This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of p-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of bis(neopentyl glycolato)diboron or bis(pinacolato)diboron and 2-I-p-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules. The DNA intercalator doxorubicin has been functionalized to enable 125I labelling. The aim of combining the DNA intercalator with 125I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation. The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective. The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid. This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

The disturbances and abnormalities occurring in the components of the Acetylcholine (ACh) neurocycle are considered one of the main features of cholinergic sicknesses like Parkinson’s and Alzheimer’s diseases. A fundamental understanding of the ACh neurocycle is therefore very critical in order to design drugs that keep the ACh concentrations in the normal physiological range. In this dissertation, a novel two-enzyme-two-compartment model is proposed in order to explore the bifurcation, dynamics, and chaotic characteristics of the ACh neurocycle. The model takes into consideration the physiological events of the choline uptake into the presynaptic neuron and the ACh release in the postsynaptic neuron. In order to approach more realistic behavior, two complete kinetic mechanisms for enzymatic processes pH-dependent are built: the first mechanism is for the hydrolysis reaction catalyzed by the acetylcholinesterase (AChE) and the other is for the synthesis reaction catalyzed by the cholineacetyltransferase (ChAT). The effects of hydrogen ion feed concentrations, AChE activity, ChAT activity, feed ACh concentrations, feed choline concentrations, and feed acetate concentrations as bifurcation parameters, on the system performance are studied. It was found that hydrogen ions play an important role, where they create potential differences through the plasma membranes. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the activity of AChE through a certain range of their concentrations, where the activity of AChE is inhibited completely after reaching certain values. A detailed bifurcation analysis over a wide range of parameters is carried out in order to uncover some important features of the system, such as hysteresis, multiplicity, Hopf bifurcation, period doubling, chaotic characteristics, and other complex dynamics. The effects of the feed choline concentrations and the feed acetate concentrations as bifurcation parameters are studied in this dissertation. It is found that the feed choline concentrations play an important role and have a direct effect on the ACh neurocycle through a certain important range of the parameters. However, the feed acetate concentrations have less effect. It is concluded from the results that the feed choline is a more important factor than the feed acetate in ACh processes. The effects of ChAT activity and the choline recycle ratio as bifurcation parameters, on the system performance are investigated. It was found that as the ChAT activity increases, ACh concentrations in compartments 1 and 2 increase continuously. The effect of the choline recycle ratio shows that choline reuptake plays a very critical role in the synthesis of ACh in compartment 1, where it supplies the choline as a substrate for the synthesis reaction by ChAT. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the choline recycle ratio through a certain range of the choline recycle ratio; then, they become constant as the choline recycle ratio increases further. It is concluded from our results that choline uptake is the rate limiting step in the ACh processes in both compartments in comparison to ChAT activity. Based on partial dissociation of the acetic acid in compartments 1, and 2 of the ACh cholinergic system, the two-parameter continuation technique has been applied to investigate the pH range to be closer to physiological ranges of pH values. In addition, static/dynamic solutions of the ACh cholinergic neurocycle system based on feed choline concentration as the main bifurcation parameter in both compartments have been investigated. The findings of the above studies are related to the real phenomena occurring in the neurons, like periodic stimulation of neural cells and non-regular functioning of ACh receptors. It was found that ACh, choline, acetate, and pH exist inside the physiological range associated with taking into consideration the partial dissociation of the acetic acid. The disturbances and irregularities (chaotic attractors) occurring in the ACh cholinergic system may be good indications of cholinergic diseases such as Alzheimer’s and Parkinson’s diseases. The results have been compared to the results of physiological experiments and other published models. As there is strong evidence that cholinergic brain diseases like Alzheimer’s disease and Parkinson’s disease are related to the concentration of ACh, the present findings are useful for uncovering some of the characteristics of these diseases and encouraging more physiological research.

The disturbances and abnormalities occurring in the components of the Acetylcholine (ACh) neurocycle are considered one of the main features of cholinergic sicknesses like Parkinson’s and Alzheimer’s diseases. A fundamental understanding of the ACh neurocycle is therefore very critical in order to design drugs that keep the ACh concentrations in the normal physiological range. In this dissertation, a novel two-enzyme-two-compartment model is proposed in order to explore the bifurcation, dynamics, and chaotic characteristics of the ACh neurocycle. The model takes into consideration the physiological events of the choline uptake into the presynaptic neuron and the ACh release in the postsynaptic neuron. In order to approach more realistic behavior, two complete kinetic mechanisms for enzymatic processes pH-dependent are built: the first mechanism is for the hydrolysis reaction catalyzed by the acetylcholinesterase (AChE) and the other is for the synthesis reaction catalyzed by the cholineacetyltransferase (ChAT). The effects of hydrogen ion feed concentrations, AChE activity, ChAT activity, feed ACh concentrations, feed choline concentrations, and feed acetate concentrations as bifurcation parameters, on the system performance are studied. It was found that hydrogen ions play an important role, where they create potential differences through the plasma membranes. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the activity of AChE through a certain range of their concentrations, where the activity of AChE is inhibited completely after reaching certain values. A detailed bifurcation analysis over a wide range of parameters is carried out in order to uncover some important features of the system, such as hysteresis, multiplicity, Hopf bifurcation, period doubling, chaotic characteristics, and other complex dynamics. The effects of the feed choline concentrations and the feed acetate concentrations as bifurcation parameters are studied in this dissertation. It is found that the feed choline concentrations play an important role and have a direct effect on the ACh neurocycle through a certain important range of the parameters. However, the feed acetate concentrations have less effect. It is concluded from the results that the feed choline is a more important factor than the feed acetate in ACh processes. The effects of ChAT activity and the choline recycle ratio as bifurcation parameters, on the system performance are investigated. It was found that as the ChAT activity increases, ACh concentrations in compartments 1 and 2 increase continuously. The effect of the choline recycle ratio shows that choline reuptake plays a very critical role in the synthesis of ACh in compartment 1, where it supplies the choline as a substrate for the synthesis reaction by ChAT. The concentrations of ACh, choline and acetate in compartments 1 and 2 are affected by the choline recycle ratio through a certain range of the choline recycle ratio; then, they become constant as the choline recycle ratio increases further. It is concluded from our results that choline uptake is the rate limiting step in the ACh processes in both compartments in comparison to ChAT activity. Based on partial dissociation of the acetic acid in compartments 1, and 2 of the ACh cholinergic system, the two-parameter continuation technique has been applied to investigate the pH range to be closer to physiological ranges of pH values. In addition, static/dynamic solutions of the ACh cholinergic neurocycle system based on feed choline concentration as the main bifurcation parameter in both compartments have been investigated. The findings of the above studies are related to the real phenomena occurring in the neurons, like periodic stimulation of neural cells and non-regular functioning of ACh receptors. It was found that ACh, choline, acetate, and pH exist inside the physiological range associated with taking into consideration the partial dissociation of the acetic acid. The disturbances and irregularities (chaotic attractors) occurring in the ACh cholinergic system may be good indications of cholinergic diseases such as Alzheimer’s and Parkinson’s diseases. The results have been compared to the results of physiological experiments and other published models. As there is strong evidence that cholinergic brain diseases like Alzheimer’s disease and Parkinson’s disease are related to the concentration of ACh, the present findings are useful for uncovering some of the characteristics of these diseases and encouraging more physiological research.

Spinal cord injuries (SCI) sever communication between supraspinal centers and the central pattern generator (CPG) responsible for locomotion. Because the CPG is intact and retains the ability to initiate locomotor activity, it can be accessed electrically and pharmacologically. The goal of this thesis was to identify and characterize a novel spinal cord surface site along the sacral dorsal column (sDC) for electrically evoking locomotor-like activity in the neonatal rat spinal cord. Stimulation of the sDC robustly activated rhythmic left-right alternation in flexor-related ventral roots that was dependent on the activation of high-threshold C fiber afferents. The C fibers synapsed onto spinal neurons, which project to the lumbar segments as part of a pathway dependent on purinergic, adrenergic, and cholinergic receptor activation. In ventral roots containing only somatic efferents, rhythmic activity was rarely recruited. However, in ventral roots containing both autonomic and somatic efferents, sacral dorsal column stimulation recruited autonomic efferent rhythms, which subsequently recruited somatic efferent motor rhythms. The efferent rhythms revealed a half-center organization with very low stimulation frequencies, and the evoked alternating bursts entrained to the stimuli. Similar entrainment was seen when sDC stimuli were applied during ongoing neurochemically-induced locomotor rhythms. The rhythmic patterns evoked by sDC stimulation operated over a limited frequency range, with a discrete burst structure of fast-onset, frequency-independent peaks. In comparison, neurochemically-induced locomotor bursts operated over a wide frequency range and had slower time to peaks that varied with burst frequency. The overall findings support the discovery of an autonomic efferent pattern generator that is recruited by sacral visceral C fiber afferents. It is hoped that this research will advance the understanding of afferent activation of the lumbar central pattern generator and potentially provide insight useful for future development and design of neuroprosthetic devices.

Hemp seed proteins (HSP) were investigated for physicochemical and functional properties in model food systems. In addition, the HSP were enzymatically digested and the released peptides investigated as potential therapeutic agents. Membrane isolated HSP (mHPC) were the most soluble with >60% solubility at pH 3-9 when compared to a maximum of 27% for isoelectric pH-precipitated proteins (iHPI). However, iHPI formed emulsions with smaller oil droplet sizes (<1 µm) while mHPI formed bigger oil droplets. The iHPI was subjected to enzymatic hydrolysis using different concentrations (1-4%) of six proteases (pepsin, pancreatin, flavourzyme, thermoase, papain and alcalase) to produce various HSP hydrolysates (HPHs). HPHs had strong in vitro inhibitions of angiotensin converting enzyme (ACE) and renin activities, the two main enzyme systems involved in hypertension. Oral administration of the HPHs to spontaneously hypertensive rats led to fast and persistent reductions in systolic blood pressure. The HPHs also inhibited in vitro activities of acetylcholinesterase (AChE), a serine hydrolase whose excessive activities lead to inadequate level of the cholinergic neurotransmitter, acetylcholine (ACh). Inadequate ACh level in the brain has been linked to neurodegenerative diseases such as dementia and Alzheimer’s disease (AD); therefore, AChE inhibition is a therapeutic target. The 1% pepsin HPH was the most active with up to 54% AChE inhibition at 10 µg/mL peptide concentration. The 1% pepsin HPH (dominated by <1 kDa) was subjected to reverse-phase HPLC peptide purification coupled with tandem mass spectrometry, which led to identification of several peptide sequences. Some of the peptides inhibited activities of both animal and human AChE forms with LYV being the most potent against human AChE (IC50 = 7 µg/ml). Thus the LYV peptide may serve as a useful template for the development of future potent AChE-inhibitory peptidomimetics. In conclusion, several novel AChE-inhibitory peptides were discovered and their amino acid sequences elucidated for the first time. Results from this work identified HSP products that could serve as functional ingredients in the food industry. The work also produced and confirmed the in vitro AChE-inhibitory activities of several new peptide sequences that may serve as therapeutic agents for AD management. / October 2015

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Recent evidence indicates that the mature neocortex retains a higher degree of plasticity than traditionally assumed. Up- and down-regulation of synaptic strength, long-term potentiation (LTP) and long-term depression (LTD), is thought to be the primary mechanism mediating experience-dependent plasticity of cortical networks. The present thesis investigate factors that regulate adult cortical plasticity, focusing on the role of neuromodulators, recent sensory experience, and different anatomical divisions of the cortex in influencing synaptic strength. First, I investigated the role of the neuromodulator histamine in gating plasticity in the primary visual cortex (V1) of urethane anesthetized adult rats. Histamine applied locally in V1 produced an enhancement of LTP elicited by theta burst stimulation (TBS) of dorsal lateral geniculate nucleus (dLGN) and allowed a sub-threshold TBS to produce stable LTP. Second, the impact of visual deprivation on LTP in V1 was assessed. Animals that received 2 and 5 hr dark exposure showed greater potentiation of field potentials when stimulated though retinal light flashes or weak TBS of the dLGN, which failed to induce LTP in control animals kept in continuous light. Third, I performed a detailed characterization of LTP induced by different TBS protocols, recording in either the monocular or binocular segment of both V1 hemispheres (i.e., ipsi- and contralateral to the stimulated dLGN). Stronger, NMDA receptor-independent LTP was found in the contralateral V1. Interestingly, weak TBS induced LTD that was NMDA receptor-dependent in the ipsilateral V1. Furthermore, a lower LTP induction threshold was observed in the binocular than the monocular segment of ipsilateral V1. Lastly, I investigated cholinergic modulation of sensory-induced activity in the barrel cortex. Basal forebrain stimulation enhanced multi-unit activity elicited by whisker deflection, an effect that was more pronounced for weaker response driven by a secondary whisker than principal whisker deflection. This thesis demonstrates that neocortical plasticity consists of multiple forms of synaptic modification. Adult cortical plasticity is greatly influenced by preceding activity of the synapse by various neuromodulator systems, and by anatomical subdivisions within primary sensory cortex fields. Together, these mechanisms may facilitate the detection, amplification, and storage of inputs to primary sensory fields of the neocortex. / Thesis (Ph.D, Psychology) -- Queen's University, 2010-04-29 14:02:30.742

The notion that learning and memory processes are highly dependent on central cholinergic neurotransmission has been widely accepted. However, studies documenting the importance of Acetylcholine (ACh) in attention have led some to suggest that attention impairments may underlie the deficits in learning and memory resulting from cholinergic disruptions. Using a visual discrimination task, I attempted to discern whether performance impairments by Scopolamine were predominantly due to the importance of muscarinic receptor integrity in attention, or memory consolidation in learning. Rats were trained in a visual discrimination task using a Y-shaped water maze apparatus. To successfully navigate to a hidden platform located in one of the two goal arms, rats learned to discriminate between 2 distinct visual cues, indicating the platform’s presence (CS+) or absence (CS-), respectively. Following task acquisition, testing continued using a combination of Regular trials (RT; both CS+ and CS- present) and Probe trials (PT; only one of the cues present). Results indicated that performance on PT was impaired due to greater task difficulty under conditions of reduced information, while Scopolamine (1 mg/kg) further impacted PT performance without affecting RTs. In a second experiment, PTs were administered with the platform present to provide reinforcement and a learning opportunity. Animals still exhibited poorer PT performance, but rapidly learned to rely on a single cue for accurate platform localization. Interestingly, this learning was not apparent under conditions of Scopolamine treatment (1 mg/kg), even though RT performance was completely unaffected. To examine experience-dependent changes in neuronal responding after visual discrimination learning, a subset of animals were anesthetised and visual evoked potentials (VEPs) in V1 and area CA1 of the hippocampus were recorded in response to CS+, CS-, and novel stimuli. In both the V1 and CA1, the VEP amplitudes elicited to familiar and novel stimuli were not significantly different. First, these experiments demonstrate the importance of the cholinergic system in sustaining visual attention and acquiring a new single-cue strategy. Furthermore, the null electrophysiology findings do not rule out the plastic response properties of the mature V1 and CA1, but remind us of the complex nature of memory encoding in the brain. / Thesis (Master, Psychology) -- Queen's University, 2011-09-16 13:50:24.045

Ethanol causes neurotoxicity via several mechanisms at different points in the cycle of dependence, including neuroinflammation and oxidative stress during ethanol exposure as well as excitotoxicity during ethanol withdrawal. The primary therapeutic implication is that ethanol-induced neurotoxicity requires multifunctional pharmacotherapies which reduce all mechanisms. Using an innovative pharmacological high throughput screening method on a large plant extract library we discovered flavonoids with alpha7 nicotinic acetylcholine receptor (nAChR) activity. In addition to their well-known anti-inflammatory and antioxidant properties, this novel activity means they can potentially reduce excitotoxicity and therefore makes them ideal for inhibition of ethanol-induced neurotoxicity. Rhamnetin, the candidate compound, was first found to inhibit lipopolysaccharide induced inflammation in immortalized BV2 microglia, in part, via alpha7 nAChRs. We then established an in vitro model of ethanol induced-neurotoxicity using organotypic hippocampal slice cultures which incorporated both neuroinflammatory and excitotoxic components. Neuroinflammation enhanced excitotoxicity under control conditions but the reverse was observed during ethanol withdrawal. Both mechanisms are important but their interaction is not simple. Finally, rhamnetin was evaluated in this model and found to reduce neuroinflammation and excitotoxicity associated with ethanol withdrawal. In conclusion, the studies herein provide strong evidence for alpha7 nAChRs selective flavonoids as potential pharmacotherapies for the treatment of ethanol-induced neurotoxicity and further implicate neuroinflammation, excitotoxicity, and their interaction as critical mechanisms in this pathology.