Nematodes are highly diverse animals capable of interacting with almost every other form of life on Earth from general trophic interactions to intimate and persistent symbiotic associations. Much of their recognition originates from their various parasitic lifestyles. From an agricultural standpoint, plant parasitic nematodes are widely known for the destruction they can cause to crop plants, such as the case of the root-knot nematode Meloidogyne incognita, or livestock animals, like the Trichinella spiralis, which infects pigs and other animals. From a human health perspective, nematodes can cause many debilitating diseases, for example Wuchereria bancrofti, which is a causative agent of lymphatic filariasis or elephantiasis. However, not all parasitic nematodes have bad implications for human health. For instance, the diverse interactions of insect parasitic nematodes can be used to our benefit. Many of these species have been considered as biological control alternatives to different insect pests that wreak havoc on human, animal, and plant health. There still remain many questions surrounding their evolution, ecology, and physiological capabilities. Many of these taxa are hard to cultivate in the lab due to their complex and intimate lifestyles. Entomopathogenic nematodes (EPNs) are of great interest in agriculture because they vector insect pathogenic bacteria, which are capable of causing death to an insect host within 48 hours post-infection. Much of the molecular underpinnings in this system still remain to be discovered, from understanding the basic ability of these two organisms to associate with one another to genetically engineering more robust and host specific pathogens for application in the field. The focus of the research presented herein is on Steinernematidae nematodes and their bacterial symbionts. Specifically, it focused on the relationship between Xenorhabdus bovienii and its Steinernema hosts. Bioassays were designed to investigate insect virulence of X. bovienii alone in two Lepidoptera insect species with known differential susceptibility to Steinernema-Xenorhabdus pairs. A comparative genomic analysis was performed to compare different Xenorhabdus bovienii strains with observed variation in insect virulence. Results from this analysis demonstrated that virulent strains possess a type VI secretion system (T6SS) locus that is completely absent in strains with attenuated virulence. Bacterial competition assays between T6SS+ and T6SS- strains suggest this locus is involved in bacterial competition. Additionally, symbiont preference assays were carried out to investigate whether Steinernema hosts are able to discern between virulent and attenuated X. bovienii strains. Results from these assays revealed that Steinernema nematodes are able to distinguish between cognate and non-cognate X. bovienii symbionts, giving preference to virulent strains over those with attenuated virulence. Altogether these results provide further evidence that supports the notion that symbiont-switching events have occurred over the Steinernema-Xenorhabdus co-evolutionary history. Specifically, the competitive virulence of certain X. bovienii strains may have conferred them the ability to be selected by different Steinernema hosts, therefore contributing to the success of the nematode-bacterium partnership in being pathogenic to diverse insect hosts.

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: The black wildebeest, Connochaetes gnou, is a terriorial animal and although it is not generally accepted, it is believed that it defines its territory by scent marking, using interdigital and preorbital secretions, faeces, and urine. The aim of this study was to characterize the chemical constituents of the interdigital secretion. Due to the complexity of the secretion, only one hundred and ten of the approximately 350 compounds could be determined with known techniques. Gas chromatography, low resolution GC-MS and retention-time comparison were the main analytical techniques used. Classes of compounds identified in the interdigital secretion include the following: • Hydrocarbons - Aliphatic (saturated and unsaturated) and aromatic • Alcohols - Aliphatic (saturated, unsaturated, cyclic and diols) • Phenols and Phenylalkanols • Aldehydes - Aliphatic (saturated and unsaturated) and aromatic • Ketones - Aliphatic (saturated, unsaturated, cyclic and diketones) and aromatic • Hydroxy ketones - Aliphatic and cyclic • Carboxylic acids - Aliphatic (saturated, unsaturated and cyclic) and aromatic • An anhydride • Esters - Methyl esters, ethyl and higher esters, unsaturated esters and aromatic esters • Lactams • A steroid Only small qualitative and quantitative differences were found between the male and female interdigital secretions. / AFRIKAANSE OPSOMMING: Die swartwildebees, Connochaetes gnou, is 'n territoriale dier en alhoewel dit nie algemeen aanvaar word nie, word vermoed dat hierdie bokke hul gebied afbaken met behulp van interdigitale en preorbitale afskeidings, en deur faeces en urine. Die doel van hierdie studie was om die chemiese samestelling van die interdigitale afskeiding te karakteriseer. As gevolg van die kompleksiteit van die afskeiding, kon slegs eenhonderd-en-tien van die ongeveer 350 verbindings met bekende bestaande tegnieke geïdentifiseer word. Gaschromatografie, lae resolusie GC-MS en retensietyd-vergelyking was die belangrikste analitiese tegnieke wat gebruik is. Klasse van verbindings wat bepaal is, sluit die volgende in: • Koolwaterstowwe - Alifaties (versadig en onversadig) en aromaties • Alkohole - Alifaties (versadig, onversadig, siklies en diole) • Fenole en Fenielalkanole • Aldehiede - Alifaties (versadig en onversadig) en aromaties • Ketone - Alifaties (versadig, onversadig, siklies en diketone) en aromaties • Hidroksiketone - Alifaties en siklies • Karboksielsure - Alifaties (versadig, onversadig en siklies) en aromaties • 'n Anhidried • Esters - Metiel esters, etiel en hoër esters, onversadigde esters en aromatiese esters • Laktame • 'n Steroïed Slegs klein kwalitatiewe en kwantitatiewe verskille is gevind tussen die bul en koei interdigitale afskeidings.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Bontebok, Damaliscus pygargus pygargus, formerly classified as D. dorcas dorcas, are territorial animals with interdigital glands between the toes of the forelegs. Males regularly defecate on dung heaps, on which they often lie, to communicate with other members of their species. They also communicate by means of visual displays, scent marking and occasionally with scraping or pawing of dung heaps. It is assumed that scent marking with the interdigital secretion serves to define territories frequented by these antelope. These glands secrete a complex mixture of volatile and non-volatile compounds and the volatile compounds in the secretion serve as a chemical signal for other bontebok. It has been suggested that the interdigital secretion is not produced in its final composition by the interdigital gland alone, but that microbial activity is responsible for many of the compounds present in the secretion. In general, many compounds can be attributed to the by-products of microbial hydrolysis of triglycerides, a common characteristic of sebum. It is well documented that micro-organisms inhabit the deep recesses of sebaceous glands and the presence of micro-organisms has been found to be consistent in all antelope exocrine glandular areas. This study involved the chemical characterisation of the volatile metabolites produced in vitro by micro-organisms from the interdigital cavity of the bontebok. Various comparative studies were made, one of which was comparison of the metabolites produced by the individual microbial species as well as the total community of bacteria incubated in different media. A comparison of the compounds identified in the interdigital secretion and the metabolites produced by the micro-organisms in the different media was also made. The volatile metabolite extracts of the individual bacterial species and of the total community were chemically characterised by low-resolution gas chromatography-mass spectroscopy. Classes of compounds identified from the volatile metabolite extracts include: • Acids - Aliphatic (saturated and unsaturated) • Alcohols - Aliphatic (saturated and unsaturated) • Aldehydes - Aliphatic (saturated and unsaturated) • Aromatic compounds • Ketones - Aliphatic (saturated and unsaturated) • Pyrazines • Dimethyldisulphide • Squalene and cholesterol Several qualitative differences were found between the compounds identified in the volatile metabolite extracts of the micro-organisms when incubated in tryptic soy broth (TSB) and minimal salt medium (MSM). In particular, when the microbes were incubated in TSB medium a number of pyrazines were found that were not present when utilising MSM as a medium. Additional qualitative differences were found between the compounds identified in the metabolite extracts of the individual bacterial species and the total community of bacteria, when incubated in both TSB and MSM media. A comparison of the interdigital secretion and the metabolite extracts of the microbial communities incubated in TSB and MSM revealed that many compounds produced in MSM corresponded to the compounds identified in the interdigital secretion. These corresponding compounds were found to be saturated and unsaturated acids, aldehydes and squalene. Furthermore, there was only one corresponding compound in the case of TSB as medium. / AFRIKAANSE OPSOMMING: Die bontebok, Damaliscus pygargus pygargus, voorheen geklassifiseer as D. dorcas dorcas, is 'n territoriale dier met interdigitale kliere tussen die kloutjies van die voorpote. Ramme ontlas gereeld op mishope, waarop hulle dikwels lê, om met ander lede van die spesie te kommunikeer. Hulle kommunikeer ook deur middel van visuele seine, reukmerking en soms deur mishope met die voorpote te kap of te skraap. Reukmerking met die interdigitale afskeiding dien klaarblyklik om gebiede wat deur hierdie diere bewoon word, af te baken. Die interdigitale kliere skei 'n komplekse mengsel van vlugtige en nie-vlugtige verbindings af en die vlugtige verbindings dien as chemiese sein vir ander bontebokke. Die vermoede bestaan dat die interdigitale klier nie alleen verantwoordelik is vir die finale samestelling van die interdigitale afskeiding nie, maar dat mikrobiese aktiwiteit bydra tot die produksie van baie van die verbindings wat in die afskeiding aanwesig is. Sekere verbindings kan in die algemeen toegeskryf word aan die vorming van die neweprodukte van mikrobiese hidrolise van trigliseriede, 'n algemene eienskap van sebum. Dit is bekend dat die diep holtes van vetkliere 'n goeie teelaarde is vir mikroorganismes en daar is gevind dat mikroorganismes feitlik deurgaans voorkom in alle anteloop eksokriene klierareas. Hierdie studie behels die chemiese karakterisering van die vlugtige metaboliete wat in vitro deur mikroorganismes van die interdigitale klierholte van die bontebok geproduseer word. Verskeie vergelykende studies is uitgevoer waarvan een die vergelyking was van die metaboliete wat deur die individuele mikrobiese spesies sowel as die totale gemeenskap van bakterieë geproduseer word tydens inkubasie in verskillende media. Vergelyking van die verbindings wat in die interdigitale afskeiding geïdentifiseer is met die metaboliete wat in verskillende media geproduseer is, het ook deel van die studie uitgemaak. Die vlugtige metaboliet ekstrakte van die individuele bakteriese spesies en van die totale gemeenskap is chemies gekarakteriseer deur middel van laeresolusie gaschromatografie-massaspektrometrie. Die volgende groepe verbindings is onder andere in die vlugtige metaboliet ekstrakte geïdentifiseer: • Sure - Alifaties (versadig en onversadig) • Alkohole - Alifaties (versadig en onversadig) • Aldehiede - Alifaties (versadig en onversadig) • Aromatiese verbindings • Ketone - Alifaties (versadig en onversadig) • Pirasiene • Dimetieldisulfied • Skwaleen en cholesterol Verskeie kwalitatiewe verskille is gevind tussen die verbindings wat geïdentifiseer is in die vlugtige metaboliet ekstrakte van die mikroorganismes onderskeidelik in TSB medium en MSM geïnkubeer. Opvallend was byvoorbeeld die voorkoms van pirasiene in gevalle waar mikroorganismes in TSB medium geïnkubeer is, terwyl hierdie groep verbindings afwesig was wanneer MSM gebruik is. Onderlinge kwalitatiewe verskille is ook gevind tussen die verbindings wat geïdentifiseer is in die metaboliet ekstrakte van die individuele bakteriese spesies en die totale gemeenskap van bakterieë, wanneer in TSB medium sowel as in MSM geïnkubeer is. Vergelyking van die verbindings in die interdigitale afskeiding en in die metaboliet ekstrakte van die mikrobiese gemeenskappe, het getoon dat 'n aantal verbindings wat in MSM geproduseer is, ooreenstem met verbindings wat in die interdigitale afskeiding geïdentifiseer is. Daar is gevind dat hierdie verbindings versadigde en onversadigde sure en aldehiede en skwaleen is. Met TSB as medium was daar slegs een ooreenstemmende verbinding.

In addition to their role in hemostasis, platelets appear to contribute to vascular inflammatory diseases. Platelets achieve this through the secretion of various prothrombotic and pro-inflammatory molecules. Platelet secretion is mediated by integral membrane proteins called Soluble NSF Attachment protein REceptors (SNAREs). SNAREs come from both granule/vesicle membranes (v-SNAREs) and target membranes (t-SNAREs) to form a trans-bilayer complex that promotes membrane fusion and subsequent granule cargo release. The work described in this dissertation dissects various, yet related aspects of platelet secretion in both physiological relevant and pathological circumstances. Atherosclerosis is a leading cause of death in the westernized countries and a major contributor to heart attacks and strokes. Given the potential involvement of platelets in atherosclerosis and previous work from our laboratory showing that VAMP-8 is the primary v-SNARE for platelet secretion, one part of this dissertation focuses on the role of VAMP-8- mediated secretion in atherosclerosis. The data showed that the deletion of VAMP-8 in the ApoE-/- null model of chronic atherosclerosis attenuated plaque development compared to the wild type littermates. Aged (50 week) VAMP-8-/-/ApoE-/- mice showed a reduction in lesion size compared to ApoE-/- controls, as measured by Oil Red-O staining of the plaques in the aortic sinus and by en face analysis of plaques in the aortic arch. These data show that the loss of VAMP-8 attenuates the development of atherosclerotic plaques and suggest that platelet secretion contributes to atherosclerosis. Considering the vital role of platelet secretion in both physiological and pathological conditions, it is essential to understand how it is regulated. SNARE proteins are controlled by a range of regulatory molecules that affect where, when, and with whom they form trans-bilayer complexes for membrane fusion. One family of such regulators is the Munc18 family: platelets contain three (Munc18a-c). The second part of this dissertation focuses on the role of Munc18b/STXBP2. Mutations in the Munc18b/STXBP2 gene underlie Familial Hemophagocytic Lymphohistocytosis type 5 (FHL5), which is a life- threatening disease caused by dysregulation of the immune system. Platelets from two biallelic FHL5 patients had almost undetectable levels of Munc18b/STXBP2 levels; the levels of Munc18a increased slightly and Munc18c levels were unaffected. Syntaxin 11 levels were affected but the levels of other secretory machinery proteins were normal. Platelet secretion from dense and alpha granule in two biallelic patients and the one heterozygous patient was decreased. The release of serotonin from dense granules, and platelet factor 4 (PF4) from alpha granules was profoundly affected in the biallelic patients and partially affected in the heterozygote heterozygous patient. Lysosome release was affected only from the platelets of the biallelic patients. These data indicate that Munc18b plays a key role in platelet secretion. Ras is the prototypical member of a family of low molecular weight, GTP-binding proteins. It affects various cellular functions by cycling between an active, guanine triphosphate (GTP) and an inactive guanine diphosphate (GDP) -bound state. Little is known about the role of Ras activation in platelets. The third part of this dissertation focuses on what could be learned about Ras’ role by analyzing platelets from patients with Noonan Syndrome. Specific mutations in the genes encoding elements of Ras signaling pathways are associated with Noonan Syndrome. Platelets from Noonan Syndrome patients with a mutation in kRas (F156V) were analyzed and shown to have a defect in aggregation in response to low levels of agonist. These data suggest that Ras may play a functionally relevant role in platelet activation. In summary, the experiments presented in investigations of this dissertation support a role for platelet secretion in several pathological conditions and suggest that platelet secretion assays may serve as useful as diagnostic tools for some genetic diseases. In addition, these studies elucidate the importance of understanding the regulation of platelet exocytosis, to drive the development of new antithrombotic therapeutics.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Horizontal gene transfer (HGT) is now indisputably the predominant driving force, if not the sole force, behind speciation and the evolution of novelty in bacteria. Of all mechanisms of horizontal gene transfer (HGT), conjugation, the contact-dependent plasmid-mediated transfer of DNA from a bacterial donor to a recipient cell, is probably the most universal. First observed between bacteria, conjugation also mediates gene transfer from bacteria to yeast, plant and even animal cells. The range of environments in which bacteria naturally exchange DNA has not been extensively explored. The interior of the animal cell represents a novel and potentially medically relevant environment for gene transfer. Since most antibiotics are ineffective inside mammalian cells, our cells may be a niche for the evolution of resistance and virulence in invasive pathogens. Invading bacteria accumulate in vacuoles inside human cells, protected from antibiotics. Herein, I demonstrate the ability of intracellular Salmonella typhimurium to meet and exchange plasmid DNA by conjugation within animal cells, revealing the animal intracellular milieu as a permissive environment for gene exchange. This finding evokes a model for the simultaneous dissemination of virulence and antibiotic resistance within a niche protected from both antibiotics and the immune system and extends the variety of environments in which bacteria are known to exchange genes. Unlike conjugation between bacteria, conjugation between bacteria and eukaryotic cells requires the import of transferred DNA into the nucleus before the transferred genes can be expressed and inherited. Plant-cell nuclear transformation by the conjugation system of the Agrobacterium tumefaciens Ti plasmid is believed to be mediated by nuclear localization sequences (NLSs) carried within the proteins that accompany the T-DNA during transfer. Whether NLSs are equally important for transmission of other conjugative plasmids to eukaryotic cells is unknown. Herein, I demonstrate nuclear localization potential within the putative conjugative escort protein TraI of the IncPa plasmid RP4. In contrast, MobA, the putative escort protein from the IncQ plasmid RSF1010, lacked any clear nuclear localization potential. It is therefore likely that specific nuclear localization signals within conjugative proteins are not essential for nuclear transformation per se, although they may assist in efficient plasmid transmission.

Organic cations (OCs) make up ~40% of all prescribed drugs and renal secretion plays a major role in clearing these (and other OCs), from the plasma. The active and rate-limiting step of renal OC secretion is mediated by luminal OC/H+ exchange, the molecular basis of which is suspected to involve two homologous transport proteins, Multidrug And Toxin Extruders 1&2-K (MATE1 and MATE2-K). This study has two aims to resolve outstanding issues dealing with the mechanism of MATE-mediated OC transport: (Aim 1) develop predictive models of ligand interaction with hMATE1; (Aim 2) establish the kinetic mechanism(s) of ligand interaction with MATE transporters and the extent to which inhibitory ligands serve as transported substrates of MATE transporters. Transport was measured using human MATE1 and MATE2-K stably expressed in Chinese Hamster Ovary cells. Both MATEs had similar affinities for the prototypic OC substrate, 1-methyl-4-phenylpyridinium (MPP), and had overlapping selectivity for most of the test inhibitors. The IC50 values for 59 structurally diverse inhibitory ligands were used to generate a common features (HIPHOP) pharmacophore and three quantitative pharmacophores for hMATE1 (each displaying a significant correlation between predicted and measured IC50 values). The models identified (i) structural features that influence ligand interaction with hMATE1, including hydrophobic regions, H-bond donor and acceptor sites and an ionizable feature; and (ii) novel high affinity inhibitors of MATE-mediated transport from 13 new drug classes. Whereas metformin and creatinine were shown to be competitive inhibitors of MPP, the inhibition of MATE1-mediated MPP transport produced by pyrimethamine (PYR) and related analogs was not competitive but, instead, had a "linear, mixed-type" inhibitory profile suggestive of a MATE binding surface rather than a singular binding site. "Competitive exchange diffusion" showed that selected inhibitory ligands (including quinidine, caffeine, and the organic anion, PAH) also serve as transported substrates for MATE1. In conclusion, these data are consistent with the presence of a MATE binding surface with multiple, non-overlapping binding sites that can display different kinetic interactions with structurally distinct substrates. The creation of hMATE1 pharmacophores offers insight into development and interpretation of predictive models of drug-drug interaction in the kidney.

Enterohaemorrhagic Escherichia coli (EHEC) are a recently emerged group of pathogens that can cause fatal infections in the young and elderly. EHEC utilize a virulence factor delivery organelle called a ’Type 3 secretion system’ that results in the formation of characteristic ‘pedestal structures’ on epithelial cells allowing colonization in the human or ruminant gastrointestinal tract. To achieve this, effector proteins have to be injected into host cells. The SepL-SepD complex has been shown to be key for controlling T3-related protein secretion in EHEC. Lack of either protein results in effector hypersecretion and strongly impaired secretion of EspADB translocon proteins. Therefore, the expression and function of SepL was the focus of my PhD research. The expression of SepL was shown to be heterogeneous and co-expressed with EspA filaments in EHEC O157 strains. My work revealed two transcriptional regulators (Ler and SepD) and two putative posttranscriptional regulators (Hfq and CsrA) of SepL expression. Further experiments mapped a key mRNA region required for heterogeneous expression of SepL. This sequence forms a predicted hairpin structure around the Shine-Dalgarno (SD) site of sepL. A model has been formed based on my data in which Hfq and CsrABCD bind to the mRNA potentially competing to control translation. Functionally, the C-terminus of SepL was found to be expendable for 1) SepD binding; 2) SepL membrane localization and 3) translocon export, however it was required for 1) limiting effector secretion via (2) a Tir interaction which might be disassociated by (3) an EscD interaction once host cell signals are sensed. Previously, the concept of two different types of T3 secretion signal were demonstrated in Yersinia spp, I tested this hypothesis in EHEC using both wild type and SepL/SepD deficient EHEC strains. SepL/SepD is required for the N-terminal signal pathway but not a chaperone binding domain signal pathway. A 12aa NleA which only contained an N-terminal signal was shown to bind to SepD and so did the multi-functional T3 chaperone ― CesT. Finally, Far-Western assays demonstrated that SepL only interacted with Tir while SepD could bind other effector proteins indicating that SepL/SepD may act as a targeting hub for effector protein secretion.

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

Tau est une protéine associée aux microtubules enrichie dans l’axone. Dans la maladie d’Alzheimer, Tau devient anormalement hyperphosphorylée, s’accumule dans le compartiment somato-dendritique et s’agrège pour former des enchevêtrements neurofibrillaires (NFTs). Ces NFTs se propagent dans le cerveau dans un ordre bien précis. Ils apparaissent d’abord dans le cortex transenthorinal pour ensuite se propager là où ces neurones projettent, c’est-à-dire au cortex entorhinal. Les NFTs s’étendent ensuite à l’hippocampe puis à différentes régions du cortex et néocortex. De plus, des études récentes ont démontré que la protéine Tau peut être sécrétée par des lignées neuronales et que lorsqu’on injecte des agrégats de Tau dans un cerveau de souris, ceux-ci peuvent pénétrer dans les neurones et induire la pathologie de Tau dans le cerveau. Ces observations ont mené à l’hypothèse que la protéine Tau pathologique pourrait être sécrétée par les neurones, pour ensuite être endocytée par les cellules avoisinantes et ainsi propager la maladie. L’objectif de la présente étude était donc de prouver la sécrétion de la protéine Tau par les neurones et d’identifier par quelle voie elle est secrétée. Nos résultats ont permis de démontrer que la protéine Tau est sécrétée par des neurones corticaux de souris de type sauvage ainsi que dans un modèle de surexpression dans des cellules HeLa et PC12. Nos résultats indiquent que la sécrétion de Tau se ferait par les autophagosomes. Finalement, nous avons démontré que la protéine Tau sécrétée est déphosphorylée et clivée par rapport à la protéine Tau intracellulaire non sécrétée. / Tau, a microtubule-associated protein, is enriched in the axon. In Alzheimer’s disease, Tau becomes hyperphosphorylated, redistributes to the somato-dendritic compartment and forms aggregates called neurofibrillary tangles (NFTs). The NFTs propagates in a predictable manner in particular neuronal networks. Indeed, they appear in the trans-entorhinal region and then propagate to the entorhinal cortex where the trans-entorhinal cortex projects. Then, the NFTs propagate to the hippocampus and to different regions of the cortex and neocortex. Recent studies have reported that Tau can be secreted by neuronal cell lines. Besides, when aggregates of Tau protein were injected in mouse brain, they could enter neurons and induced Tau pathology. Based on those observations, it was speculated that Tau could be secreted by neurons and then captured by neighbouring cells to propagate Tau pathology in the brain. The goal of the present study was to prove that Tau can be secreted by neurons and to find the secretory pathway involved in Tau secretion. Moreover, the phosphorylation state of Tau protein was examined and compared to intracellular non-secreted Tau. Our results showed that Tau is secreted by cortical neurons isolated from wild-type mice and by HeLa and PC12 cells overexpressing human Tau. Our results also indicated that autophagosomes would be involved in Tau secretion. Finally, we found that secreted Tau was dephosphorylated and cleaved compared to the non-secreted intracellular Tau.