Positron emission tomography (PET) is a quantitative and non-invasive investigative tool, which permits the identification of pivotal biomarkers and their role in disease onset, transformation and progression. Quantitative detection of these disease biomarkers has qualified PET as a pioneering approach to rapid diagnosis. The tool is a powerful approach to patient diagnosis and investigation of inter- and intra- patient disease heterogeneity thereby supporting patient stratification and forging a truly personalised medicine approach. The sensitivity of PET is complemented by use of highly selective radiopharmaceuticals; this has fuelled the popularity of radiolabelled affinity peptides and proteins (APPs). Inspired by the high selectivity and affinity of biological protein-protein interactions, APP-based radiotracers are an increasingly popular class of radiotracer. Monoclonal antibodies (mAbs) are an important category of APP-based radiotracer; their prevalence in the field is attributed to their high target selectivity and affinity characteristics, which, will likely, uphold their popularity in the field. Yet, advancements in protein engineering has transformed the landscape of APP-based radiotracers, indicated by a preference for small radiolabelled APPs which exhibit rapid pharmacokinetics whilst retaining high target specificity and affinity. Their fast pharmacokinetics is matched by the half-life of 18F which has been defined as an ideal PET radionuclide. 18F APP radiolabelling hasn't yet met its full potential due to its inherent challenges; many approaches have been adopted, ranging from non-site specific radiolabelling with amine reactive prosthetic groups to site-specific methods including oxime bond formation. The radiochemistry of 18F APP radiolabelling is diverse and evolving; yet ideal goals are apparent including site-specificity, fast reaction kinetics, mild labelling conditions and applicability to automation. Automation is challenging for multi-step 18F APP radiolabelling methods, but is a key developmental step. Automation helps with conformance to good manufacturing practice (GMP) by enhancing process robustness, consistency and reliability. It also permits the radiosynthesis of clinically relevant radiotracer doses. The development of an automated generic platform, or a method that adopts a general approach, for APP radiolabelling with 18F would assist in [18F]APPs meeting their potential in PET and, importantly, aid their translation from bench-to-bedside.

Neurodegeneration, the progressive and irrevocable loss of neuronal structure, is quickly becoming an imposing health concern in a globally ageing society. While specific neurodegenerative conditions exhibit specific clinical symptoms and progressions, a common neuropathological feature is the misfolding, oligomerisation and fibrillation of certain proteins causing neuronal stress and death. Parkinson’s disease, PD, has long been characterised by the death of nerve cells focused in the substantia nigra pars compacta region of the midbrain and deposition of large protein aggregates, called Lewy Bodies, throughout the central nervous system. More recently, the protein which forms these inclusion bodies was identified as alpha synuclein, αSyn, a ubiquitous neuroprotein with no known function. Furthermore, persons with mutations in the SNCA gene, which codes for αSyn, exhibit PD progression at a far younger age with a more severe phenotype, positively linking αSyn with PD. αSyn is an intrinsically disordered protein, IDP, and generally persists as such in solution and inside bacterial and mammalian cells. However, when in contact with a lipid bilayer the protein will embed upon the surface in an amphipathic alpha helical conformation and can also aggregate, forming toxic oligomeric and fibrillar species containing significant β-sheet identity. Its function as a helical apolipoprotein and subcellular localisation to both the nucleus and synapse has led researchers to suggest that αSyn has a role synaptic transmission and release. However, knocking out the protein does not reduce viability or produce pathological abnormalities in neuronal structure. The helical form of the protein may also persist as transient, metastable helical bundles which are non-toxic and resist aggregation. While a number of studies and tools have been reported and developed to investigate the toxic oligomeric/fibrillar forms of αSyn, very little attention has been accorded to the helical conformation. This thesis will redress this balance by producing tools which will allow us to mimic the helical form of αSyn, promote the active refolding of the full-length protein using a stable, helical peptide template and produce antibodies which recognise helical αSyn specifically for use in discovery and chaperone-like refolding. In Chapter 2 a region of αSyn (14 amino acids) was identified with a unique primary sequence located within a mutation prone section of the protein. Peptide ‘stapling’ technologies were then employed using a panel of monosubstituted ‘staple’ diastereomers, to produce a highly helical portion of αSyn. Using several other protein targets particular diastereomeric ‘staple’ combinations were analysed for obvious trends in helical content. Using solution NMR, backbone refined three dimensional structures of these helical peptides were produced which showed that they were faithful structural homologues of their parent helical proteins. In Chapter 3 the drug-like properties and therapeutic potential of stable, helical αSyn peptides were investigated. Using fluorescently labelled peptide substrates, ‘stapled’ peptides were shown to be far more cell penetrant than their wild type equivalents and demonstrated that the mechanism for cellular uptake appears to be specific. Furthermore, under harsh proteolytic conditions the ‘stapled’, helical peptides were far more resistant to hydrolysis than wild type or ‘stapled’, poorly helical peptides. The ‘stapled’ peptides were also highly soluble and did not appear to aggregate in a time-dependent manner. Using ion mobility mass spectrometry, it was shown that incubation of full-length protein with the ‘stapled’, helical peptides caused a contraction in the hydrodynamic radius of the protein. However, using solution NMR no active refolding of αSyn was observed when under the same conditions. Rather small perturbations in chemical shift were apparent which did not suggest that the αSyn protein folded into a discrete structural conformation, such as an alpha helix. In Chapter 4 the stable, helical αSyn peptide was employed as a conformational model and unique antigen in antibody discovery. Immunisation with the ‘stapled’, helical αSyn peptide initially produced a pool of polyclonal antibodies with a half log specificity for the helical peptide. After bespoke affinity chromatography this was increased to three log orders of specificity. Initial immunocytochemistry did not detect any helical αSyn protein in SH-SY5Y cells. To validate the helical epitope on the full-length protein in vitro an assay based around flow cytometry of synthetic vesicle structures was developed, with their synthesis, characterisation and binding of the αSyn protein described.

In this study, both physiological and cellular effects are elicited by natriuretic peptides (NPs), a novel type of plant hormone. It was found that rat ANP (rANP) influenced stomatal opening movement in Tradescantia sp., where a significant increase in stomatal opening was observed in the presence of 1 µM rANP. Furthermore, this effect is mediated by cGMP, a (putative) second messenger of NPs. Two inhibitors of guanylyl cyclase, LY 83583 and methylene blue, inhibited rANP-induced stomatal opening. In contrast, stomatal opening is induced in a concentration dependent manner by the cell permeant cGMP analogue 8-Br-cGMP. In addition it was found, that like in animals, the secondary structure of rANP is essential for rANP responses. Linearised rANP is biologically inactive. Since ANP elicit plant responses, an attempt was made to isolate NP analogues from plants. A protocol for partially purifying NP from plants was developed. It was found that two fractions eluted from an immunoaffinity chromatography column (0.5 M KCI eluted fraction and 0.75 M KCI eluted fraction) were biologically active. The level of cGMP in response to NPs was also tested. It is suggested that the receptor of NP is specific since only 0.75 M KCI eluted fractions increased cGMP levels in Zea mays root stele tissue. rANP did not elicit an effect on cGMP levels in this tissue and LY 83583 did not affect this response. It is therefore argued that a plant specific biologically active NP system is present in the stele and it is predicted that NPs modulate solute movement in this tissue. NPs also influence K⁺, Na⁺ and H⁺ fluxes in Zea mays root stele. Increase in both K⁺ and Na+ uptake were observed after 30 min., while H⁺ flux shifted immediately toward influx in the presence of both 0.5 and 0.75 KCI eluted fractions. Finally, a model is proposed for the effect of NPs on solute movement and its signalling system in plants.

Objectives: To study the expression of cathelicidin antimicrobial peptides (CAMP) and surfactant protein D (SP-D) in patients with chronic rhinosinusitis (CRS) and eosinophilic mucus chronic rhinosinusitis (EMCRS) and by a nasal explant in vitro model cultured with fungal allergens. Methods: Nasal biopsies from 59 CRS and EMCRS patients, stratified into Allergic fungal sinusitis (AFS), Nonallergic fungal eosinophilic sinusitis (NAFES), and Nonallergic nonfungal eosinophilic sinusitis (NANFES) were studied by quantitative real-time (q)RTPCR, Western blot, immunostaining and ELISA. Nasal tissue from CRS and EMCRS patients were cultured with increasing concentrations of fungal allergens in a nasal explant in vitro model for 24 hours and CAMP and SP-D mRNA and protein levels in response to the fungi were determined by qRT-PCR and ELISA. Results: The expression of CAMP mRNA was significantly increased in EMCRS patients compared to CRS patients (p=0.0004). By immunohistochemistry, expression of CAMP was localised to nasal epithelial, submucosal glands and inflammatory subepithelial cells. Western blotting demonstrated the presence of CAMP in the study patients. Culturing nasal explants with fungal allergens demonstrated significant upregulation of CAMP mRNA expression in CRS, but not EMCRS patients, by Aspergillus (mean 4-fold increase) and Alternaria (mean 6-fold increase) extracts with a significant dose-response effect (p<0.001). CAMP protein levels in the nasal tissue from CRS patients increased in response to Alternaria (p<0.05). In contrast, with EMCRS patients the expression of CAMP peptide in nasal tissue increased with Aspergillus (p<0.001) but decreased with Alternaria. Staining for SP-D was detected in the submucosal glands from the nasal biopsies in all patient groups except for AFS. By ELISA, SP-D was undetectable in AFS and decreased in NAFES, NANFES, and CRS compared to controls. CRS patients cultured with Aspergillus and Alternaria allergens in the in vitro nasal explant model induced significant upregulation of SP-D mRNA (p<0.0001). In contrast, NANFES nasal tissue explants cultured with Aspergillus allergens induced downregulation of SP-D and only a modest upregulation of SP-D mRNA to Alternaria allergens. Conclusion: This study demonstrates expression of cathelicidin antimicrobial peptides and surfactant proteins in nasal mucosa supporting its potential role in innate defences against inhaled pathogens. There is significant upregulation of CAMP mRNA in the EMCRS group implying an increased inflammatory state. In vitro, CAMP is significantly upregulated at the mRNA and protein level in CRS tissue explants to Aspergillus and Alternaria allergens. However, EMCRS tissue cultured with Alternaria in vitro does not demonstrate increased CAMP at the mRNA or protein level. The expression of SP-D in nasal tissue is reported for the first time. SP-D expression in the CRS, but not the EMCRS group, is upregulated in vitro by Aspergillus and Alternaria. The EMCRS group compared to CRS group demonstrate abnormal CAMP and SP-D expression to common fungal allergens. These important findings in understanding the pathogenesis of chronic rhinosinusitis are discussed in this thesis and may provide potential novel therapies for chronic rhinosinusitis in the future. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287042 / Thesis(PhD)-- School of Medicine, 2007

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

Separations coupled to mass spectrometry (MS) are widely used for large-scale protein identification in order to reduce the adverse effects of analyte ion suppression, increase the dynamic range, and as a deconvolution technique for complex datasets typical of cellular protein complements. In this work, matrix assisted laser desorption-ionization is coupled with ion mobility (IM) separation for the analysis of biological molecules. The utility of liquid-phase separations coupled to MS lies in the orthogonality of the two separation dimensions for all analytes. The data presented in this work illustrates that IM-MS relies on the correlation between separation dimensions for different classes (either structural or chemical) of analyte ions to obtain a useful separation. For example, for a series of peptide ions of increasing mass-to-charge (m/z) a plot drift time in the IM drift cell vs. m/z increases in a near-linear fashion, but DNA or lipids having similar m/z values will have very different IM drift time-m/z relationships, thus drift time vs. m/z can be used as a qualitative tool for compound class identification. In addition, IM-MS is applied to the analysis of large peptide datasets in order to determine the peak capacity of the method for bottom-up experiments in proteomics, and it is found that IM separation increases the peak capacity of an MS-only experiment by a factor of 5-10. The population density of the appearance area for peptides is further characterized in terms of the gas-phase structural propensities for tryptic peptide ions. It is found that a small percentage (~3%) of peptide sequences form extended (i.e., helical or β-sheet type) structures in the gas-phase, thus influencing the overall appearance area for peptide ions. Furthermore, the ability of IM-MS to screen for the presence of phosphopeptides is characterized, and it is found that post translationally modified peptides populate the bottom one-half to one-third of the total appearance area for peptide ions. In general, the data presented in this work indicates that IM-MS offers dynamic range and deconvolution capabilities comparable to liquid-phase separation techniques coupled to MS on a time scale (ms) that is fully compatible to current MS, including TOF-MS, technology.

A Matrix-assisted Laser Desorption/Ionization (MALDI) – Ion Mobility (IM) – Surface-induced Dissociation (SID) – Time-of-Flight (TOF) instrument with three different collision source configurations was developed in order to improve the SID performance in high throughput peptide sequencing. The first version of the instrument was equipped with an angle resolved SID source in order to maximize the collection efficiency of the SID scattering ions. An orthogonal TOF was also implemented as the second MS stage in this instrument to increase mass resolution. The second version of the instrument was developed towards simplifying the coupled configuration of the IM, SID and TOF components by using a combined SID/TOF source with a confinement ring electrode as the collision target. The fragmentation efficiency of SID in this configuration was increased up to 50% due to the surface normal impact angle used as compared with the results from a previous experiment using 45 degree impact angle. The third version of the instrument was equipped with a dual-source/dual-detector TOF to facilitate high throughput tandem analysis of peptides through simultaneous separation, fragmentation and mass analysis, while retaining precursor ion identity in the same experimental sequence. A series of small organic molecules, model peptides and tryptic peptides from a protein digest were analyzed to demonstrate the utility of these new designs for enhanced SID performance and peptide sequencing capability. Finally, a new mobility drift cell using a periodic focusing mechanism has been designed and fabricated to replace the previous uniform field drift cell. Improvement in ion transmission has been observed in the periodic focusing drift cell instrument without sacrificing the mobility resolution.

Le récepteur d'œstrogènes a (ROa), dont l'implication dans le développement du cancer du sein est clairement établie, appartient à la super-famille des récepteurs nucléaires qui sont des facteurs de transcription dont l'activité dépend d'un ligand régulateur, en l'occurrence le 17b-œstradiol. Le ROa régit l'expression de gènes nécessaires à la prolifération et à la différentiation cellulaire, ceci par association avec des séquences d'ADN situées à proximité de leur promoteur. Le ROa, une fois associé à son hormone, se défait des protéines qui le stabilisent dans une forme inactive (chaperonnes et corépresseurs) permettant ainsi le recrutement de protéines coactivatrices. Ces dissociations / associations se font de façon séquentielle selon un programme cyclique dans lequel la dégradation protéasomale du récepteur semble jouer un rôle clé. Les thérapies visant à contrecarrer la progression des cancers mammaires hormono-dépendants sont principalement basées sur l'inactivation du ROa. A cette fin et à ce jour, deux stratégies sont mises en œuvre: l'inhibition de la production d'œstrogènes (inhibiteurs d'aromatases) et l'inhibition du ROa lui-même (anti-œstrogènes). Face à l'apparition de résistances à ces traitements, la recherche de nouvelles cibles s'avère nécessaire. En effet, que ce soit de façon indirecte (inhibiteurs d'aromatases) ou directe (anti-œstrogènes), toutes les molécules utilisées en clinique ciblent la poche de liaison de l'hormone. Une autre approche visant à entraver l'action du ROa serait l'inhibition du recrutement de ses coactivateurs. Ainsi, il a été récemment rapporté que des peptides ou des mimes peptidiques inhibant de tels recrutements pourraient effectivement être d'utilité thérapeutique. Notre travail s'inscrit dans cette optique: l'étude du motif P295-T311 du ROa nous a permis de mettre en évidence une cible distincte de celles décrites ci-dessus. Ce motif semble être une région critique dans le contrôle de l'activité transcriptionnelle du ROa. Il est, en effet, sujet à phosphorylation, acétylation, méthylation, ubiquitination, SUMOylation et protéolyse. Par ailleurs, des études antérieures à ce travail ont suggéré son implication dans la liaison d'une protéine corégulatrice du ROa: la calmoduline (CaM), le principal senseur du calcium intracellulaire. Cependant, le rôle de cette association dans le processus d'activation du récepteur restait partiellement indéterminé. Afin d'étudier le rôle de la CaM dans ce processus, nous avons produit un peptide synthétique correspondant au motif P295-T311, l'ERa17p (séquence: P295LMIKRSKKNSLALSLT311). L'ERa17p se lie à une matrice de CaM-Sepharose de façon Ca2+-dépendante et inhibe la liaison du ROa à cette même matrice ce qui confirme que le motif P295-T311 est un élément essentiel pour la liaison de la CaM au ROa. De plus et au même titre que des molécules "anti-CaM", l'ERa17p est capable de dissocier les complexes ROa-CaM endogènes des cellules cancéreuses mammaires MCF-7. De façon remarquable, le peptide ERa17p présente un profil pharmacologique pseudo-œstrogénique en culture cellulaire. Il accroît, en effet, l'activité transcriptionnelle du ROa ainsi que la prolifération de lignées tumorales mammaires ROa-positives tout en étant inactif sur des lignées ROa-négatives. De plus, et de la même façon que pour les œstrogènes, ces effets sont liés à la diminution du taux de ROa. Une dégradation précoce du récepteur ainsi qu'une diminution ultérieure de sa synthèse sont à la base de cette "down regulation". L'activité pseudo-œstrogénique de l'ERa17p ne peut toutefois pas être attribuée à l'inhibition de la liaison ROa-CaM car des peptides analogues, dont certains acides aminés ont été substitués de façon à les rendre inactifs vis-à-vis de la CaM, présentent des propriétés biologiques similaires. Dans le but d'étudier les mécanismes engendrant l'œstrogénicité de l'ERa17p, nous avons synthétisé un analogue biotinylé qui nous a permis d'isoler une protéine stabilisatrice et coinhibitrice du ROa susceptible d'interagir avec l'ERa17p: l'Hsp70. La démonstration complémentaire que l'ERa17p entrave la liaison ROa-Hsp70 suggère que l'ERa17p pourrait être un inhibiteur compétitif de cette association. Par ailleurs, des études menées par modélisation moléculaire à partir de données cristallographiques du récepteur ont révélé l'existence d'une interaction intramoléculaire entre le motif S301-T311 et l'hélice 4 (H4) située dans un domaine de liaison de coactivateurs (H3-H5). Cette interaction pourrait prévenir le recrutement de ces coactivateurs, maintenant ainsi le ROa dans un état inactif. La mise en évidence de la liaison d'un ROa recombinant hautement purifié à l'ERa17p suggère que ce dernier pourrait aussi inhiber l'interaction S301-T311 – H4 et contribuer, de la sorte, à l'activation du récepteur. L'observation qu'un mutant de délétion pour le motif P295-T311 présente une activité transcriptionnelle constitutive élevée est en accord avec cette hypothèse et démontre le caractère répresseur de ce motif. Ainsi, nous avons démontré que la région P295-T311 possède une activité auto-répressive vis-à-vis de l'activation du ROa. Lorsque ce dernier se trouve dans un état inactif, l'Hsp70 serait associée à ce motif et maintiendrait le ROa dans une forme compacte (liaison intramoléculaire S301-T311 – H4) inapte au recrutement de protéines coactivatrices. En présence de son hormone, le récepteur se libérerait de l'entrave de l'Hsp70 ce qui permettrait son "ouverture" ("relaxation") et le recrutement de coactivateurs. La CaM s'associerait au ROa à ce stade, prévenant la réassociation de la chaperonne et empêchant tout retour à un état inactif. L'ERa17p jouerait donc le rôle de la CaM en inhibant, par compétition, la liaison de l'Hsp70 au ROa. D'autre part, étant donné l'implication de la dégradation du ROa dans son processus transcriptionnel, nous émettons l'hypothèse que le récepteur est régulé par des produits issus de cette dégradation; celle-ci engendrerait l'émergence de peptides régulateurs (ERa17p–like) capables d'amplifier les cycles transcription / dégradation initiés par le stimulus hormonal.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

A major problem associated with cellular cryopreservation is the recovery of cellular viability upon thawing. Current cryopreservation techniques use additives such as DMSO, sucrose and fetal bovine serum. However, each have their own respective cytotoxic issues. A significant factor in cryotoxicity is the formation of large ice crystals which can damage cellular components and cause dehydration. This has significant impacts for applications such as food preservation, scientific research, and tissue preservation. To this end, our laboratory is interested in synthesizing biologically-relevant compounds that can act as cryoprotectants by preventing the formation of large ice crystals in sub-zero temperatures. Our lab has previously synthesized structural analogues of native antifreeze glycoproteins (AFGPs, found in the blood of Antarctic cod), that possess the unique ability to inhibit ice-recrystallization. However, the mechanism by which they inhibit ice recrystallization is unclear. This thesis focuses on efforts made to understand this mechanism, and synthesize molecules that are more potent in ice recrystallization inhibition (IRI) activity compared to previously synthesized analogues. By assessing the IRI activity of various mono- and disaccharides, we have shown that the density of water molecules that surround a carbohydrate (Hydration Index) is directly proportional to the ability of sugars to inhibit ice crystal growth. In an effort to design functional AFGP analogues, various C-linked analogues were synthesized that contained different spacer lengths between the carbohydrate and the peptide backbone. Analyses of the solution conformations of these analogues showed that IRI-active AFGP analogues contain a distinct conformation in which the carbohydrate is oriented to form a hydrophobic pocket with the side chain. We hypothesize that this change in glycoconjugate hydration is responsible for disturbing its surrounding waters, thereby preventing water from adding to the ice lattice required for ice growth. Finally, SAR studies showed that threonine-containing AFGP and antifreeze proteins are more potent in antifreeze activity than serine-containing analogues. As the most potent AFGP analogue previously synthesized by our lab contains a C-linked-α-galactosyl -serine residue, we hypothesized that the analogous glycopeptide containing a C-α-galactosyl-threonine residue will be more potent in antifreeze activity. The final section describes efforts to synthesize a C-linked α-galactosyl-threonine glycoconjugate.