The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

The impact of the gut microbiota on drug metabolism and clinical outcome

The significance of the gut microbiota as a determinant of drug pharmacokinetics and accordingly therapeutic response is of increasing importance with the advent of modern medicines characterised by low solubility and/or permeability, or modified-release. These physicochemical properties and release kinetics prolong drug residence times within the gastrointestinal tract, wherein biotransformation by commensal microbes can occur. As the evidence base in support of this supplementary metabolic “organ” expands, novel opportunities to engineer the microbiota for clinical benefit have emerged. This review provides an overview of microbe-mediated alteration of drug pharmacokinetics, with particular emphasis on studies demonstrating proof of concept in vivo. Additionally, recent advances in modulating the microbiota to improve clinical response to therapeutics are explored.

Growing up in a bubble: using germ-free animals to assess the influence of the gut microbiota on brain and behavior

There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ-free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders.

Behavioural and neurochemical consequences of chronic gut microbiota depletion during adulthood in the rat

Gut microbiota colonization is a key event for host physiology that occurs early in life. Disruption of this process leads to altered brain development which ultimately manifests as changes in brain function and behaviour in adulthood. Studies using germ-free mice highlight the extreme impact on brain health that results from life without commensal microbes, however the impact of microbiota disturbances occurring in adulthood is less studied. To this end, we depleted the gut microbiota of 10-week-old male Sprague Dawley rats via chronic antibiotic treatment. Following this marked, sustained depletion of the gut bacteria, we investigated behavioural and molecular hallmarks of gut-brain communication. Our results reveal that depletion of the gut microbiota during adulthood results in deficits in spatial memory as tested by Morris water maze, increased visceral sensitivity and a greater display of depressive-like behaviours in the forced swim test. In tandem with these clear behavioural alterations we found change in altered CNS serotonin concentration along with changes in the mRNA levels of corticotrophin releasing hormone receptor 1 and glucocorticoid receptor. Additionally, we found changes in the expression of BDNF, a hallmark of altered microbiota-gut-brain axis signaling. In summary, this model of antibiotic-induced depletion of the gut microbiota can be used for future studies interested in the impact of the gut microbiota on host health without the confounding developmental influence of early-life microbial alterations.

Establishment and development of infant gut microbiota

The nascent gut microbiota at birth is established in concert with numerous developmental parameters. Here, in the INFAMTET study, we chronicled the impact of some factors which are key determinants of the infant gut microbiota, namely; mode of birth, gestational age, and type of feeding. We determined that the aggregated microbiota profile of naturally delivered, initially breastfed infants are relatively stable from one week to six months of age and are not significantly altered by increased duration of breastfeeding. Contrastingly, there is significant development of the microbiota profile of C-section delivered infants, and this development is significantly influenced by breastfeeding duration. Preterm infants, born by either mode of birth, initially have a high proportion of Proteobacteria, and demonstrate significant development of the gut microbiota from week 1 to later time-points. The microbiota is still slightly, but significantly, affected by birth mode at one year of age although no specific genera were found to be significantly altered in relative abundance. By two years of age, there is no effect of either birth mode or gestational age. However this does not preclude the possibility that symptoms developed later in life, which are associated with preterm or C-section birth, are as a result of the early perturbation of the neonatal gut microbiota. It is likely that the combination of relatively low exposure (breast fed), high exposure (formula fed) or delayed exposure (C-section and preterm) to specific antigens and the resulting inflammatory responses, in this crucial window of host-microbiota interaction, influence systemic health of the individual throughout life.

Atlantic salmon in regulated rivers : Migration, dam passage, and fish behavior

Hydropower dams block migration routes and disrupt longitudinal connectivity in rivers, thereby posing a threat to migratory fish species. Various fish passage solutions have been implemented to improve connectivity with varying success. A well-functioning passage solution must ensure safe and timely passage routes that are used by a substantial portion of the migrating fish. In this thesis, I report the results from telemetry studies where the behavior and survival of migrating Atlantic salmon spawners, post-spawners and smolts have been evaluated in relation to hydropower dam passage. I evaluate downstream passage performance at dams with no passage solutions in the River Klarälven, and with simple passage solutions in in the Winooski River. In the River Ätran, I study both upstream- and downstream passage performance at a dam with sophisticated passage solutions based on the best available technology. In addition, I have studied the survival and behavior of post-spawners and hatchery-released smolts. A substantial portion of the spawners survived spawning and initiated downstream migration. Most males migrated downstream in autumn following spawning, whereas females tended to stay in the river until spring. For hatchery-reared smolts, early release was associated with faster initiation of migration and higher survival compared to late release. Multiple dam passage resulted in high mortality for both smolts and kelts. For smolts, dam passage, even with simple passage solutions, was associated with substantial delay and mortality. High spill levels were linked to high survival and short delay for downstream migrating salmon. The best available passage solution, which consisted of a nature-like fishway and a low sloping intake rack to guide fish to a bypass, resulted in rapid passage of a large portion of the adult migrants.

Le double passage de la société à l’État et de l’État à la société (mondiale) dans la philosophie politique d’Éric Weil

Dans sa Philosophie politique, Éric Weil constate que le langage de la réflexion oppose, "comme entités autonomes et hostiles", "la civilisation et la tradition , le matériel et l’idéal, le nécessaire et le désirable, l’organisation et la justice, l’intérêt commun et la liberté des individus". Délaissant la pure réflexion, le philosophe a le devoir de montrer que la civilisation du travail se fonde en définitive sur la tradition morale porteuse de sens qui bénéficie en retour de sa rationalité et son universalité. Les couples mentionnés plus haut ne se séparent donc pas, mais s’impliquent et se confortent. L’action politique est l’illustration de cet entrelacement ou ce chiasme: elle transforme "ce qui est coexistence en conflit" ou ce qui se présente comme désaccordé et disparate en une unité indissociable, si on exclut la réflexion. Le mouvement de la philosophie politique d’Éric Weil laisse ainsi place à des rapprochements et à des réconciliations. La société, qui est l’organisation rationnelle de la lutte avec la nature et la négation de l’individualité, trouve son achèvement dans l’État moderne qu’elle rend possible et qui, conformément à son concept, est pour l’individu, le lieu du sens. Cet entrecroisement se vérifie de nouveau au plan de la politique étrangère. C’est grâce à la mise sur pied d’une société mondiale de gestion et de contrôle que les États particuliers, sans renoncer à leur souveraineté externe, en arrivent à considérer la question internationale comme d’intérêt commun et peuvent, libérés de la nécessité des préparatifs militaires, se consacrer à leur vocation essentielle: l’épanouissement de l’individu raisonnable. La violence cède peu à peu devant les institutions rationnelles et raisonnables: elle devient consciente dans la marche de l’histoire et la satisfaction des justes revendications laisse entrevoir le moment où elle sera inutile. La violence pure est certes irréductible, mais elle est démasquée comme discours incohérent ou comme silence sans pensée.

Uncovering trophic interactions in arthropod predators through DNA shotgun-sequencing of gut contents.

2016

Misunderstanding or Misdirection?: Whistleblower Protection in Queensland

In his report into corruption in Queensland, Fitzgerald listed whistleblower protection as a necessary part of a strong governance regime. "What is required is an accessible, independent body to which disclosures can be made, confidentially (at least in the first instance) and in any event free from fear of reprisals." It was one of the reforms studied by the Electoral and Administrative Review Committee, the report of which resulted in the Whistleblowers Protection Act 1994 (WPA). The need for whistleblower protection was supported by all sides of Parliament. The Premier, Wayne Goss, in his Second Reading Speech on the Public Sector Ethics Bill , said that that Act and the WPA would form a package with the former outlining required behaviour and the WPA encouraging staff to report wrongdoing. The WPA was subsequently passed and has remained virtually unamended for over a decade. Such consistency is either an indication of skilled drafting and effectiveness or the fact that the Act has been neglected. It is the hypothesis of this paper that the latter is the case. This hypothesis will be tested by examining the sincerity and diligence with which the Act has been treated during, and following, its passage.

Non-alcoholic fatty liver disease : can ultrasound assist in early diagnosis of prediabetes and delay progression to type2 diabetes mellitus

Non Alcoholic Fatty Liver Disease (NAFLD) is a condition that is frequently seen but seldom investigated. Until recently, NAFLD was considered benign, self-limiting and unworthy of further investigation. This opinion is based on retrospective studies with relatively small numbers and scant follow-up of histology data. (1) The prevalence for adults, in the USA is, 30%, and NAFLD is recognized as a common and increasing form of liver disease in the paediatric population (1). Australian data, from New South Wales, suggests the prevalence of NAFLD in “healthy” 15 year olds as being 10%.(2) Non-alcoholic fatty liver disease is a condition where fat progressively invades the liver parenchyma. The degree of infiltration ranges from simple steatosis (fat only) to steatohepatitis (fat and inflammation) steatohepatitis plus fibrosis (fat, inflammation and fibrosis) to cirrhosis (replacement of liver texture by scarred, fibrotic and non functioning tissue).Non-alcoholic fatty liver is diagnosed by exclusion rather than inclusion. None of the currently available diagnostic techniques -liver biopsy, liver function tests (LFT) or Imaging; ultrasound, Computerised tomography (CT) or Magnetic Resonance Imaging (MRI) are specific for non-alcoholic fatty liver. An association exists between NAFLD, Non Alcoholic Steatosis Hepatitis (NASH) and irreversible liver damage, cirrhosis and hepatoma. However, a more pervasive aspect of NAFLD is the association with Metabolic Syndrome. This Syndrome is categorised by increased insulin resistance (IR) and NAFLD is thought to be the hepatic representation. Those with NAFLD have an increased risk of death (3) and it is an independent predictor of atherosclerosis and cardiovascular disease (1). Liver biopsy is considered the gold standard for diagnosis, (4), and grading and staging, of non-alcoholic fatty liver disease. Fatty-liver is diagnosed when there is macrovesicular steatosis with displacement of the nucleus to the edge of the cell and at least 5% of the hepatocytes are seen to contain fat (4).Steatosis represents fat accumulation in liver tissue without inflammation. However, it is only called non-alcoholic fatty liver disease when alcohol - >20gms-30gms per day (5), has been excluded from the diet. Both non-alcoholic and alcoholic fatty liver are identical on histology. (4).LFT’s are indicative, not diagnostic. They indicate that a condition may be present but they are unable to diagnosis what the condition is. When a patient presents with raised fasting blood glucose, low HDL (high density lipoprotein), and elevated fasting triacylglycerols they are likely to have NAFLD. (6) Of the imaging techniques MRI is the least variable and the most reproducible. With CT scanning liver fat content can be semi quantitatively estimated. With increasing hepatic steatosis, liver attenuation values decrease by 1.6 Hounsfield units for every milligram of triglyceride deposited per gram of liver tissue (7). Ultrasound permits early detection of fatty liver, often in the preclinical stages before symptoms are present and serum alterations occur. Earlier, accurate reporting of this condition will allow appropriate intervention resulting in better patient health outcomes. References 1. Chalasami N. Does fat alone cause significant liver disease: It remains unclear whether simple steatosis is truly benign. American Gastroenterological Association Perspectives, February/March 2008 www.gastro.org/wmspage.cfm?parm1=5097 Viewed 20th October, 2008 2. Booth, M. George, J.Denney-Wilson, E: The population prevalence of adverse concentrations with adiposity of liver tests among Australian adolescents. Journal of Paediatrics and Child Health.2008 November 3. Catalano, D, Trovato, GM, Martines, GF, Randazzo, M, Tonzuso, A. Bright liver, body composition and insulin resistance changes with nutritional intervention: a follow-up study .Liver Int.2008; February 1280-9 4. Choudhury, J, Sanysl, A. Clinical aspects of Fatty Liver Disease. Semin in Liver Dis. 2004:24 (4):349-62 5. Dionysus Study Group. Drinking factors as cofactors of risk for alcohol induced liver change. Gut. 1997; 41 845-50 6. Preiss, D, Sattar, N. Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations. Clin Sci.2008; 115 141-50 7. American Gastroenterological Association. Technical review on nonalcoholic fatty liver disease. Gastroenterology.2002; 123: 1705-25

Vom Prosumer zum Produser : Ein neues Verständnis nutzergesteuerter Inhaltserschaffung

Alvin Tofflers Bild des Prosumers beeinflußt weiterhin maßgeblich unser Verständnis vieler heutzutage als „Social Media“ oder „Web 2.0“ beschriebener nutzergesteuerter, kollaborativer Prozesse der Inhaltserstellung. Ein genauerer Blick auf Tofflers eigene Beschreibung seines Prosumermodells offenbart jedoch, daß es fest im Zeitalter der Massenmedienvorherrschaft verankert bleibt: der Prosumer ist eben nicht jener aus eigenem Antrieb aktive, kreative Ersteller und Weiterbearbeiter neuer Inhalte, wie er heutzutage in Projekten von der Open-Source-Software über die Wikipedia bis hin zu Second Life zu finden ist, sondern nur ein ganz besonders gut informierter, und daher in seinem Konsumverhalten sowohl besonders kritischer als auch besonders aktiver Konsument. Hochspezialisierte, High-End-Konsumenten etwa im Hi-Fi- oder Automobilbereich stellen viel eher das Idealbild des Prosumers dar als das für Mitarbeiter in oft eben gerade nicht (oder zumindest noch nicht) kommerziell erfaßten nutzergesteuerten Kollaborationsprojekten der Fall ist. Solches von Tofflers in den 70ern erarbeiteten Modells zu erwarten, ist sicherlich ohnehin zuviel verlangt. Das Problem liegt also nicht bei Toffler selbst, sondern vielmehr in den im Industriezeitalter vorherrschenden Vorstellungen eines recht deutlich in Produktion, Distribution, und Konsum eingeteilten Prozesses. Diese Dreiteilung war für die Erschaffung materieller wie immaterieller Güter durchaus notwendig – sie ist selbst für die konventionellen Massenmedien zutreffend, bei denen Inhaltsproduktion ebenso aus kommerziellen Gründen auf einige wenige Institutionen konzentriert war wie das für die Produktion von Konsumgütern der Fall ist. Im beginnenden Informationszeitalter, beherrscht durch dezentralisierte Mediennetzwerke und weithin erhaltbare und erschwingliche Produktionsmittel, liegt der Fall jedoch anders. Was passiert, wenn Distribution automatisch erfolgt, und wenn beinahe jeder Konsument auch Produzent sein kann, anstelle einer kleinen Schar von kommerziell unterstützten Produzenten, denen bestenfallls vielleicht eine Handvoll von nahezu professionellen Prosumern zur Seite steht? Was geschieht, wenn sich die Zahl der von Eric von Hippel als ‚lead user’ beschriebenen als Produzenten aktiven Konsumenten massiv ausdehnt – wenn, wie Wikipedias Slogan es beschreibt, ‚anyone can edit’, wenn also potentiell jeder Nutzer aktiv an der Inhaltserstellung teilnehmen kann? Um die kreative und kollaborative Beteiligung zu beschreiben, die heutzutage nutzergesteuerte Projekte wie etwa die Wikipedia auszeichnet, sind Begriffe wie ‚Produktion’ und ‚Konsum’ nur noch bedingt nützlich – selbst in Konstruktionen wie 'nutzergesteuerte Produktion' oder 'P2P-Produktion'. In den Nutzergemeinschaften, die an solchen Formen der Inhaltserschaffung teilnehmen, haben sich Rollen als Konsumenten und Benutzer längst unwiederbringlich mit solchen als Produzent vermischt: Nutzer sind immer auch unausweichlich Produzenten der gemeinsamen Informationssammlung, ganz egal, ob sie sich dessens auch bewußt sind: sie haben eine neue, hybride Rolle angenommen, die sich vielleicht am besten als 'Produtzer' umschreiben lassen kann. Projekte, die auf solche Produtzung (Englisch: produsage) aufbauen, finden sich in Bereichen von Open-Source-Software über Bürgerjournalismus bis hin zur Wikipedia, und darüberhinaus auch zunehmend in Computerspielen, Filesharing, und selbst im Design materieller Güter. Obwohl unterschiedlich in ihrer Ausrichtung, bauen sie doch auf eine kleine Zahl universeller Grundprinzipien auf. Dieser Vortrag beschreibt diese Grundprinzipien, und zeigt die möglichen Implikationen dieses Übergangs von Produktion (und Prosumption) zu Produtzung auf.

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the Kd of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar Kd values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.