Vascular calcification is a prominent cardiovascular condition found worldwide. This condition is predominantly found in the elderly population, and patients who suffer from chronic kidney disease, due to an imbalance of serum phosphate and calcium levels. For many years, vascular calcification was believed to be a passive pathological process which develops with ageing and/or lifestyle. Little has been documented about the disease until the 20th century, when interest in cardiovascular research grew amongst scientists. Indeed, vascular calcification underpins severe clinical outcomes and cardiovascular diseases have been labelled the global leading cause of death. Calcific aortic valve diseases (CAVD) is a progressive degenerative condition characterised by the development of lipo-calcification around the aortic valve leaflets leading to severe aortic stenosis and aortic regurgitation, which may ultimately lead to heart failure. At present there are no pharmaceutical therapies that can stop its progression and its molecular mechanisms are not fully understood. Recent findings have suggested that vascular smooth muscle cell (VSMC) calcification shares many common features with physiological skeletogenesis via the release of matrix vesicles (MVs), which are specialised structures that initiate mineralisation during bone formation. The ability for MVs to nucleate calcium and phosphate highly depend on their protein composition, as this may vary depending on active cell signalling and the microenvironment. This mechanism involving MV-regulated calcification has yet to be examined in CAVD. In this study, examined whether calcium and/or phosphate regulate VIC-derived MVs to induce calcification in the aortic valve. I used a primary rat valve interstitial cell (VIC) model, coupled with stenotic human valve tissues to characterise and study the mechanisms underpinning CAVD. X-ray fluorescence and diffraction analysis showed the mineral found in calcified human aortic valves to be hydroxyapatite (HA), the main component in bone. Additional imaging studies employing transmission electron microscopy (TEM) revealed particles that were similar in size and morphology to skeletal MVs. To further characterise VIC-derived MVs in vitro, I harvested MVs from rat VICs, and subsequently studied their protein composition using Isobaric tag for relative and absolute quantitation (iTRAQ) mass spectrometry. The data obtained from the proteomics analysis was compared to previous published studies on MV proteins derived from osteoblasts and VSMCs. The results showed the upregulation of numerous calcification regulators in MVs isolated from all 3 cell types, in particular, the Annexin family, which are known calcium binding proteins. Further studies conducted with Annexin 6, an established calcium regulator in arterial calcification, revealed its colocalisation with MV-enriched areas in calcified human aortic valve tissue suggesting it may play an important role in calcium regulation during CAVD.

O Trypanosoma cruzi libera para o meio, vesículas contendo material de sua superfície e aparentemente estas vesículas seriam uma maneira de ativar e preparar a célula hospedeira para a invasão, além de induzir um aumento na expressão de alguns componentes da matriz extracelular. Neste trabalho demonstramos que essas vesículas aumentam a expressão de fibronectina na matriz, sendo este aumento dose-dependente e linear ao longo do tempo. Porém, para laminina não conseguimos observar o mesmo comportamento. Nossos resultados também mostram que os constituintes lipídicos das vesículas podem ser os responsáveis pelo aumento da expressão de fibronectina em cultura de células epiteliais. Os dois grandes grupos de glicoproteínas encontradas na superfície do parasita e nas vesículas, mucinas e Tc85 não parecem estar envolvidos no processo. As culturas celulares tratadas com os lipídeos extraídos das vesículas apresentaram um aumento de fibronectina também dose-dependente, porém, com uma resposta linear ao longo do tempo e uma expressão máxima atingida em tempos menores / Trypanosoma cruzi releases to the environment plasma membrane vesicles. Apparently, these vesicles could be a signal released by the parasite to prepare the host cell for the invasion. This study demonstrated that these vesicles induce a dosisdependent expression of fibronectin, linear over time. The same behavior has not been observed for laminin. Our results also show that lipids from the vesicles are involved in the increase of expression of fibronectin by epithelial cells. The two major surface membrane glycoproteins, Tc85 and mucins, also present in the vesicles do not participate in this phenomenon. Cell cultures that have been treated with lipids extracted from T. cruzi membrane vesicles also provoked a dosis-dependent increase in fibronectin and linear over time

Human Pegivirus (HPgV; originally called GB virus C) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are infected with HPgV. No causal association between HPgV and disease has been identified; however, several studies found an association between persistent HPgV infection and prolonged survival of HIV-infected individuals that appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (>10 million genome copies/ml plasma) but grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. The overall goals of my thesis were to characterize HPgV tropism, effects of HPgV infection on host immune response and mechanisms of viral persistence. Previous studies found HPgV RNA in T and B lymphocytes and ex vivo infected lymphocytes produce viral particles. To further characterize HPgV tropism, we quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naïve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) obtained from persistently infected humans using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 9), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naïve (CD45RA+) CD4+ cells than in central memory and effector memory cells (p<0.01). HPgV sequences were highly conserved between patients (0.117 ± 0.02 substitutions per site) and within subjects (0.006 ± 0.003 substitutions per site). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-positive microvesicles (SEV) from serum delivered CFSE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells (PBMCs) with evidence of subsequent viral replication. Thus, HPgV RNA-positive SEV may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus. Although HPgV infection reduces NK cell activation in HIV-infected individuals, the mechanism by which this occurs is not characterized. We studied HPgV effects on NK cell non-cytolytic function in HIV-infected people by measuring expression of IL-12 induced interferon gamma (IFNg) and cytolytic function by measuring K562 target-cell induced CD107a and granzyme B. IFNg expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects treated with combination antiretroviral therapy (p=0.02). In contrast, cytolytic NK cell functions were not affected by HPgV. Inhibition of IFNg was due to inhibition of tyrosine kinase (Tyk2) by HPgV envelope protein E2. HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited IL-12-mediated IFNg release by NK cells. Thus HPgV-E2 inhibits NK cell non-cytolytic functions. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV's ability to persist with high viral loads (>10 million genome copies/ml plasma) and reduce immune cell activation. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.

Neste trabalho foram analisadas sob o ponto de vista físico-químico, as interações entre DNA e lipossomos catiônicos de brometo de dioctadecildimetilamônio (DODAB). Foram utilizados os seguintes modelos: 1) 2\'-desoxiadenosina 5\'-monofosfato (DMP), um modelo de unidade monomérica do polímero que é o DNA; 2) DNAs de bacteriófagos T2, T4, T5, T7 e &#955;; 3) vesículas pequenas de DODAB preparadas por sonicação; 4) vesículas grandes de DODAB preparadas por aquecimento (56 ºC/30 minutos) ou vaporização clorofórmica. A interação entre 2\'-desoxiadenosina 5\'-monofosfato (DMP) e lipossomos de DODAB resultou em adsorção máxima de DMP ao lipossomo catiônico com uma proporção molar DODAB : DMP de 2:1 e mobilidade eletroforética (ME) mínima mas positiva dos lipossomos. Em força iônica de 5 mM, a adsorção máxima de DMP sobre os lipossomos aproxima-se de zero, mostrando que a formação do complexo DODAB/DMP é essencialmente dirigida pela atração eletrostática. A adição de nucleotídeo na faixa de milimolar (0,4 - 1,5 mM) induz a um aumento de turbidez da dispersão de lipossomos (DODAB 0,08 mM) em função do tempo. Ocorrem velocidades de floculação muito maiores que as obtidas por adição de NaCl (40 120 mM). O nucleotídeo comporta-se como um ânion hidrofóbico com uma afinidade pela membrana muito maior que a exibida por um ânion simples como o cloreto. DMP induz ruptura dos lipossomos contendo [14C]sacarose, sugerindo que a interação DMP/bicamada não é superficial. Embora a interação preserve a carga positiva do liposomo, a integridade lipossomal não é preservada. Na adsorção máxima, a inserção de DMP na bicamada é a explicação mais razoável para manutenção da carga positiva da vesícula, proporção molar DODAB : DMP de 2:1 e extravasamento de conteúdo interno lipossomal. A interação DODAB/DNA também é dirigida por atração eletrostática entre o DNA e a bicamada catiônica. Marcadores incorporados ao DNA ou a sítios da bicamada são deslocados para a água devido à interação. Sob condições de excesso de DODAB, na situação de adsorção máxima de DODAB sobre DNA, existem cerca de 70 moléculas de DODAB por nucleotídeo de DNA e esta proporção não depende do tipo de DNA. A interação DODAB/DNA leva à formação de glóbulos visíveis por microscopia óptica de campo escuro e ocorrência de uma dependência linear entre turbidez e 1/&#955;2, onde &#955; é o comprimento de onda da luz incidente. Na condição de adsorção máxima de DODAB, a formação de complexos globulares DODAB/DNA causa perda de hipocromismo da dupla fita de DNA conforme detectado por efeitos de temperatura sobre absorbância em 260 nm de misturas DNA/DODAB. Em suma, a interação de DODAB/DNA não é superficial: o lipossomo perde sua integridade e o DNA perde sua estrutura em dupla hélice, tornando-se fita simples. A atração hidrofóbica entre bases nitrogenadas de DNA e cadeias hidrocarbônicas dos lipídios catiônicos tem papel importante na determinação da estrutura do complexo. / In this work, interactions between DNA and cationic liposomes made up of dioctadecyldimethylammonium bromide (DODAB) are evaluated from a physicochemical point of view. The following models were used: 1) 2\'-deoxyadenosine 5\'-monophosphate (DMP), a model of monomeric unity of polymer, the DNA; 2) DNAs of T2, T4, T5, T7 and &#955; bacteriophages; 3) small vesicles of DODAB prepared by sonication; 4) large vesicles of DODAB prepared by heating (56 ºC/30 minutes) or by chloroform vaporization. The interaction between 2\'deoxyadenosine 5\'-monophosphate (DMP) and cationic liposomes made up of dioctadecyldimethylammonium bromide (DODAB) in water is described. At maximal adsorption, the molar ratio DODAB/DMP is 2:1 and electrophoretic mobility for the liposomes attains a minimum at a positive value. At 5 mM ionic strength, maximal DMP adsorption on the liposome becomes close to zero, demonstrating that the electrostatic attraction essentially drives the DODAB/DMP complexation. Over the millimolar range of DMP concentrations (0.4-1.5 mM), upon nucleotide addition, turbidity of the liposome dispersion (0.08 mM DODAB) steeply increases as a function of time in contrast with the much smaller flocculation rates upon NaCl addition over a much higher range of NaCl concentrations (40-120 mM). The nucleotide behaves as a hydrophobic anion with an affinity for the membrane that is much higher than that exhibited by a simple anion as chloride. DMP-induced rupture of liposomes containing [14C]sacarose was evaluated from dialysis of DMP/liposomes mixtures. In water, DMP-induced leakage of radioactive liposomal contents suggests that the DMP/bilayer interaction is not superficial. Although the interaction preserves the positive liposome charge, it doesn\'t preserve its integrity. At maximal adsorption, DMP insertion in the cationic bilayer is the most reasonable explanation for the remaining positive charge on the vesicle, the 2:1 DODAB : DMP molar ratio, and leakage of internal contents from the liposome. The DODAB/DNA interaction is also driven by the electrostatic attraction between DNA and bilayer. Probes located on DNA or in the bilayer are displaced from their DNA or bilayer sites. Under conditions of DODAB excess, at maximal DODAB adsorption on DNA, there are ca. 70 DODAB molecules adsorbed per nucleotide on DNA, a molar proportion (MP) that does not depend on DNA type. The DODAB/DNA interaction led to formation of globules as visualized from dark-field optical microscopy and to occurrence of a linear dependence between turbidity for the mixture and 1/&#955;2, where &#955; is the wavelength of incident light. At maximal DODAB adsorption, the formation of DODAB/DNA globular complexes causes loss of doublestranded DNA hypochromism as detected from temperature effects on DNA absorbance at 260 nm in the presence or absence of DODAB. In summary, liposome loses its integrity and DNA loses its double helix becoming single-stranded. The hydrophobic attraction between nitrogenous bases on DNA and hydrocarbon chains on liposome bilayers plays an important role in determining structure of the complex.

Les syndécans forment une famille de quatre protéines transmembranaires qui sont substituées par l'héparane sulfate. Grâce à ces chaînes glucidiques extracellulaires, les syndécans contrôlent la signalisation d'une pléthore de facteurs de croissance et de molécules d'adhésion. Une autre caractéristique remarquable des syndécans est la conservation de leur domaine intracellulaire au cours de l'évolution. Ce domaine contient un motif C-terminal qui peut induire une interaction avec les protéines dites «PDZ». Les interactions PDZ sont promiscues et les protéines PDZ contrôlent divers aspects de la signalisation cellulaire et de la communication cellule-cellule. Quatre interactions syndecan-PDZ ont été décrites à ce jour et toutes ces interactions ont des effets drastiques sur le comportement des cellules. En particulier, il a été documenté que l'interaction syndécan-synténine a un impact sur le trafic intracellulaire de molécules de signalisation liant l’héparan sulfate. De plus, les syndécans et la synténine coopèrent dans le contrôle la biogenèse des exosomes, organites extracellulaires fonctionnant comme des médiateurs importants de la communication cellule-cellule (y compris dans différentes maladies systémiques comme le cancer). Le protéome humain compte 150 protéines PDZ qui contiennent 266 domaines PDZ. Dans ce travail, nous avons mis à jour la complexité de l'interactome syndecan-PDZ et testé son impact sur le trafic membranaire et sur la composition des vésicules extracellulaires. Notre travail ouvre la voie à une meilleure compréhension des réseaux moléculaires contrôlant la communication cellule-cellule en physio-pathologie. / Syndecans form a family of four transmembrane proteins that are substituted with heparan sulfate. By virtue of these extracellular carbohydrate chains, syndecans control the signaling of a plethora of growth factors and adhesion molecules. Another remarkable feature of syndecans is the conservation of their intracellular domain through evolution. This domain contains a C-terminal motif that can mediate interaction with PDZ proteins. PDZ interactions are promiscuous and PDZ proteins control various aspects of cell signaling and cell-cell communication. Four syndecan-PDZ interactions have been described so far and all these interactions have broad effects on cell behavior. In particular, it was documented that syndecan-syntenin interaction has impact on the intracellular trafficking of heparan sulfate cargo. Moreover syndecan-syntenin controls the biogenesis of exosomes, extracellular organelles emerging as important mediators of cell-cell communication in health and diseases. The human proteome contains 150 PDZ proteins and 266 PDZ domains. Here we started addressing the complexity of the syndecan-PDZ interactome and tested for its impact on membrane trafficking and on the composition of extracellular vesicles. Our work paves the way for a better understanding of the molecular mechanisms and networks controlling cell-cell communication in health and disease.

In der vorliegenden Arbeit werden die Eigenschaften geschlossener fluider Membranen, sogenannter Vesikeln, bei endlichen Temperaturen untersucht. Dies beinhaltet Betrachtungen zur Form freier Vesikeln, eine Untersuchung des Adhäsionsverhaltens von Vesikeln an planaren Substraten sowie eine Untersuchung der Eigenschaften fluider Vesikeln in eingeschränkten Geometrien. Diese Untersuchungen fanden mit Hilfe von Monte-Carlo-Simulationen einer triangulierten Vesikeloberfläche statt. Die statistischen Eigenschaften der fluktuierenden fluiden Vesikeln wurden zum Teil mittels Freier-Energie-Profile analysiert. In diesem Zusammenhang wurde eine neuartige Histogrammethode entwickelt.<br><BR> Die Form für eine freie fluide Vesikel mit frei veränderlichem Volumen, die das Konfigurationsenergie-Funktional minimiert, ist im Falle verschwindender Temperatur eine Kugel. Mit Hilfe von Monte-Carlo-Simulationen sowie einem analytisch behandelbaren Modellsystem konnte gezeigt werden, daß sich dieses Ergebnis nicht auf endliche Temperaturen verallgemeinern lässt und statt dessen leicht prolate und oblate Vesikelformen gegenüber der Kugelgestalt überwiegen. Dabei ist die Wahrscheinlichkeit für eine prolate Form ein wenig gröoßer als für eine oblate. Diese spontane Asphärizität ist entropischen Ursprungs und tritt nicht bei zweidimensionalen Vesikeln auf. Durch osmotische Drücke in der Vesikel, die größer sind als in der umgebenden Flüssigkeit, lässt sich die Asphärizität reduzieren oder sogar kompensieren. Die Übergänge zwischen den beobachteten prolaten und oblaten Formen erfolgen im Bereich von Millisekunden in Abwesenheit osmotisch aktiver Partikel. Bei Vorhandensein derartiger Partikel ergeben sich Übergangszeiten im Bereich von Sekunden.<br><br> Im Rahmen der Untersuchung des Adhäsionsverhaltens fluider Vesikeln an planaren, homogenen Substraten konnte mit Hilfe von Monte-Carlo-Simulationen festgestellt werden, dass die Eigenschaften der Kontaktfläche der Vesikeln stark davon abhängen, welche Kräfte den Kontakt bewirken. Für eine dominierende attraktive Wechselwirkung zwischen Substrat und Vesikelmembran sowie im Falle eines Massendichteunterschieds der Flüssigkeiten innerhalb und außerhalb der Vesikel, der die Vesikel auf das Substrat sinken lässt, ndet man innerhalb der Kontakt ache eine ortsunabhangige Verteilung des Abstands zwischen Vesikelmembran und Substrat. Drückt die Vesikel ohne Berücksichtigung osmotischer Effekte auf Grund einer Differenz der Massendichten der Membran und der umgebenden Flüssigkeit gegen das Substrat, so erhält man eine Abstandsverteilung zwischen Vesikelmembran und Substrat, die mit dem Abstand vom Rand der Kontaktfläche variiert. Dieser Effekt ist zudem temperaturabhängig.<br><br> Ferner wurde die Adhäsion fluider Vesikeln an chemisch strukturierten planaren Substraten untersucht. Durch das Wechselspiel von entropischen Eekten und Konfigurationsenergien entsteht eine komplexe Abhängigkeit der Vesikelform von Biegesteifigkeit, osmotischen Bedingungen und der Geometrie der attraktiven Domänen.<br><br> Für die Bestimmung der Biegesteifigkeit der Vesikelmembranen liefern die existierenden Verfahren stark voneinander abweichende Ergebnisse. In der vorliegenden Arbeit konnte mittels Monte-Carlo-Simulationen zur Bestimmung der Biegesteifigkeit anhand des Mikropipettenverfahrens von Evans gezeigt werden, dass dieses Verfahren die <i>a priori</i> für die Simulation vorgegebene Biegesteifigkeit im wesentlichen reproduzieren kann.<br><br> Im Hinblick auf medizinisch-pharmazeutische Anwendungen ist der Durchgang fluider Vesikeln durch enge Poren relevant. In Monte-Carlo-Simulationen konnte gezeigt werden, dass ein spontaner Transport der Vesikel durch ein Konzentrationsgefälle osmotisch aktiver Substanzen, das den physiologischen Bedingungen entspricht, induziert werden kann. Es konnten die hierfür notwendigen osmotischen Bedingungen sowie die charakteristischen Zeitskalen abgeschätzt werden. Im realen Experiment sind Eindringzeiten in eine enge Pore im Bereich weniger Minuten zu erwarten. Ferner konnte beobachtet werden, dass bei Vesikeln mit einer homogenen, positiven spontanen Krümmung Deformationen hin zu prolaten Formen leichter erfolgen als bei Vesikeln ohne spontane Krümmung. Mit diesem Effekt ist eine Verringerung der Energiebarriere für das Eindringen in eine Pore verbunden, deren Radius nur wenig kleiner als der Vesikelradius ist. / In this thesis, the properties of closed fluid membranes or vesicles are studied at finite temperatures. The work contains investigations of the shape of free vesicles, studies of the adhesion behavior of vesicles to planar substrates, and investigations of the properties of fluid vesicles in confined geometries. The investigations have been performed with Monte Carlo simulations of triangulated vesicles. The statistical properties of fluctuating vesicles have been analyzed in detail by means of free energy profiles. In this context, a new histogram method was developed. <br><br> The shape of minimum configurational energy for a free vesicle without volume constraint at zero temperature is a sphere. It is shown by means of Monte Carlo simulations and a model which can be analyzed analytically, that this result does not apply to finite temperatures. Instead, prolate and oblate shapes prevail and the probability for a prolate shape is slightly larger than that for an oblate shape. This spontaneous asphericity is of entropic origin and cannot be observed in two dimensions. Osmotic pressures inside the vesicle that are larger than in the surrounding liquid may reduce or even compensate the asphericity. The transitions between the observed prolate and oblate states occur on the time scale of milliseconds in the absence of osmotically active particles and on the time scale of seconds in the presence of osmotically active particles. <br><br> As far as the adhesion behavior of fluid vesicles to planar homogeneous substrates is concerned, Monte Carlo simulations reveal a strong dependence of the properties of the contact area on its driving force. In the case of a dominating attractive interaction between vesicle membran and substrate as well as for a mass density difference of the liquids inside and outside the vesicle, which push the vesicle against the substrate, the distribution of the distance between the vesicle membrane and the substrate is homogenous. If the vesicle is pushed against the substrate by a difference of the mass densities of the membrane and the surrounding liquid, neglecting all osmotic effects, one gets a distance distribution between the vesicle membrane and the substrate which varies with the distance from the rim of the contact area. Moreover, this effect is temperature-dependent. <br><br> Furthermore, the adhesion of fluid vesicles to chemically structured planar substrates has been studied. The interplay between entropic effects and configurational energies causes a complex dependence of the vesicle shape on the bending rigidity, osmotic conditions, and the geometry of the attractive domains. <br><br> There are several experimental methods for measuring the bending rigidity of vesicle membranes which lead to rather different results for the numerical value. Monte Carlo simulations of Evans' micropipette method show that the difference between the measured bending rigidity and the a priori chosen bending rigidity is small. <br><br> The passage of fluid vesicles through narrow pores has some relevance to medical/pharmaceutical applications. In Monte Carlo simulations it is shown that a spontaneous transport of vesicles can be induced by a concentration gradient of osmotically active particles which corresponds to the physiological conditions. The necessary osmotic conditions and the charateristic time scales are calculated. For real experiments, penetration into the pore should occur within a few minutes. Moreover, it was observed that vesicles with a homogeneous positive spontaneous curvature can be deformed more easily into prolate shapes than vesicles with zero spontaneous curvature. This effect leads to a decrease of the energy barrier for the penetration into a wide pore, which has a radius slightly smaller than that of the vesicle.

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into &gt;200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.

The antimicrobial properties of dextrose encapsulated gold nanoparticles (dGNPs) with average diameters of 25 nm, 60 nm, and 120 nm (± 5 nm) synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of the dGNPs on the growth, morphology and the ultrastructural properties of bacteria. dGNPs were found to have significant dose dependent antibacterial activity which was directly proportional to their size and also their concentration. The microbial assays revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action through the disruption of the bacterial cell membrane causing leakage of cytoplasmic content. The overall outcomes of this study suggest that dGNPs hold promise as a potent antimicrobial agent against a wide range of disease causing bacteria and can control and prevent possible infections or diseases.