Dissertations / Theses on the topic 'Therapeutics, pharmacological'

Malaria remains a major public health concern for billions of people worldwide. Achieving the ambitious goal of malaria eradication requires co-ordination of control strategies dealing with a range of parasite, vector, human, social and environmental factors. Availability of effective antimalarial treatment is a key component in malaria control. However the number of drugs available is limited and drug resistance, particularly in Plasmodium falciparum, has now been reported for all currently available antimalarials. Mathematical models provide the opportunity to explore key features underlying antimalarial drug action, effectiveness and resistance. They further allow investigation into questions that cannot otherwise be easily addressed, either because they are too expensive, unethical or logistically too complex. This thesis aims to develop pharmacological models to investigate antimalarial drug treatment. In Chapter 2 we develop a pharmacokinetic-pharmacodynamic (PK/PD) model of antimalarial drug treatment (calibrated using published data) and use it to investigate the efficacy of artemisinin combination therapies (ACTs). Chapter 3 addresses two assumptions built into the methodology that limit the models future application. The model now allows for (i) time lags and drug concentration profiles for drugs absorbed across the gut wall and, if necessary, converted to another active form (ii) multiple drugs within a treatment regimen (iii) differing modes of drug action in combinations (iv) modelling drugs converted to an active metabolite with similar modes of action. In Chapter 4 we extend the methodology to allow for i) the presence of more than one clone when treatment begins (ii) the acquisition of new clones during treatment follow-up (iii) the tracking of individual clones using molecular markers. We then use these extensions to simulate clinical trial data to determine the best methods of analysis. Chapter 5 details how the drug action components of the extended PK/PD model were incorporated into OpenMalaria; a mathematical model of malaria epidemiology allowing investigation of the effects of various intervention strategies including malaria vaccines, vector control strategies and antimalarial drug treatment. In Chapter 6 we investigate the ability of clinical trials to accurately estimate (WoS) using the extended PK/PD model. Windows of selection (WoS) are often used to quantify the genetic process whereby parasites evolve increasing tolerance to antimalarial drugs. We noted a conspicuous lack of comprehensive, good-quality PK datasets currently available in the literature. Despite this, the models produced results highly consistent with field data. They were applied to investigate the potential implications of drug resistance and to make predications about the future effectiveness of antimalarials. We emphasise the value of mathematical models by simulating ‘field data’ to assess the best methods of analysing clinical trials and to investigate the predictive ability of WoS. While we do not suggest models can replace the information gained in clinical trials, this work does demonstrate the importance of mathematical models capable of generating results consistent with field data.

Molecular and Cellular Physiology
Ph.D.
The field of cancer therapeutic development has been dominated by two research and discovery paradigms, the cytotoxicity-based or phenotype driven strategy and the target-based rational approach. This thesis describes the standardization of novel assays used in both approaches and the discoveries made using these processes. Rational drug design or the target-based approach to discovering novel anti-cancer agents requires a basic understanding of the oncogenic signals that induce uncontrolled cellular proliferation. c-MET is a proto-oncogene, linked to a number of different cancers, that encodes a receptor tyrosine kinase. As an oncogene, c-MET has been shown to transform cells in the laboratory setting and is dysregulated in number of malignancies. Thus, we sought to discover a small molecule inhibitor of c-MET kinase activity by screening a novel library of small molecules. In the second part of this dissertation, we describe the standardization of a high-throughput assay to identify putative c-MET inhibitors and the results of our screening attempt. Cytotoxicity-based screening is another validated approach that is used to discover anti-cancer agents. As a parallel program to our c-MET discovery effort, we designed a high-throughput cytotoxicity assay to identify a novel small molecule with high cytotoxic activity towards tumor cells. The result of this screen was the identification of ON015640, a novel anti-cancer therapeutic with tubulin-depolymerizing activity. Throughout the course of this project, we tried to discern the advantages and disadvantages of the two predominant paradigms in cancer therapeutic research. Both strategies require careful assay design and an acute understanding of the molecular and genetic underpinnings of cancer. While it is clear that structure-based rational drug design has its merits and its success stories, it has become increasingly clear that seeking out a desired biological effect may serve as a more effective staring point when dealing with cancers for which no clear oncogene addiction phenotype has been observed.
Temple University--Theses

Nrf2 is a transcription factor that, under conditions of chemical stress, is able to evade its cytosolic repression and translocate to the nucleus to initiate the transcription of a battery of cytoprotective genes, such as those involved in the detoxication of xenobiotics. Nrf2 has previously been shown to afford protection against chronic and acute renal injury, yet, relatively little is known about the mechanism by which Nrf2 affords this protection, and the extent of its transcriptional roles in the kidney. This thesis seeks to further our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney. Using an iTRAQ-based proteomic approach to quantify protein expression levels in the kidneys of Nrf2+/+ and Nrf2-/- mice, acutely treated with vehicle or the potent Nrf2 inducer CDDO-Me (3 mg/kg), we demonstrated that 189 proteins were differentially expressed in the Nrf2-/- mouse kidney, compared to Nrf2+/+, and 42 proteins were differentially expressed in the CDDO-Me treated Nrf2+/+ mouse kidney, compared to vehicle. The key finding was that the kidneys of Nrf2-/- mice are deficient in proteins that mediate cellular redox balance, the metabolism of a range of xenobiotics, and the regulation of core metabolic processes, including energy metabolism and the synthesis and recycling of amino acids. Functional demonstration of a reduction in energy metabolism was demonstrated by assessing total NADPH and GSH, of which Nrf2-/- mouse kidneys had 35% and 30% less than their Nrf2+/+ counterparts, respectively. A single acute dose of CDDO-Me failed to augment the expression of proteins, other than Nqo1, that were shown to be regulated by Nrf2 at the basal level in the mouse kidney, however qPCR analysis of these kidneys revealed that CDDO-Me has an effect at the transcriptional level which has not fully translated within the timeframe of this study. In summary, we have provided evidence that Nrf2 regulates the expression of an array of proteins that contribute to cell defence and the maintenance of homeostasis in the kidney, supporting current interest in Nrf2 as a novel therapeutic target in a number of renal diseases. MicroRNAs are a recently discovered RNA-regulatory element that show promise in their use as biomarkers of physiological and pathological events. In order to provide insight into the microRNAs under Nrf2 control in the kidney, we performed an unbiased microRNA array analysis on kidney homogenates from Nrf2+/+ and Nrf2-/- mice, treated with vehicle or CDDO-Me, and then validated several promising microRNA candidates using targeted qPCR analysis. Of particular note are miR-466h-3p, the expression of which was significantly increased in the CDDO-Me treated Nrf2+/+ mouse kidney and decreased in the Nrf2-/- mouse kidney, compared to their respective controls, and miR-28c and 144, which were both significantly decreased in the CDDO-Me treated Nrf2+/+ mouse kidney, and increased in the Nrf2-/- mouse kidney. This novel analysis represents the first step in characterising the renal Nrf2 microRNA-ome, which could reveal novel mechanisms of Nrf2 function and markers of its activity that could translate to the clinic. Recent interest in the use of CDDO-Me as a therapeutic intervention for late-stage chronic kidney disease has culminated in a phase III clinical trial (BEACON), which was subsequently terminated due to unforeseen adverse cardiac events, of which the cause has yet to be identified. In order to determine whether the drive to produce more potent Nrf2 inducers has inadvertently led to the generation of inherently more toxic compounds, the relationship between potency towards Nrf2 and toxicity was evaluated for CDDO-Me and related triterpenoids, and other classes of Nrf2 inducer. Using a rat H4IIE-ARE8L luciferase reporter cell line to determine in vitro therapeutic indices, it was discovered that within the compounds tested an increase in potency toward Nrf2 of four magnitudes results was associated with an increase in toxicity of only two magnitudes, resulting in a relative increase in in vitro safety. This data indicates that it is possible to generate potent Nrf2-inducers that are not inherently toxic, and suggests that therapeutic targeting of Nrf2 continues to hold promise as a novel treatment for a range of diseases. In summary, by using a proteomic approach we have identified an array of renal Nrf2-regulated proteins that contribute to various cytoprotective and metabolic processes in the kidney, supporting current interest in the therapeutic targeting of Nrf2 as treatment for renal disease. Additionally, the microRNAs under Nrf2 regulation in the kidney have also been identified, and represent the first step in fully characterising the Nrf2 microRNA-ome. Finally, it was shown that the drive to produce more potent Nrf2 inducers has not led to the generation of inherently more toxic compounds; indeed an increase in potency is associated with a relative increase in in vitro safety, suggesting that the targeting of Nrf2 is still a promising therapeutic route. Overall, the work presented in this thesis has furthered our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney.

Corn Hageman factor inhibitor (CHFI) is a bifunctional serine protease / α-amylase inhibitor protein having 127 residues and a molecular weight of 13.6-kDa. CHFI is selective toward FXIIa without affecting the function of the other coagulation factors. Coagulation FXII is a serine protease recognized to cause kinin generation and blood coagulation, cleaving plasma kallikrein and FXI. Results from FXII-deficient animal models proposed that this protein contributes to stable thrombosis that can cause obstruction of the blood vessels and its subsequent complications such as ischemic stroke. In contrast to other blood coagulation factors, deficiency in FXII is not related with haemorrhage in patients or in animals. These findings propose that specific inhibition of FXII could be an attractive medicine and a new method of anticoagulation to treat or prevent pathological thrombosis that could have a lower risk for bleeding and a safer anticoagulation profile than the currently available anticoagulants. Therefore, the current PhD project aimed at pharmacological investigation into CHFI-FXII interaction and FXII function at molecular level through the following objectives: first, developing an efficient expression and purification system for generating soluble and functional recombinant native type CHFI and establishing an inhibitory activity assay against FXIIa to verify the proper function of the recombinant protein. The second objective was testing the different recombinant variants of CHFI with the desired point mutations guided by a proper prediction study of CHFI-FXII interaction. The third was to investigate into the hypothesis of the tight-binding property of CHFI via different approaches of enzyme inhibition mechanisms and kinetic data analysis. The last objective was to investigate into the function of FXII by examining theeffect of Cys466 and glycosylated peptide remnant from the proline-rich region on the function of the catalytic domain via characterizing the different recombinant variants of the catalytic domain of FXIIa, FXIIc, FXIIac, HISTF-β FXII, and MBP-β-FXIIa. In the current study, an efficient system for soluble expression, single step purification and proper storage of functional, wild type rHIS-GST-CHFI was, for the first time, identified. The fully functional recombinant protein was verified via developing an inhibition test against FXIIa. The established expression, purification and inhibition assays were used as a fundamental guide to both generating and characterizing mutant proteins of interest that were made on the basis of an appropriate docking model of CHFI-FXIIa interaction. For the first time, the current investigation into the question of specificity of CHFI against FXII revealed that the central Arg34 at the very top of the fully exposed region of CHFI inhibition loop play a central role in the inhibition function of CHFI toward FXIIa. In addition, this study identified Trp22 at the N-terminus and Arg43 at the C-terminus of the central inhibition loop as two key interaction residues with FXIIa. It was also observed that, in the preinhibition test, CHFI behaves as a noncompetitive inhibitor. In contrast, it acts as a competitive inhibitor in the acute inhibition test, proposing that CHFI is a competitive inhibitor with slow degree of reversibility due to tightness of binding. Reversibility assay showed that CHFI is an inhibitor with slow degree of dissociation. The tight-binding property of CHFI could be due to a non-active site interaction and or numerous hydrogen bonds between the III key interaction residues and their potential targets on FXIIa. With respect to the investigation into FXII function, It was observed that both Cys340-Cys466 and glycosylated peptide fragment of the proline-rich region have a functional role for the full catalytic activity of FXII protease domain. Cumulatively, the current study identified the key important residues on the exposed surface of CHFI and their potential target residues on the surface of FXIIa that would be highly informative and important factors helping to understand the mechanism of selective and tight binding interaction of CHFI with FXIIa. This project can be considered as an early, necessary approach to design novel, specific and safe anticoagulants for the treatment of thrombosis and its complications.

Chronic cough is defined as a cough that lasts for greater than 8 weeks in duration and has been estimated to have a prevalence of 11-13% of the population. Limited research has been conducted exploring its impact on the wider community. In up to 42% of chronic cough cases, the cough persists despite medical management, these cases tend to be labelled as refractory chronic cough. Pharmacological treatments are limited often with undesirable side effects. Research into non-pharmacological treatments for refractory chronic cough has been limited. An internet based European survey explored the impact of chronic cough (January 2012 - April 2013). A systematic review investigated the effectiveness of non-pharmacological interventions for refractory chronic cough. A single blinded multi-centre randomised controlled trial (RCT) investigated the efficacy of a non-pharmacological intervention (Physiotherapy, Speech and Language Therapy Interventions, - PSALTI) on cough related quality of life (QoL), cough frequency, severity, sensitivity, vocal performance, anxiety and depression alongside a control intervention. In total 1120 responses were collected and analysed from the European survey. Findings identified that cough impacted upon QoL, mood and ability to undertake activities and limited/ no effectiveness of medication; also a wish for more patient information to be available. PSALTI trial showed statistically significant differences between groups for the outcomes; QoL, cough frequency and urge to cough, improvements were significantly greater in the PSALTI group compared with control. There were no significant changes in outcomes from 4 weeks to 3 months suggesting that observed improvements were maintained. This thesis has identified the impact of chronic cough in Europe. It identified the need to improve the management of chronic cough and the information available for patients. This thesis also provides the first evidence within a single blinded multi-centre RCT that PSALTI is an effective treatment option for people with refractory chronic cough.

Muscle tension recordings were made from earthworm body wall muscle and the action of acetylcholine, cholinomimtics and cholinolytics examined. The effect of cholinergic agents on muscle twitches induced following field stimulation was also investigated. Carbachol was 8 times more active than acetylcholine in the presence of physostigmine. α-Bungarotoxin, d-tubocurarine, gallamine, atropine, mecamylamine and hexamethonium reduced both electrical stimulation of the muscle and the acetylcholine response while β-bungarotoxin reduced the electrically induced twitch but enhanced the acetylcholine contraction. 0.4wM Hemicholinium abolished the electrically induced twitch while having no effect on the acetylcholine response, but at 4.0uiM, did reduce the acetylcholine response. These results provide further evidence for cholinergic excitatory innervation of earthworm body wall muscle. Intracellular recordings were made from identifiable central neurones of Helix aspersa and the action of anthelmintic compounds investigated. The anthelmintics pyrantel, morantel and deacylated amidantel mimicked acetylcholine induced excitation"D" and inhibition"H" had the same ionic mechanism and were blocked by d-tubocurarine. This suggests these compounds interact with acetylcholine receptors on Helix neurones. Levamisole only inhibited the activity. A series of glutamate analogues was tested on Helix neurones which were either excited or inhibited by Lglutamate. The only analogue with clear glutamate-1ike activity was thio-glutamic acid. In normal saline Lglutamate hyperpolarises the membrane potential of cell F-1. This event is chloride mediated and is reversed to a depolarisation followed by hyperpolarisation in low external chloride. This afterhyperpolarisation is reduced in sodium or potassium free saline or following application of strophanthidin, l.O-lOOyM. The local anaesthetics procaine and tetracaine mimicked the "H" and "D" effects of acetylcholine on certain neurones. Tetracaine, O.OlyM, gradually and reversibly reduced both "H" and "D" responses of acetylcholine and the "H" response to dopamine. This provides evidence that local anaesthetics can interact with responses linked to chloride, sodium and potassium ion channels.

Thesis (M.D.) - University of Glasgow, 2007.
M.D. thesis submitted to the Faculty of Medicine, Dept. of Medicine and Therapeutics, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

Tuberculosis (TB) remains one of the most devastating diseases in humans. Nowadays, tuberculosis therapy is not sufficient to control the TB epidemic and only lasts for 6 months to cure patients and prevent relapse; therefore, the treatment of Mycobacterium tuberculosis (Mtb) is particularly challenging (1). New antibiotics, mainly those that are derived from new chemical classes, are more likely to be more effective against resistant strains. Moreover, expanding the knowledge of the mode of action of drugs has important implications in tackling TB. Only empirical approaches can be adopted in the journey of discovering new anti-tubercular drugs until a clear picture of latency and persister cells’ physiology is achieved. Mtb has the extraordinary ability to survive under hypoxia, suggesting a high degree of metabolic plasticity. The flexibility conferred by a modular respiratory system is critical to the survival of Mtb, thereby also making it a promising area of research for new drug targets. This thesis aimed towards the characterisation of cytochrome bd-I quinol oxidase (bd-I), a respiratory component that is believed to operate during both the replicative and “dormant” Mtb phenotypes. The essential nature of Mtb bd-I, which has no human homologue, has been confirmed in a recent deep sequencing study of genes required for Mtb growth by Griffin et al. (2), further confirming its potential as a novel target. Recombinant Mtb bd-I was successfully expressed under the control of the pUC19 lac promoter in the Escherichia coli ML16 bo3/bd-I and MB44 bo3/bd-I/bd-II knockout strains, allowing “noise-free” measurement of the enzyme. Initial steady-state kinetics of the enzyme was presented using a range of quinol substrates, revealing a substrate preference for dQH2 over Q1H2 and Q2H2. A number of bd-I inhibitors were identified and their pharmacodynamic profiles against Mtb H37Rv were determined. In addition, a pharmaco-metabolomics platform was initiated to explore the cellular response of Mtb to current first-line TB drugs as well as in house bd-I and type II NADH inhibitors. The initial findings are discussed in the context of the known mode of action of the drugs and future research needs in drug discovery of this devastating disease.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.

Orientador: Kleber Gomes Franchini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-20T23:08:02Z (GMT). No. of bitstreams: 1 Cardoso_Leandro_M.pdf: 3831901 bytes, checksum: 40e5df211d7e6fd923f654bd8d3a1ec1 (MD5) Previous issue date: 2012
Resumo: Nas doenças do coração ocorre hipertrofia e remodelamento do ventrículo esquerdo com impacto negativo na evolução clínica. Esses fatores influenciam independentemente o risco cardiovascular por elevarem a predisposição a infartos do miocárdio, ao desenvolvimento de insuficiência cardíaca e ao aparecimento de arritmias ventriculares graves. Estruturalmente ocorrem crescimento e degeneração dos miócitos cardíacos, fibrose e alterações da microcirculação. Estas alterações comprometem irreversivelmente a histoarquitetura do miocárdio, limitando a eficácia dos tratamentos convencionais, principalmente no estágio mais avançado caracterizado pela insuficiência cardíaca. A rede de sinalização celular envolvida na resposta das células miocárdicas (miócito e não miócitos) a forças mecânicas tem papel crítico na compreensão da patogênese da hipertrofia. A quinase de adesão focal (FAK), uma enzima associada à sinalização por integrinas, é ativada precocemente e exerce influência no desenvolvimento e sustentação da resposta hipertrófica do miocárdio à sobrecarga pressórica. Nesse estudo foi avaliado o efeito de um composto inédito derivado de quinazolina (BZLO) com potencial ação inibitória da FAK no coração de camundongos submetidos à sobrecarga pressórica por coarctação da aorta. In vitro, esse composto inibiu a atividade catalítica da FAK purificada com IC50 de 1nM. In vivo, o tratamento dos camundongos submetidos à coarctação da aorta com o composto (BZLO) provocou inibição do resíduo Tyr397 da FAK. Demonstrando que o composto não só preveniu, mas também promoveu regressão da hipertrofia do ventrículo esquerdo típica do modelo, acompanhada de atenuação da hipertrofia dos cardiomiócitos e da fibrose intersticial, e preservação da função ventricular. Esses resultados indicam que o tratamento com o composto (BZLO), presumivelmente por inibir a FAK, atenua as alterações estruturais e funcionais provenientes da sobrecarga pressórica crônica no ventrículo esquerdo de camundongos, sugerindo seu potencial como alternativa na prevenção e tratamento da insuficiência cardíaca
Abstract: Cardiac remodeling and hypertrophy have negative impact in the evolution of heart disease. They raise predisposition to myocardium infarction, heart failure and fatal ventricular arrhythmias, being independent markers of cardiovascular prognosis. Cardiomyocyte growth followed by degeneration, interstitial fibrosis and alterations in the microcirculation can be detected structurally in hypertrophic hearts. These alterations jeopardize myocardium tissue architecture irreversibly limiting therapeutic efficacy especially in advanced stages of disease with heart failure. The understanding of signaling network in response to mechanical stimuli is crucial in the study of cardiac hypertrophy. Focal adhesion kinase (FAK) an enzyme linked to signaling by integrins is activated in response to pressure overload and influences the development of myocardial hypertrophic response. In the present study, a compound based on quinazoline scaffold which is a FAK inhibitor, named BZLO, is examined for its effects on cardiac hypertrophy of mice model of aortic constriction. BZLO strongly inhibits catalytic activity of purified FAK in vitro (IC50 = 1nM). Satisfactory bioavailability of BZLO was observed after oral administration. BZLO treatment decreased FAK phosphorylation at Tyr397 and attenuated cardiomyocyte growth and interstitial fibrosis preserving cardiac function in the mice model of chronic pressure overload induced by aortic constriction. These findings suggest that BZLO is effective in attenuating the deleterious structural and functional consequences of chronic pressure overload in mice heart, suggesting that it has the potential to be considered as an option for the prevention and treatment of heart failure
Mestrado
Fisiopatologia Médica
Mestre em Ciências

The finding that NAD+ plays a role in a variety of signalling pathways, including gene expression, Ca2+ signalling and DNA repair mechanisms, has sparked interest in the proteins involved in these pathways as potential pharmacological targets for drug development. Recently, FK866, a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt) an important enzyme in the NAD+ rescue pathway, has been evaluated in clinical trials against cancer. The aim of this study is to further investigate the mechanisms and therapeutic characteristics of FK866 in different cancer cell lines and to determine if decreasing intracellular NAD+ levels can be used as a co-therapy strategy to improve the efficacy of current and new chemotherapy treatments. Experiments measuring cell vitality showed that FK866 dose-dependently decreased cell vitality. To investigate NAD+ consumption during Nampt inhibition, NAD+ levels were measured in cells treated with FK866 and inhibition of each of the main NAD+ consuming enzymes (PARP, sirtuins or CD38). This revealed differential NAD+ consumption rates by the different NAD+ consuming enzymes in MDA-MB-231 cells, with sirtuins being the major NAD+ consuming enzyme. The glycolytic effects of Nampt inhibition was measured using SEAHORSE assays; which measured the oxygen consumption and extracellular acidification rates as well as measuring NAD+/ NADH ratios. In the MCF-7 and MDA-MB-231 cell lines, FK866 had no effect on the oxygen consumption rates; however there was a general decrease in extracellular acidification rates indicating an effect on glycolytic activity. When measuring the NAD+/NADH ratio however, there was only a decrease in the MDA -MB-231 cells but no change in the MCF-7 cell line. Cell vitality and NAD+ levels were measured after treatment with FK866 in addition to NAD+ consuming enzyme inhibitors or the alkylating agent, Temozolomide to see if combination therapy would have more cytotoxic potential. This co-treatment indicated that there was no real positive effect on either the MCF-7 or MDA-MB-231 cells in either the cell vitality or NAD+ levels. Finally the effects of FK866 and the oral PARP inhibitor, Olaparib, were investigated using 3D cell culture (spheroids) and compared with 2D monolayer cultures. The effects of FK866 showed little difference in spheroid or monolayer culture. However, when treating with Olaparib there was higher level of cell viability and NAD+ levels with the cells grown in spheroid culture in comparison to cells grown in monolayer. In conclusion, this study has shown that FK866, as a single treatment decreases cell vitality, NAD+ levels and glycolytic activity. However as a co -therapy with PARP or Sirtuin inhibitors there is an increase in the cell vitality and NAD+ levels. Although similarities have been seen between spheroid culture and monolayers as a single treatment, FK866 does not seem to have the beneficial effects as a therapeutic.

Nrf2 (Nuclear factor erythroid 2-related factor 2), a redox-sensitive transcription factor, plays a critical role in the regulation of cellular defence and contributes to a number of cellular processes. Nrf2 is regulated through an interplay of complex transcriptional and post-translational mechanisms that modulates its activity during cellular perturbations or other biological processes thereby ensuring cellular homeostasis is maintained through the orchestration of adaptive responses. However, there is mounting evidence that constitutive upregulation of the Nrf2 pathway drives the enhanced proliferation and chemoresistance of various cancers. Therefore, an ability to modulate the activity of the Nrf2 pathway holds promise as a therapeutic strategy in certain disease settings. The work presented in this thesis showed that CDDO-Me provoked the induction of the Nrf2 pathway in C57BL6J WT and Nrf2 KO mice and CD1 WT mice. Analysis of CDDO-Me induced gene expression changes in both WT and Nrf2 KO mice showed a significant increase in the relative mRNA levels of ARE-dependent genes in the livers of CDDO-Me treated WT animals. Notably, CDDO-Me also provoked the accumulation of Nrf2 and NQO1 in human PBMCs and PHHs demonstrating its translational relevance. The mechanism of action of CDDO-Me as an inducer of Nrf2 is poorly understood. It was shown here that CDDO-Me post-transcriptionally evoked concentration and time-dependent, accumulation of Nrf2 protein in Hepa1c1c7 cells. Furthermore, CDDO-Me was shown to stabilize Nrf2 protein independently of the modulation of protein kinases and other signalling pathways that are purported to regulate Nrf2 activity. The work here also provides in vitro insights into the molecular mechanism of Nrf2 inhibition by the quassinoid brusatol. Brusatol post-transcriptionally evoked concentration- and time-dependent, yet transient, depletion of basal and inducible protein levels of Nrf2 in Hepa-1c1c7 cells. Furthermore, the ability of brusatol to inhibit Nrf2 was not affected by siRNA depletion of Keap1. In keeping with the latter observation, brusatol induced the depletion of Nrf2 independently of the proteasome and autophagic degradation machineries. Thus, these findings indicate that brusatol exploits a previously unknown mechanism of Nrf2 degradation. By examining the molecular mechanisms underlying the activation of Nrf2 by CDDO-Me and its inhibition by brusatol, this work reveals novel aspects of regulation within this important cellular pathway, and informs the design of new pharmacological inducers and inhibitors, which hold promise as therapeutic agents in a number of diseases.

Dementia is the top global public health threat of the twenty first century. Within the dementia spectrum, Alzheimer’s disease (AD) is the most common type of dementia that occurs with aging and accounts for about 60% - 80% of diagnosed cases. But currently available discoveries failed to develop disease-modifying therapies for all patients living with AD. Recent discoveries can only partially slow down cognitive decline in a small subset of patients with limited effectiveness. The heterogeneity and complexity of the pathophysiology of AD indicate that a single drug approach may not be sufficient to prevent disease onset and progression. Human umbilical cord blood cells (HUCBC) and lithium treatment have shown promise against numerous neurological conditions, including AD. Yet, they also show significant unwanted, adverse effects. To address this barrier to yield successful treatments, we employed two key modifications to these two treatment strategy. We used human umbilical cord blood derived serum (HUCBS, also labeled as CBS) rather than HUCBC. We also utilized ionic cocrystal of lithium salicylate l-proline (LISPRO, also labeled as LP) instead of usual lithium salt. Both HUCBS and LISPRO have been shown to have strong neuroprotective, anti-inflammatory properties in separate studies conducted in transgenic AD mouse models. The studies detailed herein independently investigated the effectiveness of biological (HUCBS) and pharmacological (LISPRO) approaches in modulating the pathology and cognitive impairments in AD mouse models (e.g., 5XFAD, 3XTg-AD, APPswe/PS1dE9, and Tg2576). While administration of HUCBC stimulate anti-inflammatory pathways shown in previous studies, we found that HUCBS markedly promoted neurotrophic soluble amyloid precursor protein alpha (sAPPα) through non-amyloidogenic amyloid precursor protein (APP) processing pathway compared to adult (ABS) and aged blood serum (AgBS) in Chinese hamster ovary cells expressing wild type APP (CHO/APPwt). Using chromatographic fractionation, mass-spectrometry, and targeting complement proteins in cord blood serum fraction (αCBSF), we discovered the source of sAPPα in HUCBS as C1 complement protein. Further, intraperitoneal administration of αCBSF via osmotic minipump for 6 weeks showed prevention of cognitive impairment in 5XFAD mice assessed by novel object recognition, and Y-maze test. A series of recent studies have shown that lithium can prevent both AD- and age-associated cognitive decline. But, current United States Food and Drug Administration-approved lithium pharmaceutics (carbonate and citrate forms) have a narrow therapeutic window and unstable pharmaceutics that can cause toxicity without monitoring. Here we investigated the safety, pharmacokinetics, and therapeutic efficacy of LISPRO (LP), lithium salicylate (LS), and lithium carbonate (LC) in cell culture and mouse (B6129SF2/J, Tg2576, and 3xTg-AD) models. Cytokine profiles from the brain, plasma and splenocytes demonstrated that 8-week oral treatment with LISPRO downregulates pro-inflammatory cytokines, upregulates anti-inflammatory cytokines and suppress renal cyclooxygenase 2 (COX2) expression in Tg2576 mice. Pharmacokinetic studies indicated that LISPRO provides significantly higher brain and more steady plasma lithium levels in both B6129SF2/J and transgenic Tg2576 mice compared with lithium carbonate. Oral administration of LISPRO for 28 weeks significantly reduced β-amyloid plaques and tau phosphorylation. In addition, LISPRO significantly elevated pre-synaptic (synaptophysin) and post-synaptic protein (post synaptic density protein 95) expression in brains from transgenic 3XTg-AD mice. Moreover, female APPswe/PS1dE9 mice at 4 months of age were orally treated with LP, LS, or LC for 8- to 9- months at 2.25 mmol lithium/kg/day followed by measuring body weight, internal organs’ growth, and cognitive and non-cognitive function. LISPRO treatment prevented cognitive decline compared with transgenic APPswe/PS1dE9 cohort, as shown by shorter escape latency during training and probe trials in the Morris water maze and longer contextual freezing time during fear conditioning. As expected, LISPRO treatment also reduced depression assayed by tail suspension test and irritability assessed with the touch escape test. But, lithium treatment did not alter anxiety or locomotor activity as assessed by open field, elevated plus maze or accelerated rotarod tests. Taken together, these data indicate that both biological HUCBS and pharmacological LISPRO treatment may prove to be viable effective strategy for ameliorating Alzheimer’s like pathology and cognitive impairment in preclinical models.

Gold complexes have a long history of being used as therapeutic agents, especially in applications against immune diseases such as rheumatoid arthritis. In 1979, an oral gold(I) drug – auranofin (AuRF, brand name as Ridaura®) – was demonstrated to exhibit anticancer properties. Since then, a considerable number of gold(I) complexes have been reported to show remarkable anticancer activities, but the understanding of their mechanism of actions is limited. In the present study, AuRF and several other anticancer gold(I)-phosphine complexes including AuPEt ([Au(triethylphosphine)Cl]) were demonstrated to induce autophagy – a cellular catabolic process of macromolecules and organelles through lysosomal degradation. The induced autophagy involved the accumulation of autophagosomes, which was mediated by the enhancement of autophagy initiation rather than by the blockage of autophagosomes maturation. Moreover, the AuRF and AuPEt induced autophagy was demonstrated to have a pro-survival effect for the cancer cells. To better explore the mechanism of actions of AuRF and other anticancer gold(I) complexes, a subcellular fractionation-based proteomic approach has been developed and optimized. This approach combined the use of subcellular fractionation, protein extraction, HPLC-LTQ-Orbitrap mass spectrometry, and bottom-up protein identification and quantification. By using this approach, the proteome coverage was increased, the complexities of the sub-proteomes were reduced, and the low-abundant organelle proteins were enriched. The nuclear sub-proteomes of AuRF-treated or AuPEt-treated cells were analyzed to identify the significantly regulated transcription regulators and the signaling pathways involved. The analysis delineates the possible AuRF-activated anticancer pathways involving up-regulation of the tumor suppressor cyclin-dependent kinase inhibitor 2A (〖p14〗^ARF), inhibition of the E2F transcription activity, blocking of the translocation of E3 ubiquitin-protein ligase (MDM2) from nucleus to cytoplasm and induction of the tumor suppressor p53. Furthermore, the KeyNode-based pathway analysis was applied to analyze the whole proteomes obtained from merging the sub-proteomes. Alongside the p53 pathway and E2F network, the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR, the rate-limiting enzyme of cholesterol biosynthesis) is one of the most up-regulated pathways of AuRF treatment. AuRF also showed significant inhibition to HMGCR activity in vitro with an IC50 value at the micromolar level. The effects of AuRF and AuPEt on the high mobility group box-1 protein (HMGB1), which exhibits distinct functions dependent on its cellular locations, were investigated. Treatment of cells with AuRF or AuPEt resulted in down-regulation of nuclear HMGB1, which is associated with p53-dependent cytotoxicities. The cytoplasmic HMGB1, which can induce autophagy, was found to be up-regulated. The levels of secreted HMGB1, which exhibits pro-inflammatory properties, were reduced, possibly contributing to anti-rheumatoid arthritis actions of AuRF. Collectively, the pharmacological and proteomic analyses in this research of AuRF and other anticancer gold(I) complexes supplement the current knowledge of their mechanism of actions.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

PRN psychotropic medication administration is a common practice used by psychiatric RNs in acute inpatient mental health settings to manage anxiety and agitation in patients, and as a result, there are high incidences of PRN psychotropic medication administration (Mugoya & Kampfe, 2010). There is limited documentation indicating that therapeutic non-pharmacological interventions are utilized prior to PRN psychotropic medication administration (Curtis et al., 2007). The purpose of this scholarly project was to determine what non-pharmacological interventions are used by mental health RNs prior to or in lieu of administering PRN psychotropic medication to manage anxiety and agitation in adult psychiatric hospitalized patients. Another aim of this project was to determine what factors influence mental health RNs' decisions to administer PRN psychotropic medications. A descriptive study was conducted. Two survey-type self-reporting tools were used. Twenty RNs completed Nursing Surveys. Thirteen RNs completed both Nursing Surveys and Nursing Intervention Tracking Forms. Findings from this study revealed that non-pharmacological interventions successfully resolved problems over 1/3 of the time and may prevent the use of PRN psychotropic medications. In cases where PRN psychotropic medication was not administered, distraction was the non-pharmacological intervention most often used. The most common factor influencing RNs' decisions to administer PRN psychotropic medications was a high level of anxiety persistent after non-pharmacological interventions were used. This study reinforces the importance of RNs using non-pharmacological interventions to manage patients exhibiting signs or symptoms of anxiety or agitation.

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models.

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models.
Zarqa University
The full text was made available at the end of the embargo, 3rd December 2019

Processing of Chinese medicinal materials (CMMs) is a unique technique for preparing decoction pieces. According to the traditional Chinese medicine (TCM) theory, processing can reduce the toxicity, alter the indications and enhance the efficacy of the herbs. Pinelliae Rhizoma (PR), the dried tuber of Pinellia ternata (Thunb.) Breit., is a traditional Chinese medicinal herb. Although toxic, it is commonly used for treating cancer, cough and phlegm. TCM doctors usually prescribe raw PR to manage cancer and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRZA), the product of raw PR processed with ginger juice and alumen, for treating cough and phlegm. To guarantee the quality of a processed herb, standardized processing procedure is critical. However, the current manufacturing protocol of PRZA varies greatly among different places in China. In addition, the mechanisms involved in raw PR's toxicities, the toxicity-reducing effect of processing, and the anticancer effects of raw PR are still not fully understood. In this study, we standardized the manufacturing procedure for PRZA, and explored the mechanisms involved in raw PR-induced cardiotoxicity, the toxicity-reducing effect of processing, and the anti-liver cancer effects of raw PR.;Our results showed that the standardized manufacturing procedure for PRZA is as follows: soak raw PR in water until the center of the cut surface is devoid of a dry core, boil for 6 h after adding 12.5 kg alumen and 25 L freshly squeezed ginger juice for each 100 kg of raw PR, then take out and dry. The toxicity and bioactivity assays demonstrated that PRZA produced using our optimized protocol could reduce the cardiotoxicity, and enhance the antitussive and expectorant efficacies of raw PR, supporting the traditional processing theory; and raw PR exhibited more potent anti-liver cancer efficacy than PRZA, supporting the common clinical practice. Moreover, as expected raw PR and PRZA showed different chemical profiles. These results suggest that our optimized protocol for producing PRZA is appropriate. The optimized protocol, shown to be applicable for PRZA industrial production, will be included in the upcoming "National Standards for Processing CMMs" (全國中藥炮製規範) to update the 1998 edition of this China national standard handbook.;Using a comprehensive metabolomics approach, we explored the underlying mechanisms of raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing. Results showed that inhibition of mTOR signaling and activation of the TGF-b pathway may contribute to raw PR-induced cardiotoxicity, and free radical scavenging may be responsible for the toxicity-reducing effect of processing.;In Summary, in this study we achieved the follows: 1) standardized the manufacturing procedure for PRZA; 2) found that processing with ginger juice and alumen reduced the toxicity of raw PR, and discovered the potential mechanisms for raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing; 3) demonstrated the anti-liver cancer activities and some underlying mechanisms of action of raw PR. Our findings provide a standardized manufacturing procedure for PRZA, help in the understanding of the mechanisms involved in raw PR-caused cardiotoxicity and the toxicity-reducing effect of processing, and provide a pharmacological basis for the clinical application of raw PR in liver cancer treatment. The outcome of this study should guarantee the safety and efficacy of PRZA, and provide scientific justifications for the traditional processing theory of PR.

The renin-angiotensin system plays a central role in the maintenance of blood pressure. Angiotensin II, the main effector of this system, results from the action of angiotensin-converting enzyme (ACE) on angiotensin I. Angiotensin II, maintains vasomotor tone via its vasoconstrictor action, and also increases salt and water retention by stimulating the release of aldosterone. ACE inhibitors, such as captopril, enalapril and lisinopril, are highly effective in the treatment of hypertension and congestive cardiac failure. Previous studies have suggested that angiotensin converting enzyme (ACE) production may be enhanced during pharmacological inhibition of the enzyme. Little is known, however about the mechanism of this induction. After demonstrating increases in circulating ACE protein in cardiac failure patients receiving the ACE inhibitor captopril, a rat model was used to study this effect. A sensitive enzyme linked immunosorbent assay for rat ACE was developed and a partial cDNA for rat ACE cloned to enable examination of ACE mRNA and protein expression during enzyme inhibition with enalapril. Rat lung ACE mRNA increased by 156% (p<0.05) and ACE protein doubled within 3 hours of administering a single dose of enalapril. Testicular ACE mRNA also increased by 300% (p<0.05) within 2 hours and returned to pretreatment levels by 6 hours. The angiotensin II antagonist saralasin similarly caused a significant (p<0.0001) 800% enhancement of mRNA expression. Aldosterone pretreatment of rats prior to enalapril administration was found to abolish this mRNA induction. These findings indicate that increased ACE expression during inhibition results from reduced levels of angiotensin II with consequent reduced stimulation of the angiotensin 11 receptor and its effects, such as aldosterone release. This suggests that ACE levels are regulated by a negative feedback loop involving the distal components of the renin-angiotensin system, namely angiotensin II and aldosterone. In situ hybridisation and immunohistochemical techniques were employed to localise the site of this inductive response in rat tissue sections. It was found that lung macrophages were markedly induced to produce ACE, as was ACE in seminiferous tubules. ACE induction was also noted in the expected sites of renal tubular epithelium and glomerular tissue. Interestingly, ACE expression was also enhanced in cardiac valves. In these studies it has been conclusively demonstrated that new ACE expression is induced by enzyme inhibitor therapy. A variety of techniques have been developed that will allow futher study of ACE in rat tissues.

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as compared to the mammalian control, rat cb5r. We have observed two key structural differences between the fungal and mammalian proteins that may account for decreased thermal stability and inhibitor specificity of C. albicans Cbr1. Substrate binding affinity and catalytic efficiencies, as well as investigation in the flavin-binding environment, were comparable between the fungal and rat enzymes. In S. cerevisiae, CBR1 and MCR1 knockout strains have been challenged with environmental stressors and subsequently shown to have a role in azole and amphotericin B resistance. Our results of potential protein interactions of C. albicans Cbr1 describe proteins involved in the weak acid stress response, implying a novel role of the protein in pathogenicity. Conclusively, this report describes potential inhibitors of the fungal protein, as well as elaborating upon its important role in ergosterol biosynthesis and possible mechanisms of CYP450-mediated drug detoxification.

Epilepsy is a neurological disorder that is characterised by spontaneous seizures. After various epileptogenic injuries, astrocytes become dysfunctional and experimental evidence indicates that these cells contribute to seizure mechanisms. The generation of inflammatory molecules in astrocytes appears to play a key pathogenic role in seizures. However, astrocytes may also contribute to repair the hyperexcitable neuronal networks underlying seizures. We focused our studies on understanding the role of astrocytes in epilepsy by (1) developing a new in vivo imaging method to monitor astrocytic cell activation during epileptogenesis and coupled this with their phenotypic characterization; (2) studying the role of Toll-like receptor 3 (TLR3) signaling in seizure mechanisms. We report that in vivo bioluminescence imaging is a powerful tool for monitoring astrocytic activation in diseased conditions. Characterization of astrocytic activation during epileptogenesis showed a rapid cell activation corresponding to their inflammatory phenotype while homeostatic (neuroprotective) mechanisms were activated with a delay. Moreover, we demonstrate that in vivo imaging of astrocyte activation allows to study the potential involvement of these cells in the therapeutic effects of anti-inflammatory drugs. We also show that priming TLR3 activation in astrocytes with the use of a synthetic agonist results in remarkable anti-inflammatory and anti-ictogenic effects. Mechanistic studies revealed that interferon regulatory factor (IRF)-3/Interferon-β mediated cascade is likely responsible for the inhibitory effects of TLR3 priming on seizures and neuronal excitability. In summary, astroglia activation during the critical epileptogenesis phase provides a potential target for interfering in a timely manner with the inflammatory phenotype of these cells contributing to seizures. Importantly, there are astrocytic cell functions that mediate decreased neuronal excitability and they should be carefully considered when developing treatments targeting these cells for therapeutic purposes.

Sea bass (Lateolabrax maculatus) has been used for dietary therapy practice in China. In traditional Chinese medicinal books, it has been indicated that sea bass can be applied for managing many inflammation associated conditions. However, the studies on the pharmacological mechanisms of anti-inflammation of sea bass remain scarce. Hence, this study aims to illustrate the pharmacological and chemical basis for the folk use of sea bass in managing inflammation- associated conditions. For in vivo studies, dietary effect of sea bass on inflammation-associated conditions in ulcerative colitis, skin wounds, and intestinal dysbiosis were evaluated. A series of inflammatory mediators associated with wound healing and ulcerative colitis, and the proliferation effects of fibroblasts upon treatments were studied via Western blotting, enzyme-linked immunosorbent assay (ELISA), hematological parameters, histopathological and Immunofluorescence analysis, using cutaneous wound model and DSS induced colitis model, respectively. β-diversity analysis and species variance statistics were conducted to evaluate the effect of ASB on the microbial communities with colitis and discovered the high dimensional biomarkers. Results showed that ASB could significantly ameliorate several pathophysiological and morphological features in DSS induced colitis. ASB has a potential in accelerating the proliferation phase of wound healing via well-organized abundant collagen deposition, angiogenesis, strengthening the skin contraction and skin organ maturation in wounds. Moreover, the study also found that ASB could significantly down-regulated the expression levels of inflammatory associated mediators in colitis and skin wound. Additionally, principal coordinate analysis (PCoA) and relative abundance at phylum level among groups were indicated that ASB possess a potential amelioration on intestinal dysbiosis in colitis. Histogram of linear discriminant analysis (LDA) scores and Cladogram as the results of LEfSe analysis identified that Christensenellaceae might be treated as the the biomarker for treating colitis. For in vivo studies, macrophages and fibroblasts were used for further evaluation. Result showed that ASB could significantly inhibited the production of pro-inflammatory mediators in lipopolysaccharide (LPS) induced macrophages. The mRNA and protein expression level of inflammatory associated mediators were significantly down-regulated upon ASB treatment. Moreover, results also suggested that ASB treatment has a closely link to accelerate the wound healing through migration and proliferation enhancement. Furthermore, the characterization of the aqueous extract of sea bass (ASB) was conducted. Six kinds of peptides and two protein identified from fraction F1 by LC-QE-HF-MS might be responsible for anti-inflammatory activity. It confirmed that Fraction F1 could be treated as the main component for contributing the potential anti-inflammatory activities to ASB. Current results illustrated that fraction F1 (kDa) is a kind of nanoparticles with stability separated from ASB. It can be treated as a promising candidate for treating inflammation associated conditions, providing the chemical basis for the folk use of sea bass in managing inflammation-associated conditions. Current studies established a pharmacological and chemical basis for the folk use of sea bass in managing inflammation-associated conditions. A further justification for the clinical application of sea bass in treating inflammation associated conditions is necessary. Keywords: sea bass; inflammation; ulcerative colitis; wound healing; gut microbiota; peptides

Parkinson’s disease (PD) is the second most common neurodegenerative disease affecting 2% of the population over 65 years old that lacks effective cure. The current available treatments for PD are largely symptomatic and palliative. Tianma Gouteng Yin (TGY) is a traditional Chinese medicine (TCM) formula belongs to the formulas that dispel wind. Nowadays, it has been a commonly prescribed formula to treat Parkinsonian-like symptoms such as tremor and paralysis in some of the patients. However, just as most of the TCM formula, the material basis and the underlying pharmacological effects of TGY are still lacking experimental evidence. In this study, a method using UHPLC/Q-TOF-MS and HPLC-ELSD has been developed and successfully applied to qualitatively and quantitatively determine the complex phytochemicals of TGY. Totally 28 phytochemicals were identified, of which 20 were simultaneously quantified. The material basis profile of TGY decoction was delineated for the first time. After full component analysis of TGY, the neuroprotective activity of TGY was verified both in vivo and in vitro. In Drosophila PD models, TGY mitigated rotenone induced toxicity and promoted α-synuclein clearance. In stereotaxic rotenone intoxication rats, TGY exerted neuroprotective effects in terms of preventing dopaminergic neurons loss and alleviating neuroinflammation. TGY alleviated rotenone induced apoptosis in SH-SY5Y cells. Furthermore, we discovered that Geniposide, an important component of TGY, is an autophagy inducer both in vivo and in vitro and is neuroprotective in transgenic Drosophila PD model. In general, our study proves that TGY is neuroprotective in PD models. In addition to the efficacy study, safety of TGY application in terms of TGY-drug interaction was also evaluated. In our study, herb-drug interactions between TGY and one of the most popular drugs used in PD treatment, Sinemet, were studied. The pharmacokinetics data showed that co-administration of TGY could suppress the absorption of Levodopa, the main component of Sinemet, for the first time. This information suggest that in clinical practice, TGY should avoid been administrated with Levodopa containing medicaments at the same time. In conclusion, the data of this study provides valuable information on the material basis, efficacy and safety of TGY. This information is useful reference for the clinical application of TGY in PD treatment. Keywords: Tianma Gouteng Yin, Parkinson’s disease, Drosophila, Rotenone, -synuclein, Geniposide, Autophagy, Sinemet, Herb-drug interaction

INTRODUCTION: In 1966, Rett syndrome (RTT, OMIM#312750) was for the first time described as a clinical issue by Dr. Andreas Rett, an Austrian pediatrician. He has observed in 22 patients similar unique symptoms. A few years later Hagberg and colleagues described further the syndrome in 35 girls. Rett syndrome is the cause of mental retardation that affects 1 in 10.000 female births, which makes it the second cause of mental retardation in girls. In 1999 Zoghbi lab found out the genetic basis of Rett disease. Mutation in MeCP2 is in 95% cases the reason of classical Rett. MeCP2 is a nuclear protein, expressed widely in different tissues, but is most abundant in neurons of the mature nervous system. A few years earlier Bird and coworkers indentified MeCP2 as a new protein that binds to the methylated CpG dinucleotides. Although the function of the MeCP2 is still unknown, it is considered likely to regulate gene expression, either through the silencing or activation of the specific genes or by more global regulation of transcriptional processes. RTT was the first neurodevelopmental disorder related to epigenetics. AIM OF THE STUDY Hypotesis: The main aim of this thesis is to perform preclinical evaluation of drugs that are known to target pathways which are altered in Rett syndrome. It is also necessary to investigate new mechanisms associated with the development of Rett syndrome, aiming to find new pathways related to Rett phenotype that can be manipulated through the pharmacological approach. Aims: 1. Determine tests that can reflect the difference at the behavioral and molecular levels between the Mecp2 KO and WT littermate mice. 2. Optimize study design protocol for evaluation of in vivo drug treatments. 3. Identify candidate drugs against selected targets in order to improve Rett disease, with the goal to reverse the symptoms, prolong the life span or ameliorate dysfunctions based on inflammation and neural mechanisms. 4. Determine the potential effects of novel therapeutic approaches for newly discovered pathways dysregulated in Rett syndrome. Methods: Animals knockout (KO) to the MeCP2 gene are a well-established murine model that mimics RTT human disease. This model is an excellent tool for the study of the consequences of the loss of MeCP2 in neuronal function. Once we know the dose administered, use two experimental groups of treated and untreated animals. Treatment is initiated when the animals are 4 weeks old and start showing symptoms characteristic of this model: reduced mobility, retraction of the hind legs, tremors, hair frizzing and respiratory abnormalities, difficulty walking, retraction of the rear legs. During the test, the mice will be evaluated according to a set of symptoms resulting from MeCP2 deficiency. Results: Part 1 • We performed a comprehensive panel of experiments investigating the differences between Mecp2 KO and wt littermate mice at the behavioral and molecular levels. This was further extended to evaluate the specific drug treatments efficacy. Part 2 • Combined administration of Levodopa and a Dopa decarboxylase inhibitor in Rett syndrome mouse models was well tolerated, diminished Rett syndrome-associated symptoms and increased lifespan. • The use of L-Dopa + Ddci in the Mecp2 KO mice induced dendritic growth mediated by dopaminergic neurons. • L-Dopa + Ddci-treated group exhibited higher Th and pTh expression and dopamine levels in comparison to the vehicle treated group. Part 3 • The results presented here reveal an important role for the relationship between Mecp2 and GSK3 signaling in Rett syndrome disease • Inhibitor of GSK3, SB216763, improved life span and reduced single and total symptoms scores, as well as motor deficiency in Mecp2 KO mice • Inhibition of GSK3 is a possible line of stimulation of neuronal dendritic growth, supported by elevated levels of D2- receptor after drug administration. • Treatment with SB216763 decreased inflammation and strengthened antioxidant defense in the brain of Mecp2KO mice Part 4 • Treatment with copaxone, a BDNF analogue reflect rather low improvement in Mecp2 KO studied mice, displaying also very variable results between studied animals. • Treatment with dexamethasone, an example of glucocorticoids intervention display rather low improvement – approximately 20% when compared to untreated mice. • Ropirinol treatment confirmed that dopminergic pathway is dysregulated in Rett. Both life span and the phenotype were improved. However, the efficiency was lower compared to L-dopa + Ddci treatment. • Injections of bromperidol, a serotonin modulator, were toxic for Mecp2 KO mice. Even though the selected doses were very low, they caused significant reduction of body weight in the KO treated group. • Previously described positive effect of cysteamine was not reproduced in our laboratory (Roux et al., 2012). • Gabapentin, one of GABA modulators, improved the phenotype, but not life span of Mecp2 KO animals, being more efficient with the low dose. • TDZD8 treatment confirmed previously a described role of GSK3 inhibition in Rett. Both life span and phenotype were improved. However, the efficacy was lower than after SB216763 treatment. • Antioxidants studied in this thesis displayed improvement of 30% in Rett syndrome phenotype and some of them prolonged the life span by 25% when compared to the vehicle group. • Epigenetics drugs evaluated in this thesis, even though some of them were suggested to be relevant for Rett syndrome, did not show any specific improvement. Conclusions: The treatment with L-Dopa + Ddci is promising to overcome the dopaminergic defects observed in the preclinical model of Rett syndrome (Szczesna et al., 2014) The new data indicate selected inhibitor, SB216763, as a being potential drug treatment for Rett disease. The drug can be adressed to have neuroprotection properties via the improvement of synaptic plasticity, reduced oxidant and inflammation damage or partial rescue of mobility dysfunctions (Szczesna et al in preparation).
INTRODUCCIÓN: En 1966, el síndrome de Rett (RTT, OMIM#312750) fue por primera vez descrito como un problema clínico por el pediatra austriaco Andreas Rett. Observó síntomas similares en 22 pacientes. Pocos años después, Hagberg y colaboradores describieron el síndrome en 35 niñas más. El síndrome de Rett causa retraso mental en 1 de cada 10000 niñas, lo que hace que sea la segunda causa de retraso mental en niñas. En 1999 en el laboratorio de Huda Zoghbi descubrieron las bases genéticas de la enfermedad. El 95% de los casos de Rett clásico se produce por mutaciones en MeCP2. MeCP2 es una proteína nuclear, que se expresa en diferentes tejidos, pero es especialmente abundante en neuronas del sistema nervioso maduro. Pocos años antes Bird y colaboradores habían identificado MeCP2 como proteína con capacidad para unirse a dinucleótidos CpG. Aunque la función de MeCP2 todavía no se conoce con exactitud, se considera que probablemente actúa como regulador de la expresión génica, tanto mediante el silenciamiento o activación de genes específicos como actuando de manera más global sobre la transcripción. El síndrome de Rett fue la primera enfermedad del desarrollo neuronal relacionada con la epigenética. OBJETIVOS DEL ESTUDIO Hipótesis: El principal objetivo de esta tesis es realizar la evaluación preclínica de fármacos que actúan sobre las diferentes rutas alteradas en el síndrome de Rett. Es necesario también investigar nuevos mecanismos asociados al desarrollo de la enfermedad, con el fin de descubrir nuevas rutas que puedan estar relacionadas con la patología y que sean susceptibles de ser manipuladas mediante la utilización de fármacos específicos. Objetivos: 1. Determinar qué tests pueden reflejar las diferencias tanto a nivel conductual como molecular entre ratones knockout de MeCP2 y salvajes de la misma camada. 2. Optimización de protocolos de evaluación de tratamientos farmacológicos in vivo. 3. Identificación de fármacos dirigidos a dianas específicas para la mejora del curso de la enfermedad, con la finalidad de revertir la sintomatología, aumentar la supervivencia o mejorar disfunciones relacionadas con mecanismos neuronales o inflamación. 4. Determinar el potencial de las nuevas terapias desarrolladas para las rutas que se descubran alteradas en el síndrome de Rett. Métodos: El knockout de MeCP2 es un modelo murino bien establecido que mimetiza el síndrome de Rett humano. Este es un excelente modelo para el estudio de las consecuencias relacionadas con la pérdida de MeCP2 en las funciones neuronales. Una vez conocida la dosis que se ha de administrar, se utilizaron dos grupos experimetales, el grupo de tratados y no tratados. El tratamiento se inicia cuando los animales tienen 4 semanas y empiezan a mostras síntomas característicos del modelo: movilidad reducida, retracción de las patas traseras, temblores, pelo encrespado y anormalidades respiratorias, dificultas al andar, retracción de las patas delanteras. Durante el test, los ratones serán evaluados según los síntomas derivados de la deficiencia de MeCP2. Resultados: Parte 1 • Hemos realizado una serie de experimentos investigando las diferencias entre ratones MeCP2 KO y sus hermanos de camada salvajes, tanto a nivel de comportamiento como molecular. Posteriormente, se evaluó la eficacia de una serie de tratamientos. Parte 2 • La administración combinada de Levodopa y un inhibidor de la Dopa decarboxylasa en ratones con síndrome de Rett fue bien tolerada por los animales, disminuyendo los síntomas asociados al síndrome y aumentando la supervivencia. • El uso de L-Dopa + Ddci en el los ratones Mecp2 KO indujo el crecimiento dendrítico mediado por neuronas dopaminérgicas. • El grupo tratado con L-Dopa + Ddci muestra exhibió un aumento en los niveles de expression Th y pTh y de los niveles de dopamina en comparación con el grupo tratado con el vehículo. Parte 3 • Los resultados presentados revelan que la relación entre Mecp2 y la ruta de señalización de GSK3 juega un papel importante en el síndrome de Rett. • El inhibidor de GSK3, SB216763, mejoró la supervivencia y redujo la gravedad de los síntomas, así como la deficiencia motora ,de los ratones Mecp2 KO • La inhibición de GSK3 es una posible vía de estimulación del crecimiento de las neuronas dendríticas, avalado por los elevados niveles del receptor D2 encontrados tras la administración de la droga. • El tratamiento con SB216763 disminuyó la inflamación y reforzó las defensas antioxidantes en el cerebro de los ratones Mecp2 KO. Parte 4 • El tratamiento con copaxona, un análogo de BDNF dio lugar a una menor mejora de los ratones Mecp2 KO, mostrando además una gran variabilidad entre los ratones estudiados. • El tratamiento con dexamethasona, un ejemplo de tratamiento con glucocorticoides, mostró una pequeña mejora, aproximadamente un 20% cuando se comparan los ratones tratados con los no tratados. • El tratamiento con Ropirinol confirmó que la vía dopaminérgica está alterada en Rett. Tanto la supervivencia como la sintomatología mejoró tras la administración de la droga. Sin embrago, la eficiencia fue menor a la obtenida con la tratamiento combinado de Dopa + Ddci. • Las inyecciones de bromperidol, un modulador de la serotonina, resultaron ser tóxicas en ratones Mecp2. Incluso aunque las dosis administradas fueran muy bajas, los ratones sufrían pérdida de peso acusado. • Resultados previos describiendo un efecto positive en los ratones Mecp2 tras la administración de cysteamina no pudieron ser reproducidos en nuestro laboratorio (Roux et al., 2012). • Gabapentin, un modulador de GABA, mejoró los síntomas pero no tuvo ningún efecto en la supervivencia de los animales Mecp2. • El tratamiento con TDZD8 confirmó resultados los previos describiendo el efecto positivo de la inhibición de GSK3 en Rett. Se mejoró tanto la supervivencia como la sintomatología. Sin embrago, la eficiencia fue menor a la obtenida con SB216763. • Los antioxidantes estudiados en esta tesis mostraron una mejora del 30% en la sintomatología de Rett, siendo algunos también eficaces en la mejora de la supervivencia. • Las drogas epigenéticas evaluadas en esta tesis no mostraron ninguna mejora significativa en el tratamiento del síndrome de Rett. Conclusiones: El tratamiento con L-Dopa + Ddci es muy prometedor para sobreponer los defectos dopaminérgicos observados en el modelo preclínico del síndrome de Rett utilizado en esta tesis (Szczesna et al., 2014). Los nuevos datos indican que el inhibidor de GSK3, SB216763, es un posible fármaco para el tratamiento del síndrome de Rett. La droga ha mostrado propiedades neuroprotectoras mediante la mejora de la plasticidad sináptica y la reducción del daño oxidativo e inflamación, así como la mejora de las disfunciones motoras (Szczesna et al, artículo en preparación).

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: In traditional medicine, there is a long-standing culture of combining herbal drugs to increase the therapeutic efficacy. The improved medical action is thought to be due to synergistic interactions between different plant bioactive components. The aim of this study was to test the pharmacological interactions in a medicinal plant combination which consisted of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus. The rationale for the analysis of this particular mixture is that it had noteworthy antibacterial activity and exhibited the highest activity out of seven medicinal plant mixtures previously investigated. Using chromatographic analysis, the phytochemistry of the plants was also assessed. The chloroform: methanol (1:1; v/v) extracts or hydo-distilled essential oils (A. crenulata and E. globulus) were screened individually and in combinations (double and triple plant combination) for activity against five respiratory pathogens using a microdilution assay. The antimicrobial interactions in combinations were assessed with the fractional inhibitory concentration (FIC) and the isobolograms. The organic extracts generally showed the highest antimicrobial activity with E. globulus having the highest activity with MIC values below 1 mg ml-1 representing noteworthy activity. The overall activity of the aqueous extracts was poor. The essential oil activity of E. globulus was mostly noteworthy (0.5 to 2 mg ml-1) while A. crenulata essential oil displayed moderate activity (1 to 4 mg ml-1). The ΣFIC values for double combinations (1:1) of A. crenulata with D. viscosa, A. crenulata with E. globulus and D. viscosa with E. globulus were calculated from the minimum inhibitory concentration (MIC) data and the interactions were classified as synergistic, additive, indifferent and antagonistic. The highest synergistic interactions observed were for a 1:1 combination of A. crenulata with E. globulus against K. pneumoniae, S. aureus and B. subtilis with ΣFIC values of 0.07. There was only one incident of antagonism noted in the study for D. viscosa with E. globulus (1:1) against C. neoformans with ΣFIC value of 4.25. The double combinations against selective pathogens (K. pneumoniae, S. aureus and E. coli) were further analysed for interactions using isobolograms. Mostly, the antimicrobial interactions as presented by the isobolograms were congruent with FIC results which further validated the occurrence of relevant antimicrobial interactions in those combinations. The ΣFIC values for triple combinations (1:1:1) revealed mostly synergistic interactions. When the triple combinations were analysed further against certain pathogens based on the predictions of the Design of Experiments software program (MODDE 9.1®), the MIC values remained the same despite the different combinations that were tested Thin layer chromatography (TLC) was used for a quick chemical fingerprinting of the plant extracts. This was followed by a bio-autographic assay. The chemical profiles of the organic extracts and essential oils from two of the study aromatic plants (A. crenulata and E. globulus) were further analysed with liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS) respectively. For combined plant extracts, a multivariate data analysis using principal component analysis (PCA) and hierarchical clustering analysis (HCA) was used to determine the relationship of the chemical make-up of combinations with that of individual plant extracts. According to the TLC analysis, E. globulus extracts had more compounds than the other two plants in the study. For the bio-autographic assay, E. globulus and combinations that included this plant showed greater inhibition zones than A. crenulata and D. viscosa. For the LC-MS analysis, PCA and HCA showed a close relationship between A. crenulata with D. viscosa, D. viscosa with E. globulus and the triple combination. Twenty one components were identified in the essential oil of A. crenulata representing 88.83% of the total oil composition. The oil was dominated by oxygen-containing monoterpenes (46.25%). In the essential oil of E. globulus, twenty six compounds were identified making up to 95.62% of the oil composition. Oxygen-containing monoterpenes (32.98%) also dominated the E. globulus essential oil. There was no great variation in essential oil metabolites of the individual plants and their combination as shown by both PCA and HCA. The enhanced in vitro antimicrobial activity and pharmacological interactions (synergy and additivity) in some of the combinations (double and triple) that were tested in this study adds scientific support to the use of medicinal plant combinations in Western Cape traditional medicine. The metabolic profiles of plants in combination might be unique due to interaction of the different plant bioactive molecules and thus result into defined antimicrobial activity.
AFRIKAANSE OPSOMMING: In tradisionele geneeskunde is dit ’n lank bestaande kultuur om kruiemiddels te kombineer om die terapeutiese werking daarvan te verhoog. Dié verbeterde mediese werking word toegeskryf aan die oënskynlik sinergistiese interaksies tussen verskillende bioaktiewe plantkomponente. Die doel van hierdie studie was om die farmakologiese interaksies in medisinale plantkombinasies van Agathosma crenulata, Dodonaea viscosa en Eucalyptus globulus te bestudeer. Daar is op die ontleding van hierdie spesifieke mengsel besluit omdat dit oor beduidende antibakteriese waarde beskik en omdat dit uit sewe medisinale plantmengsels wat voorheen bestudeer is, as die doeltreffendste een aangewys is. Die fitochemie van die plante is ook met behulp van chromatografiese ontleding beoordeel. Deur middel van ’n mikroverdunningstoets is die chloroform:metanol- (1:1; v/v-)ekstrakte of hidrogedistilleerde vlugtige olies (A. crenulata en E. globulus) individueel sowel as in kombinasie (dubbele en drievoudige plantkombinasies) nagegaan vir hul werking met betrekking tot vyf respiratoriese patogene. Die gekombineerde antimikrobiese interaksies is met behulp van fraksioneel stremmende konsentrasie (FIC) en isobologramme ondersoek. Die organiese ekstrakte het oor die algemeen die meeste antimikrobiese aktiwiteit by E. globulus getoon, met MIC-waardes onder 1 mg ml-1 wat as noemenswaardige aktiwiteit beskou is. Die algehele aktiwiteit van die waterekstrakte was swak. Die vlugtige-olieaktiwiteit van E. globulus was merendeels noemenswaardig (0,5 tot 2 mg ml-1), terwyl die vlugtige olie van A. crenulata matige aktiwiteit getoon het (1 tot 4 mg ml-1). Die ΣFIC-waardes vir dubbelkombinasies (1:1) van A. crenulata en D. viscosa, A. crenulata en E. globulus, en D. viscosa en E. globulus is uit die minimum stremmende konsentrasie (MIC) bereken en die interaksies is as sinergisties, additief, neutraal en antagonisties geklassifiseer. Die sterkste sinergistiese interaksies is by ’n 1:1-kombinasie van A. crenulata en E. globulus met betrekking tot K. pneumoniae, S. aureus en B. subtilis opgemerk, met ΣFIC-waardes van 0,07. Die studie het slegs een geval van antagonisme opgelewer, naamlik by D. viscosa en E. globulus (1:1) met betrekking tot C. neoformans, wat ’n ΣFIC-waarde van 4,25 geregistreer het. Die werking van die dubbelkombinasies met betrekking tot gekose patogene (K. pneumoniae, S. aureus en E. coli) is voorts met behulp van isobologramme vir interaksies nagegaan. Die antimikrobiese interaksies wat uit die isobologramme geblyk het, was meestal in pas met FIC-resultate, wat die bestaan van tersaaklike antimikrobiese interaksies in daardie kombinasies verder bevestig het. Die ΣFIC-waardes vir die drievoudige kombinasies (1:1:1) het meestal sinergistiese interaksies aan die lig gebring. Toe die drievoudige kombinasies verder op grond van die voorspellings van die sagteware Design of Experiments (MODDE 9.1®) met betrekking tot sekere patogene ontleed is, het die MIC-waardes onveranderd gebly, ondanks verskillende toetskombinasies. Dunlaagchromatografie (TLC) is vir ’n vinnige chemiese ontleding van die plantekstrakte gebruik en is gevolg deur ’n bio-outografiese toets. Die chemiese profiele van die organiese ekstrakte en vlugtige olies van twee van die aromatiese plante in die studie (A. crenulata en E. globulus) is verder met vloeistofchromatografie-massaspektrometrie (LC-MS) en gaschromatografie-massaspektrometrie (GC-MS) onderskeidelik ontleed. Vir gekombineerde plantekstrakte is veelveranderlike-ontleding in die vorm van hoofkomponentontleding (PCA) en hiërargiese groepsontleding (HCA) gebruik om die verhouding van die chemiese samestelling van kombinasies in vergelyking met dié van individuele plantekstrakte te bepaal. Volgens die TLC-ontleding beskik E. globulus-ekstrakte oor meer verbindings as die ander twee plante in die studie. Vir die bio-outografiese toets het E. globulus en kombinasies daarmee groter stremmingsones as A. crenulata en D. viscosa getoon. In die LC-MS-ontleding het PCA en HCA op ’n hegte verhouding tussen A. crenulata en D. viscosa, D. viscosa en E. globulus, en die drievoudige kombinasie daarvan gedui. Een-en-twintig komponente is in die vlugtige olie van A. crenulata gevind, wat 88,83% van die algehele oliesamestelling uitmaak. Die olie is deur suurstofhoudende monoterpene (46,25%) oorheers. Die vlugtige olie van E. globulus het 26 verbindings opgelewer, wat 95,62% van die oliesamestelling uitmaak. Suurstofhoudende monoterpene (32,98%) het ook die vlugtige olie van E. globulus oorheers. Nóg PCA nóg HCA het op enige beduidende variasie in die metaboliete van die vlugtige olies van die individuele plante en hul kombinasies gedui. Die verhoogde in vitro- antimikrobiese aktiwiteit en farmakologiese interaksies (sinergie en additiwiteit) in van die kombinasies (dubbel én drievoudig) wat in hierdie studie getoets is, bied wetenskaplike stawing vir die gebruik van medisinale plantkombinasies in Wes-Kaapse tradisionele geneeskunde. Die metaboliese profiele van plantkombinasies kan verander weens die interaksie van die verskillende bioaktiewe plantmolekules, en kan baie bepaalde antimikrobiese aktiwiteit tot gevolg hê.

Par définition, les maladies mitochondriales résultent d’un défaut dans le processus des oxydations phosphorylantes (OXPHOS). Celui-ci permet aux cellules de se fournir en ATP, soit la principale source d’énergie qu’elles peuvent utiliser. Dans ce processus, quatre complexes (I-IV) insérés dans la membrane mitochondriale interne transfèrent à l’oxygène moléculaire les équivalents réducteurs libérés par l’oxydation de carbohydrates et d’acides gras. Cette activité génère une force proton motrice utilisée pour la synthèse d’ATP à partir d’ADP et de phosphate inorganique par le complexe V ou ATP synthase.Des maladies dont NARP (Neuropathy Ataxia Retinitis Pigmentosa) et MILS (Maternally Inherited Leigh Syndrome) ont été associées à des mutations de la sous-unité a de l’ATP synthase. Son gène (ATP6) est dans le génome mitochondrial. Celui-ci est présent jusqu’à plusieurs milliers de copies par cellule. Les mutations du gène ATP6 coexistent souvent avec des copies sauvages du génome mitochondrial dans les cellules et tissus des patients, ce qui rend leur étude difficile. La levure Saccharomyces cerevisiae dont on peut modifier à loisir le génome mitochondrial permet de s’affranchir de cette hétérogénéité génétique (appelée hétéroplasmie). De plus, grâce à sa bonne capacité fermentaire, elle est capable de survivre à l’inactivation du système OXPHOS.J’ai au cours de ma thèse exploité ces caractéristiques pour mieux définir les conséquences sur l’ATP synthase de cinq mutations du gène ATP6 identifiées chez des patients : m.8993T>G, m.9191T>C, m.8969G>A, m.8909T>C, et m.9166T>C. Le pouvoir pathogène des trois premières a été établi. Les deux dernières sont des nouveaux variants de l’ADN mitochondrial. Via l’identification de suppresseurs intragéniques, et à la lumière de structures à haute résolution de l’ATP synthase décrites récemment, j’ai pu définir les bases moléculaires des mécanismes pathogènes induits par les mutations m.8993T>G, m.9191T>C, et m.8969G>A. Le variant m.8909T>C a été identifié en combinaison avec une mutation pathogène bien connue dans un ARN de transfert (m.3243A>G). Nous avons trouvé qu’un équivalent de cette nouvelle mutation a en levure des effets délétères sur l’assemblage ou la stabilité de la sous-unité a comparables à ceux induits par des mutations du gène ATP6 (m.8993T>C, m.9176T>C) dont le pouvoir pathogène a été établi, et qu’elle a donc potentiellement la capacité d’affecter seule la santé humaine. Mes études en levure sont cohérentes avec des études ayant conclu récemment à la pathogénicité du variant m.9166T>C et permettent de mieux comprendre comment il impacte l’ATP synthase.J’ai identifié un mécanisme de suppression actif sur des modèles levure de mutations pathogènes de la sous-unité a. Il implique le transporteur des oxodicarboxylates (Odc1) localisé dans la membrane mitochondriale interne. J’ai trouvé que la surexpression d’Odc1 permet une plus grande activité du cycle de Krebs (ou TCA). Ce cycle intervient dans l’oxydation de substrats organiques dont les équivalents réducteurs sont ensuite transférés à l’oxygène par la chaîne respiratoire. Il tourne à bas régime dans les mutants de l’ATP synthase dont l’activité canal à protons est altérée. La suppression-Odc1 dépendante entraîne un découplage partiel de la membrane interne, de sorte que le cycle TCA est stimulé malgré le défaut en ATP synthase. Cet effet permet une plus grande production d’ATP via la phosphorylation d’ADP couplée directement à une des réactions du cycle de Krebs. Ces résultats ouvrent des perspectives intéressantes pour le traitement des maladies associées à des altérations de l’ATP synthase, et possiblement d’autres désordres métaboliques. Cette étude apporte de plus un éclairage nouveau sur le contrôle de la biogenèse du complexe IV par l’ATP synthase
By definition, mitochondrial diseases result from a defect in the process of oxidative phosphorylation (OXPHOS). This is responsible for the production of ATP, the main source of cellular energy. In this process, four multiprotein complexes (I-IV) inserted into the inner mitochondrial membrane transfer to molecular oxygen the reducing equivalents released by the oxidation of carbohydrates and fatty acids. This activity generates a proton motive force used for the synthesis of ATP from ADP and inorganic phosphate by the Complex V or ATP synthase.Diseases including NARP (Neuropathy Ataxia Retinitis Pigmentosa) and MILS (Maternally Inherited Leigh Syndrome) have been associated with mutations in the subunit a of ATP synthase. Its gene (ATP6) is in the mitochondrial genome. This genome is present in up to several thousand copies per cell. Mutations in the ATP6 gene often coexist with wild-type copies of the mitochondrial genome in patients' cells and tissues (heteroplasmy), which makes their study difficult. The yeast Saccharomyces cerevisiae, whose mitochondrial genome can be modified at will, makes it possible to overcome this genetic heterogeneity owing to its incapacity to stably maintaining heteroplasmy. In addition, thanks to its good fermentation capacity, this organism is able to survive mutations that inactivate the OXPHOS system.During my thesis, I exploited these characteristics to better define the consequences on ATP synthase of five ATP6 gene mutations identified in patients: m.8969G>A, m.9191T>C, m.8993T>G, m.8909T>C, and m.9166T>C. The pathogenicity of the first three has been established. The last two are new mitochondrial DNA variants. Through the identification of intragenic suppressors, and in the light of high-resolution structures of ATP synthase described recently, I was able to define the molecular bases of the pathogenic mechanisms induced by the m.8993T>G, m.9191T>C and m.8969G>A mutations. The m.8909T>C variant was identified in combination with a well-known pathogenic mutation in tRNALeu (m.3243A>G). We have found that an equivalent of this new mutation in yeast has deleterious effects on the assembly/stability of the subunit a comparable to those induced by mutations of the ATP6 gene (m.8993T>C, m.9176T>C) with a well-established pathogenicity, and therefore has the potential to affect human health on its own. My studies in yeast are consistent with studies that recently concluded on the pathogenicity of the m.9166T>C variant and allow to better understand how it impacts ATP synthase.I have identified an active suppressor mechanism in yeast models of pathogenic subunit a mutations. It involves the oxodicarboxylate transporter (Odc1) located in the inner mitochondrial membrane. I have found that artificially overexpressing Odc1 allows for greater Krebs cycle (or TCA) activity. This cycle is involved in the oxidation of organic substrates whose reducing equivalents are then transferred to oxygen by the respiratory chain. It runs low in ATP synthase mutants with impaired proton channel activity. The Odc1-dependent suppressor activity results from a partial uncoupling of the inner membrane so that the TCA cycle is stimulated despite the presence of defect in ATP synthase. This effect allows a greater production of ATP via ADP phosphorylation coupled with one of the reactions of the Krebs cycle. These results open interesting perspectives for the treatment of diseases associated with alterations in ATP synthase, and possibly other metabolic disorders. This study also sheds new light on the control of complex IV biogenesis by ATP synthase

In humans and other organisms, monoaminergic systems are crucial in neuronal function and behavior. The monoamine transporters (MATs), which can be found on the presynaptic plasma membrane of neurons in the central nervous system (CNS), are crucial in the regulation of neurotransmitter concentration in the synaptic cleft. As the duration and concentration of neurotransmitters in the cleft affect further downstream signaling responses, these proteins are important targets for both understanding neuronal physiology and compounds of interest. Multiple theories exist proponing the contribution of MATs to a variety of mental and neurological disorders, including depression. This theory establishes that depression is caused by imbalances in monoamine neurotransmitters. Compounds such as Fluoxetine (FLX) are classified as selective serotonin reuptake inhibitors (SSRIs), these drugs selectively block the reuptake of neurotransmitters at the serotonin transporter (SERT). Since differences in MAT selectivity of inhibitory compounds are influential to selecting efficacious antidepressant treatments, we utilized a unique fluorescent analysis technique to explore three therapeutic compounds of interest (in-vitro) which contain structural similarity to FLX. Our results confirm the selectivity of FLX at SERT, and classify the novel compounds studied into different potential categories of reuptake inhibitors. We hope these compounds will be studied further to elucidate their potentially therapeutic roles and mitigation of undesired side effects seen in other medications.

The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target.

The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target.
Jordan University of Science and Technology

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.

The purpose of this current study is to examine the impact of non-pharmacologic pain interventions administered by trained Child Life professionals in an emergency department on pain perception in children. Results showed no significant decrease in children's pain report during the medical procedure compared to before the medical procedure. However, pain after the medical procedure is significantly less than pain during the medical procedure.