовування нових поколінь споживачів. Таким чином, підприємцям запропонована бізнес-ідея зі заснування стартап-проекту у вигляді школи моди в Гані. Цей стартап-проект представлений як результат практичного впровадження міждисциплінарного підходу до створення та управління інноваційною діяльністю в умовах «режимів із загостреннями» в інформаційному суспільстві, при яких усі процеси в економіці і звичайному житті людей прискорюються за законом експоненти. Це вимагає оперативного реагування підприємців і інвесторів на зміни у глобальному маркетинговому середовищі. Аналіз результатів маркетингових досліджень щодо самооцінки студентів шкіл моди в Гані представлено у статті у якості ефективного напрямку побудови набору конкурентних переваг для нової школи моди. Була запропонована інноваційна можливість просування навчальних робіт студентів за межами Гани.In this article the development of the fashion market in Ghana was investigated. It is shown that the Ghanaian economy is going through a period of significant changes driven by a number of factors, including new and emerging technology; international trade and finance; the global competition and Ghana competitive advantage integrated into the global economy; the innovation and technical progress. It was highlighted that in Ghana fashion designers realized that the old rules didn’t work on the fashion market any more like it had already happened in most other fields of people activity. Some market opportunities and market problems for local fashion designers were shown. On the one hand it is highlighted in the article that talented fashion designers don’t have an ability to promote their works abroad and don’t have relevant education for this. On the other hand it is shown that the fashion market needs new marketing tools to be implemented that also ensures rising the demand on special education on marketing, promotion, branding fashion clothes and rebranding to win new generations of consumers Therefore a business idea of setting up a startup-project of a fashion school in Ghana is proposed for entrepreneurs. This startup-project is presented as a practical implementation result of the interdisciplinary approach to creating and managing an innovative activity under the ‘blow-up regimes’ in the information society on the basis of the four-dimensional thinking. These ‘blow-up regimes’ assume that all processes are accelerating economics and people life in accordance with the exponential law. It makes entrepreneurs and investors react address a challenge of global marketing environment rapidly. The analysis of marketing researches results of self-evaluation by students, conducted in the fashion schools in Ghana, is represented in the article as an effective order for building up a set of competitive advantages of the new fashion school. The innovative opportunity of promotion training works of the students abroad is proposed

Body Marking As Identification among the Dangmes in Ghana

Body marking is an intangible and a sensitive cultural variable in Ghana. Unfortunately, most individuals do not want to associate themselves with people who have body marks. This obvious misunderstanding of the art has led to stigmatization of the bearers of these marks. Body markings have been used as a form of identification among many cultures through several generations, Ghana has a rich culture of body marking among the various ethnic groups practice it, yet; the purpose and connotations of these marks have not been uncovered in certain parts of the country. As the study is centered on identification, it will help recognize the cultural identity of the nation by comprehending and embracing the custom of the various ethnic groups of Ghana where the need be. This research is therefore geared towards understanding the purposes and reasons for making body marks. Symbolic and interpretative anthropology and diffusionism were the theories that guided the study and are explained as such. Three ethnic groups from Ga-Dangme were selected for the study and these are Ningo, Prampram and Ada. The qualitative research method was used, with interviews and observations being the main form of data collection instruments. Out of the overall accessible population of 90 respondents, a sample size of 60 was purposively selected based on their knowledge on the subject matter. The results of the study showed that there are different body marks that are made for different cultural reasons. Keywords: Body marks, identification, scarification, cultural interpretatio

The effect of reducing the direct cost of care on health service utilization and health outcomes in Ghana : a randomized controlled trial

Aim: To improve malaria control in children less than five years of age. Objectives: To assess the impact of reducing the direct cost of health care on utilization of health services and morbidity associated with malaria among children under 5 years old. Primary objective To compare the prevalence of anaemia (Hb<8g/dl) among children six to fifty-nine months of age, from households with and without improved financial access. Secondary objectives included: To compare the health service utilization rate among children from households with or without improved financial access; to compare household all-cause mortality between the trial arms, to examine primary and major secondary outcomes by socio-economic status of households, to compare outcomes among children from self-enrolled and trial-enrolled households; and to document community knowledge and attitudes on user fees. Methods: 2332 households containing 2757 children less than 5 years of age in Dangme West, Ghana were randomised either to be enrolled into a pre-payment scheme operating allowing free access to primary care, including drugs or to a control which paid user fees for their health care as pertained normally. These included 138 households, comprising 165 children, who had enrolled in the pre-payment scheme prior to the closure of the registration window were included in the study as an observational arm. 2 Results: 2194 households comprising 2592 children were allocated randomly into control and intervention groups. 138 households, comprising 165 children had themselves enrolled in the pre-payment scheme. At baseline, the two randomised groups were the same, but the group who self-enrolled were significantly less poor and had better health outcome measures. Introducing free primary healthcare significantly altered the healthcare seeking behaviour of households, with those randomly allocated to the intervention arm using formal healthcare more (95% Cl 1.04-1.20; p=O.OOI) and home treatment and chemical sellers less than those in the control group. This did not, however, lead to any measurable difference in any of the health outcomes at the end of the six-month trial period. For the primary outcome of moderate anaemia there was no difference seen OR 1.05 (0.66-1.67). Conclusions: This study suggests that reducing the direct cost of health care has a significant impact on healthcare-seeking behaviour but cannot, on its own, be assumed to have an impact on health outcomeEThOS - Electronic Theses Online ServiceGBUnited Kingdo

Social media in Ghana's legacy media industry: Collecting "likes" or building a new revenue source?

Thesis submitted to the Department of Business Administration, Ashesi University College, in partial fulfillment of Bachelor of Science degree in Management Information Systems, April 2014Today, Ghana has over fourteen percent of its population using the internet. Social Media is one of the main platforms that these users interact with online. This has presented an opportunity for companies to gain business benefits by reaching their clients online. For others, it has provided a means to reach new audience. Social media has greatly affected the legacy media industry, especially regarding content delivery and creation. This has caused legacy media companies to look into the feedback and interactions of audience on the social media sites of their brands, in order to gain insights to improve their core business. This thesis presents a study into how legacy media brands in Ghana are harnessing social data from social media and how these actions can be improved to achieve performance goals.Ashesi University Colleg