Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Bacterial chemotaxis, as observed for Escherichia coli, in a field of chemoattractant molecules is characterised by a run-and-tumble motion. The motion is effected by the clockwise (CW) or counter-clockwise (CCW) rotation of flagella; filamentous appendages attached to molecular motors on the cell body. Runs appear when all flagella turn in the CCW-direction and are used to maintain a favourable direction. Tumbles emerge as soon as one flagellum starts to turn CW and are used for reorientation. Because of the variation observed between individual bacteria displaying run-and-tumble motion, we choose to model this behaviour within a probabilistic framework. An important feature of the chemotactic ability of E.coli is that the cell increases run while moving in the right direction and shortens it in the opposite case. This underlines that tumbles are used for reorientation. It has been found from experiments that there can be significant variation in the tumble fashion depending on the fraction of CW-rotating motors (Turner et al., 2000). The change in angle produced when fewer flagella are rotating CW was found to be smaller when compared to the case for many CW-rotating flagella. In addition, the change of direction contributed by a small portion of CW-rotating flagella is rarely significant for bacteria with many flagella. Based on these observations, we have distinguished between models for the one-flagellated and the multi-flagellated cases. Furthermore, since the tumbling angle change increases with the fraction of CW-rotating motors, it would not be impossible to have some cases where the amount of turn produced by the CW-rotating motors induces the bacterium to have a change of direction greater than 2π. But, this feature could not have been observed because when the bacterium tumbles it can effectuate several revolutions before resuming to a new direction. Therefore, we do not restrict our change of direction to (0,2π) to allow the bacteria to have the possibility to effectuate change of directions of magnitude greater than 2π. To this end, we differentiate between the probability of having directional change of magnitude α and α +2π . Thus we do not use angle change distributions that are defined modulo 2π such as the von Mises distribution or the wrapped normal distribution. The chemotactic ability of the bacterium is modelled by representing the CCW-bias of a single flagellum as a function of the chemoattractant concentration. The model includes the temporal memory of chemoattractant concentration that the bacterium has, which usually spans about 4s. The information about the quality of the current direction of the bacterium is transmitted to the flagellar motor by assuming that this one varies with the chemoattractant concentration level. In addition, the saturation of the bias is incorporated by assuming that the bacterium performs a temporal comparison of the receptor occupancy. The present CCW-bias-Model accounts for the chemotactic ability of the bacterium as well as its adaptation to uniform chemoattractant environment. The models of one-flagellated and multi-flagellated bacterial motion, are used to investigate two main problems. The first one consists of determining the optimal tumbling angle strategy of the bacteria. The second one consists of looking at the effects of the tumble variation on the chemotactic efficiency of the bacteria. In order to address these questions, the chemotactic efficiency measure is defined in such a way that it reflects the ability of the bacteria to converge and to stay in a near neighbourhood of the source so that they gain more nutrients. Since its movement is entirely governed by its single flagellum, the one flagellated bacterium is more able to effectuate a run motion. Tumbling events are modelled to be all equivalent because there is not any fraction of flagella to consider. On the other hand, the tumble variation of the multi-flagellated bacteria is modelled by assuming that the directional change during a tumble is a function of the fraction of CW-rotating motors. By assuming that the number of CW-rotating flagella follows a binomial distribution, we suppose that the multi-flagellated bacteria are less able to effectuate a run motion. This also implies that the change of direction produced by fewer CW-rotating flagella are more likely to happen, and this compensates the lack of run. The models show that the optimal tumbling angle change for the bacteria is less than 2π and that higher flagellated bacteria have higher chemotacitc efficiency. As the number of flagella of the bacteria increases, there can be more tumble variation, in this case the bacteria are more capable of adjusting their direction. There could be some situation were the bacteria are not moving to the right direction, but do not require a large change of direction. This ability to adjust their direction accordingly allows them to converge nearer to the source and to gain more nutrients. In addition, the dependence of the tumbling angle on the fraction of CW-rotating flagella of the mutli-flagellated bacteria, implies that there is a correlation between the tumbling angle deviation and the external environment, because the rotational states CCW-CW of the flagella depends on the external cue. Consequently, it would not be impossible that the average magnitude of tumbling angle change depends on the external environment. To investigate this possibility we analyse the distribution of the tumbling tendency of a single bacterium over time, which is the distribution over time of the average positive tumbling change of the bacterium, within zerogradient environment and within non-zero-gradient environment. We defined the average of these tumbling tendency over time as the directional persistence. We observe that the directional persistence within these different nonzero- gradient environment remains the same. However, the difference between the directional persistence within zero-gradient and non-zeros gradient environment gets larger as the number of flagella of the cell increases. There is more correlation between the external environment and the tumbling tendency of the bacterium. Which is the reason why the higher flagellated bacteria responds the best to the external environment by having the higher chemotactic performance. Finally, the total directional persistence generated by the optimal tumbling angle change of the bacteria is the average directional persistence of the bacteria regardless of their number of flagella. Its value, predicted by the model is 1.54 rad within a non-zero-gradient environment and 1.63 rad within a zero-gradient environment. / AFRIKAANSE OPSOMMING: Bakteriese chemotakse, soos waargeneem word vir Escherichia coli, in ’n veld van chemiese lokmiddel molekules word gekenmerk deur ’n hardloopen- tuimel beweging. Die beweging word bewerkstellig deur die regsom of linksom rotasie van flagella; filamentagtige aanhangsels geheg aan molekulêre motors op die selliggaam. ’n Hardloop aksie kom voor as al die flagella linksom roteer en word gebruik om ’m voordelige koers te handhaaf. Tuimels kom voor sodra een van die flagella regsom draai en word gebruik vir heroriënteering. Van wee die variasie wat waargeneem word tussen individuele bakterieë wat hardloop-en-tuimel bewegiging vertoon, verkies ons ’n probabilistiese raamwerk om in te werk. ’n Belangrike eienskap van die chemotakse vermoë van E. coli is dat die sel meer gereeld hardloop terwyl dit in die regte rigting beweeg en minder gereeld in die teenoorgestelde geval. Dit beklemtoon dat tuimels gebruik word vir heroriënteering. Dit is al eksperimenteel vasgestel dat daar betekenisvolle variasie kan wees in die tuimel wyse, wat afhang van die breukdeel regsom roterende motors (Turner et al., 2000). Die hoekverskil afkomstig van minder regsom roterende flagella was vasgestel om kleiner te wees in vergelyking met die menig regsom roterende geval. Verder word die bydrae tot die hoekverskil van ’n klein breukdeel regsom roterende flagella selde beduidend vir bakterieë met baie flagella. As gevolg van hierdie waarnemings, tref ons onderskeid tussen modelle vir een-flagella en multiflagella gevalle. Aangesien die tuimel hoeksverskil vergroot saam met die breukdeel regsom roterende motore, is dit nie onmoontlik om gevalle te hê waar die hoeveelheid draaiaksie gegenereer deur die regsom roterende motore ’n rigtingsverskil groter as 2π kan bewerkstellig nie. Dit was nie moontlik om hierdie eienskap waar te neem nie aangesien die bakterieë ’n paar keer kan tuimel voordat ’n nuwe rigting vasgestel word. Vir hierdie rede beperk ons nie die hoeksverskil tot (0,2π) nie om die bakterieë toe te laat om rigtings veranderinge groter as 2π te ondergaan. Vir hierdie doel, onderskei ons tussen die waarskynlikheid van ’n rigtinsverskil met grootte α en α + 2π. Dus, gebruik ons nie hoekverskil verspreidings wat modulo 2 gedefinieer is nie, soos die von Mises verspreiding of omwinde normaalverdeling. Die chemotakse vermoë van die bakterium word gemodelleer deur die linksom sydigheid van ’n enkele flagellum as ’n funksie van die chemotakse lokmiddel konsentrasie voor te stel. Die model sluit in die tydelike geheue wat die bakterium besit oor chemotakse lokmiddel konsentrasie, wat gewoonlik oor 4s strek. Die informasie oor die kwaliteit van die huidige rigting van die bakterium word deur gegee na die flagella motor toe deur die aanname te maak dat dit wissel met die chemotakse lokmiddel konsentrasie vlak. Die versadiging van die sydigheid word geinkorporeer deur aan te neem dat die bakterium ’n temporale vergelyking maak tussen reseptor okkupasie. Die huidige linksom sydige model neem die bakterium chemotakse vermoë in ag, as ook aanpassing tot ’n uniforme chemotakse lokmiddel omgewing. Die modelle van een-flagella en multi-flagella bakteriële beweging word gebruik om twee hoof probleme te bestudeer. Die eerste, bestaan daaruit om vas te stel wat die optimale tuimel hoek strategie van die bakterieë is. Die tweede kyk na die uitwerking van tuimel variasie op chemotakse effektiwiteit. In orde om hierdie vra te adreseer word die chemotakse effektiwiteit op so mannier gedefinieer dat dit die bakteriese vermoë om die buurt om die oorsprong te nader en daar te bly. Aangesien die beweging heeltemal vasgestel word deur een flagella, in die een-flagella geval, is ’n bakterium meer in staat daartoe om ’n hardloop aksie te bewerkstellig. Tuimel voorvalle word as ekwivalent gemodeleer omdat daar geen breukdeel roterende flagella is om in ag te neem nie. In teenstelling, word die tuimel variasie van multi-flagella bakterieë gemodeleer deur die aanname te maak dat rigtingsverandering gedurende ’n tuimel ’n funksie is van die breukdeel regsom roterende motore. Deur die aanname te maak dat die getal regsom roterende flagella ’n binomiese verspreiding volg, veronderstel ons dat multi-flagella bakterieë minder in staat daartoe is om ’n hardloop aksie te onderneem. Hierdie impliseer ook dat rigtingverandering wat geproduseer word deur minder regsom roterende flagella meer geneig is om voor te kom en dan kompenseer vir ’n tekortkoming aan hardloop gebeure. Die modelle wys dat die optimale tuimelhoek verandering minder as 2 is en dat bakterieë met meer flagella meer chemotaksies effektief is. Soos die getal flagella vermeder, kan daar meer tuimel variasie wees, en in die geval is die bakterieë meer in staat om hul rigting te verander. Daar kan omstandighede wees waar die bakterieë nie in die regtige rigting beweeg nie, maar nie ’n groot rigtingsverskil nodig het nie. Hierdie vermoë om hul rigting byvolglik te verander stel hul in staat om nader aan die oorsprong te konvergeer en dus meer voedingstowwe op te neem. Die afhanklikheid van die tuimel hoek op die breukdeel regsom roterende flagella van multi-flagella bakterieë dui daarop dat daar ’n korrelasie is tussen die tuimel hoek afwyking en die eksterne omgewing, omdat die roterings toestand, regs- of linksom, van die flagella afhanklik is van die eksterne sein. As ’n gevolg, is dit nie onmoontlik dat die gemiddelde grootte van die tuimel hoek verandering van die eksterne omgewing afhang nie. Om hierdie moontlikheid te bestudeer, analiseer ons die verspreiding van die tuimel neiging van ’n enkele bakterium oor tyd, wat die verspreiding oor tyd van die gemiddelde positiewe tuimel verandering is, in ’n nulgradient en nie-nul-gradient omgewing. Ons het hierdie gemiddelde tuimel neigings oor tyd gedefinieer as die rigtings volharding. Ons het waargeneem dat die rigtings volharding binne verskillende nienul- gradient omgewings dieselfde bly. Nogtans is die verskil tussen die rigtings volharding binne nul-gradient en nie-nul-gradient omgewings groter soos die getal flagella vermeder. Daar is meer korrelasie tussen die eksterne omgewing en tuimel neiging van die bakterium. Dit is die rede hoekom bakterieë met meer flagella die beste reageer op die eksterne omgewing deur beter chemotakse effektiwiteit. Ten slotte, die totale rigtings volharding gegenereer deur die optimale tuimel hoek verandering is die gemiddelde rigtings volharding ongeag van die getal flagella. Die waarde wat deur die model voorspel word is 1.54 rad binne ’n nie-nul-gradient omgewing en 1.63 rad binne ’n nul-gradient omgewing.

The dopamine signalling pathway has been implicated in the pathophysiology of neuropsychiatric conditions including bipolar disorder and schizophrenia. A detailed analysis of this pathway is essential for understanding these conditions. Previous work (Zhan et al., 2008) has identified eleven novel human dopamine receptor interacting proteins (DRIPs), but their role in cell signalling remains unclear. In this project we have employed a biomedical model, Dictyostelium discoideum, to help elucidate the cellular signalling of two DRIPs, the Zizimin GEF (DRIP2) and MARK (DRIP9) proteins. Bioinformatics analysis of these proteins shows conservation of the domain structure in the human and Dictyostelium gene products. To investigate the function of these proteins during development, two Dictyostelium homologues within each family (ZizA and ZizB; MrkA and MrkC) were ablated and changes in developmental for resulting null mutants were analysed. Development was unaltered following ablation of zizA, mrkA and mrkC, however, ablation of zizB gave rise to a clear change in developmental morphology. To further understand the developmental defect of zizB, directional cell movement (chemotaxis) was analysed in the zizA and zizB null mutants. Ablation of zizA caused no gross phenotypic change in chemotaxis, whereas zizB ablation gave rise to a reduction in cell speed, directionality and aspect (roundness). Furthermore, expression studies showed zizA and zizB were constantly expressed throughout development. Overexpression of each gene (labelled with the fluorescent tag, GFP) demonstrated a cytosolic localisation the gene products, with the ZizB-GFP fusion protein additionally exhibiting enrichment of the cortex, causing a large increase in filopodia formation and a partial inhibition of cytokinesis. Analysis of protein binding partners for ZizB indicates specific interaction with Rac1 A and a range of actin-interacting proteins. In conclusion this project provides the first insight into the molecular and cellular functions of Zizimin proteins, potential dopamine receptor interacting protein.

The control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques. High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively. Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively). To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad). Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units.

Exogenous and endogenous cannabinoids have been reported to modulate functional activities of macrophages. It is recognized that macrophages express primarily the CB2 cannabinoid receptor, but recent studies indicate that its expression is differential in relation to activation state with maximal levels occurring when cells are in "responsive" and "primed" states. The functional activities of macrophages when in these states of activation are the most susceptible to the action of cannabinoids, at least in terms of a functional linkage to the CB2. To assess the effect of cannabinoid treatment on macrophage chemotaxis and test the hypothesis that cannabinoids inhibit the chemotactic response of macrophages and microglia to endogenous and exogenous, pathogen-derived stimuli, primary murine peritoneal macrophages and neonatal rat microglia were used. Chemotaxis assays and scanning electron microscopy studies demonstrated that cannabinoids inhibit chemotaxis, a signature activity attributed to "responsive" macrophage-like cells, to the endogenous chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) and to Acanthamoeba conditioned medium containing secreted proteases. The partial agonist delta-9-tetrahydrocannabinol (THC), administered in vitro, inhibited the chemotactic response of peritoneal macrophages to the chemokine RANTES and to Acanthamoeba conditioned medium. In vivo treatment with THC also resulted in inhibition of the in vitro chemotactic response of murine peritoneal macrophages to RANTES and amoebic conditioned medium. Pharmacological studies employing cannabinoid receptor agonists and antagonists demonstrated the involvement of CB2 in cannabinoid-mediated inhibition of peritoneal macrophage chemotaxis to RANTES and Acanthamoeba conditioned medium, implying that signaling through cannabinoid receptors may desensitize chemokine receptors. Treatment with cannabinoids had no apparent effect on chemokine receptor mRNA levels, but did enhance CCR5 protein phosphorylation. Macrophage migration to Acanthamoeba conditioned medium may involve activation and signaling through protease activated receptors (PARs), as pathogen-derived proteases have been shown to activate PARs and initiate cellular migration; however, further studies are required to demonstrate PAR activation by amoebic conditioned medium and to assess the effects of cannabinoids on PAR signaling. Acanthamoeba are opportunistic pathogens that cause Granulomatis amoebic encephalitis, an infection of the CNS that is often fatal. THC treatment has been shown to increase mortality to Acanthamoeba infections and is characterized by an absence of granuloma formation. We hypothesize that inhibitory effect of THC on macrophage migration may be a key factor in cannabinoid-mediated immunosuppression. To assess the effect of cannabinoids on microglial migration to Acanthamoeba conditioned medium, chemotaxis assays were performed using primary rat microglia treated with cannabinoids. These studies demonstrated that cannabinoids inhibit microglial chemotaxis to amoebic conditioned medium. Furthermore, the studies demonstrate that cannabinoids, acting through cannabinoid receptors, may cross-talk with a diverse array G-protein coupled receptors so as to modulate responsiveness of macrophage and macrophage-like cells.

Apoptosis is a form of programmed cell death that is essential in such processes as organ and tissue remodeling and maturation of hematopoietic cells. The clearance of apoptotic cells is essential to prevent autoimmune responses to sequestered antigens. This process is mediated by phagocytes of the monocyte lineage. Before phagocytosis can occur, macrophages must be recruited to the apoptotic cells through chemotaxis. Products of the reaction catalyzed by the phospholipases A2 (PLA2) have been shown to induce monocyte chemotaxis either directly or indirectly. Some investigators have implicated a cytosolic calcium-independent PLA2 (iPLA2) in the production of these products during apoptosis. However, a recent report suggests that the secreted group IIa (sPLA2) binds to surfaces of apoptotic cells. The "receptor" for this pool of sPLA2 is the rod domain of vimentin, an intermediate filament protein that is exposed by caspase activity when cells undergo apoptosis. Based on these observations, we hypothesize that the exposure of vimentin on apoptotic cells traps a pool of catalytically active sPLA2 that then generates the bioactive lipids that induce macrophage chemotaxis. In our methods, [3H]-oleate labeled E-coli is used as a substrate for sPLA2 and enzyme activity is quantified by scintillation counting of released radiolabeled oleic acid. Apoptosis is induced with anti-fas (CD-95) on Jurkat cells and monitored through annexin-V binding and propidium iodide(PI) staining followed by flow cytometric analyses. THP-1 monocytes are employed in chemotaxis assay with monocyte chemotactic protein (MCP-1) as a positive control. The preliminary data show equivalent group IIa sPLA2 association with anti-fas treated and control cells, and the enzyme remains catalytically active when bound. In line with the hypothesis, trapped sPLA2 generated soluble molecules that directly or indirectly induced migration of THP-1 monocytes. However, the similar binding effect observed with apoptotic or control cells is surprising and experiments are being planned to determine if the interaction between IIa sPLA2 and apoptotic cells is vimentin dependent.

Bacterial chemotaxis is the movement of bacteria towards or away from chemical stimuli in the surrounding media. Bacteria respond to chemotactic signals through chemoreceptors which bind specific ligands and transduce signals through a modified two-component system. Typical chemoreceptors bind a ligand in the periplasm and signal across the inner membrane to the cytoplasmic chemosensory array through the inner membrane. Bacterial chemoreceptors must integrate multiple signals within an array of different receptor homologues to a single output. Chemoreceptors act cooperatively to allow a rapid signal spread across the array and large signal gain. Chemoreceptors adapt to a signal by chemical modification of their cytoplasmic domains in order respond across a wide range of effector concentrations. How bacterial chemoreceptors transduce signals through the inner membrane, integrate multiple effector responses, signal cooperatively and adapt to result in a single output signal is not currently fully known. In Rhodobacter sphaeroides, additional complexity arises from the presence of multiple homologues of various chemotactic components, notably the array scaffold protein CheW. Decoding this signalling mechanism and heterogeneity involved in this system is important in decoding the action of a biological system, with implications for biotechnology and synthetic biology. This study used the two model systems Escherichia coli and R. sphaeroides to analyse the mechanism of signalling through bacterial chemoreceptors. Rational design of activity-shifting chemoreceptor mutations was undertaken and these variants were analysed in phenotypic and fluorescence localisation studies. Molecular-dynamics simulations showed an increase in flexibility of chemoreceptors corresponds to a decrease in kinase output activity, which was determined by the computational tracking of bacteria free-swimming in media. Fluorescence recovery after photobleaching was used to show that this increase in flexibility results in a decrease in binding of receptors to their array scaffold proteins. A two-hybrid screen also suggested that inter-receptor affinity is also likely to decrease. These results show that signalling through chemoreceptors is likely through a mechanism involving the selective flexibility of chemoreceptor cytoplasmic domains. Analysis of R. sphaeroides chemoreceptors and CheW scaffold proteins in E. coli showed that it should be possible to design, from the bottom-up, a functional bacterial chemotaxis system in order to analyse individual protein specificity. Expression of R. sphaeroides MCPs in this E. coli system show the reconstitution of a chemotactic array, but not one capable of signalling specifically to proposed attractants. Results gained from this system suggest the R. sphaeroides CheW proteins are not homologous and their differential binding affinities may allow array activity 'fine-tuning'.

ErbB2 est un récepteur à activité tyrosine kinase dont la surexpression dans le cancer du sein est corrélée à un mauvais pronostic. Son activation induit de nombreuses voies de signalisation. L'objectif de notre travail était d'étudier le réseau de signalisation associé à la migration dépendante d'ErbB2 et de déterminer la contribution des microtubules à ce processus.ErbB2 recrute un module de signalisation qui comporte l'effecteur Memo, la GTPase RhoA, et la formine mDia1. Ce module réprime GSK3, pour permettre la localisation à la membrane plasmique d'un complexe de capture des microtubules comprenant le suppresseur de tumeur APC et la spectraplakine ACF7.La voie Memo/ACF7 est impliquée dans le chimiotactisme via la capture des microtubules ainsi que la phospholipase PLC&#947;1, un autre effecteur d'ErbB2 qui participe également à la capture des microtubules. Sa signalisation rejoint la voie Memo en amont de GSK3 via les PKC classiques. PLC&#947;1 agit aussi via aPKC&#950;.PI3K est également impliquée dans le chimiotactisme grâce à la stabilisation des microtubules. Elle implique l'inhibition de GSK3 et la phosphorylation de la Stathmine par la kinase PAK1.Sur la base de ces résultats, nous proposons un modèle, basé sur un processus en deux étapes. Tout d'abord, les microtubules sont capturés lors de la formation de la protrusion cellulaire. Puis, ils sont stabilisées à l'avant des cellules. Ces deux étapes sont régies par des voies de signalisation différentes qui coordonnent la capture des microtubules et la stabilité des microtubules pour contrôler la réponse chimiotactique. / ErbB2 is a receptor tyrosine kinase who's over expression in breast cancer correlates with poor prognosis. Upon activation, ErbB2 induces numerous signaling pathways. Our aim is to investigate the signaling network associated with ErbB2-driven migration and to determine the contribution of microtubules to migration.ErbB2 recruits a signaling module including the ErbB2 effector Memo, the GTPase RhoA, and the formin mDia1. It represses GSK3 activity, to allow the localization to the plasma membrane of a microtubule capture complex comprising the tumor suppressor APC and the spectraplakin ACF7.Memo/ACF7 pathway is involved in chemotaxis via microtubule capture. PLC&#947;1, another effector of ErbB2, also participates in microtubule capture. It joins Memo pathway via classic PKCs upstream GSK3, and also acts via aPKC&#950;. PI3K is involved in chemotaxis through microtubule stabilization. Our results suggested that PI3K-dependent microtubules stabilization involves inhibition of GSK3 activity and phosphorylation of Stathmin via PAK1 activity.Defects in microtubule capture/stability are closely correlated with chemotaxis disturbances and rescue of microtubules within cell protrusion re-establishes cell orientation.We propose a model based on a two-step process to explain regulation of microtubule dynamics downstream of ErbB2. First, microtubules are captured during the formation of cell protrusions. Then they are stabilized at the cell front. These two steps are governed by different signaling pathways that coordinate microtubule capture and microtubule stability to control chemotaxis.

A pancreatite aguda (PA) é uma doença que frequentemente está associada a uma inflamação local e sistêmica não controlada que é responsável pela alta morbidade e mortalidade desta doença. Independentemente do mecanismo inicial, o processo inflamatório ocorre após uma lesão nas células acinares, levando ao recrutamento de células inflamatórias que produzem citocinas e quimiocinas que são capazes de desempenhar um papel importante na patogênese dessa doença. A inflamação pancreática induz distúrbios profundos na homeostase levando ao dano tecidual em outros órgãos, tais como o intestino e o pulmão. Por outro lado, órgãos como o cérebro têm mecanismos estruturais e celulares de proteção contra a inflamação. Tem sido descrito, que pacientes com insuficiência cardíaca apresentam lesão cardíaca, levando a produção de citocinas pró-inflamatórias, ativação de células T e do sistema complemento. Além da cascata inflamatória, durante todo o estágio da PA o sistema cardiovascular pode sofrer alterações cardíacas e histológicas compatíveis com o processo inflamatório desencadeado pela PA. A intensidade da inflamação sistêmica devido à PA é semelhante em ratos jovens e idosos, porém, a duração da inflamação sistêmica parece ser muito maior nos idosos. A idade avançada é considerada um fator prognóstico independente para um pior prognóstico na PA, mas os mecanismos envolvidos não são totalmente compreendidos. Decidimos analisar a expressão gênica de diversas citocinas, quimiocinas e fatores de crescimento no coração de ratos idosos e jovens submetidos ao modelo animal de PA, com o objetivo de investigar os efeitos da inflamação sistêmica no coração e determinar se esses efeitos sofrem algum tipo de modificação durante a senilidade. Ratos jovens e idosos foram submetidos à PA utilizando-se ácido taurocólico 2,5% e, após 11 horas, o tecido cardíaco foi coletado para histologia e para a extração de RNA. A determinação do RNAm de citocinas e quimiocinas foi feita através do PCR Array. Como resultado, a expressão das interleucinas IL-6 E IL-10, e as quimiocinas CCL7 e CCL19 nos ratos idosos com PA foi menor do que nos ratos jovens com PA. Entretanto, os níveis de CXCL3 e CCL20 aumentaram nos idosos com PA. A expressão de CCL20 e CCL19 foi significativamente alta em ambos os grupos jovens e idosos com PA quando comparados com seus respectivos controles. Nos idosos com PA houve uma redução na expressão de CXCL1, CCL1, CCL11 e IL-10 comparados com seus controles, já entre os grupos saudáveis os idosos expressaram mais do que os jovens. Na análise histológica, a infiltração dos neutrófilos foi significativamente maior no tecido cardíaco dos ratos jovens e idosos com PA, comparado com seus controles. Esse estudo indica que a inflamação sistêmica pode mostrar características únicas para diferentes órgãos do corpo, e a diminuição da expressão gênica de algumas quimiocinas no tecido cardíaco de ratos mais velhos com PA pode sugerir um possível mecanismo cardioprotetor em animais mais velhos / Acute pancreatitis (AP) is a disease frequently associated with uncontrolled local and systemic inflammation that is responsible for the morbidity and mortality of this disease. Regardless of the initial mechanism, the inflammatory process is triggered by injury to the pancreatic acinar cells, leading to recruitment of inflammatory cell that produce cytokines and chemokines, which play an important role in the pathogenesis of this disease. Pancreatic inflammation induces profound disturbances in homeostasis, leading to tissue injury in other organs such as the intestine and lung. On the other hand, organs such as the brain have structural and cellular mechanisms of protection against inflammation. It has been described that patients with heart failure exhibit cardiac injury, leading to the production of pro-inflammatory cytokines, activation of T cells and activation of the complement system. In addition to inflammatory cascade during all stage of the PA, the cardiovascular system may suffer cardiac and histological changes compatible with inflammatory process triggered by the AP. The intensity of systemic inflammation associated with AP is similar in young and old rats, however, the duration of systemic inflammation is much longer in older animals. Advanced age is considered to be an independent prognostic factor for a poorer prognosis in AP, but the mechanisms involved are not fully understood. We analyzed the gene expression of several cytokines, chemokines and growth factors in young and aged rats hearts in an animal model of AP to investigate the effects of systemic inflammation on the heart, and to determine whether the effects are modified with age. Young and old rats were subjected to AP using taurocholic acid (2.5%) and after 11 hours, the cardiac tissue was collected to histology and RNA extraction. The determination of mRNA of cytokines and chemokines was performed by PCR array. The expression of interleukins IL-6 and IL-10 and the chemokines CCL7 and CCL19 in the aged AP rats was lower than young AP rats. However, CXCL3 and CCL20 levels showed opposite results, with increased levels in the aged AP group. The expression of CCL20 and CCL19 was greater in both young and aged AP rats compared with their respective controls. In aged AP rats there were a reduction in the expression of CXCL1, CCL1, CCL11 and IL-10 compared to their controls, and among healthy groups, the elderly rats expressed more than the young rats. In histological analysis, the neutrophils infiltration was significantly higher in the heart tissue of AP rats, both young and old, compared with their controls. This study indicates that systemic inflammation may show unique features for different organs in the body and decreased gene expression of some chemokines in the heart tissue of older AP may suggest a possible cardioprotective mechanism in older animals

Angiogenesis is the process by which new blood vessels develop from existing vessels. Angiogenesis is important in a number of conditions such as embryogenesis, wound healing and cancer. It has been modelled phenomenologically at the macroscale, using the well-known 'snail-trail' approach in which trailing endothelial cells follow the paths of other, leading endothelial cells. In this thesis, we systematically determine the collective behaviour of endothelial cells from their behaviour at the cell-level during corneal angiogenesis. We formulate an agent-based model, based on the snail-trail process, to describe the behaviour of individual cells. We incorporate cell motility through biased random walks, and include processes which produce (branching) and annihilate (anastomosis) cells to represent sprout and loop formation. We use the transition probabilities associated with the discrete model and a mean-field approximation to systematically derive a system of non-linear partial differential equations (PDEs) of population behaviour that impose physically realistic density restrictions, and are structurally different from existing snail-trail models. We use this framework to evaluate the validity of a classical snail-trail model and elucidate implicit assumptions. We then extend our framework to explicitly account for cell volume. This generates non-linear PDE models which vary in complexity depending on the extent of volume exclusion incorporated on the microscale. By comparing discrete and continuum models, we assess the extent to which continuum models, including the classical snail-trail model, account for single and multi-species exclusion processes. We also distinguish macroscale exclusion effects introduced by each cell species. Finally, we compare the predictive power of different continuum models. In summary, we develop a microscale to macroscale framework for angiogenesis based on the snail-trail process, which provides a systematic way of deriving population behaviour from individual cell behaviour and can be extended to account for more realistic and/or detailed cell interactions.

A pancreatite aguda (PA) é uma doença que frequentemente está associada a uma inflamação local e sistêmica não controlada que é responsável pela alta morbidade e mortalidade desta doença. Independentemente do mecanismo inicial, o processo inflamatório ocorre após uma lesão nas células acinares, levando ao recrutamento de células inflamatórias que produzem citocinas e quimiocinas que são capazes de desempenhar um papel importante na patogênese dessa doença. A inflamação pancreática induz distúrbios profundos na homeostase levando ao dano tecidual em outros órgãos, tais como o intestino e o pulmão. Por outro lado, órgãos como o cérebro têm mecanismos estruturais e celulares de proteção contra a inflamação. Tem sido descrito, que pacientes com insuficiência cardíaca apresentam lesão cardíaca, levando a produção de citocinas pró-inflamatórias, ativação de células T e do sistema complemento. Além da cascata inflamatória, durante todo o estágio da PA o sistema cardiovascular pode sofrer alterações cardíacas e histológicas compatíveis com o processo inflamatório desencadeado pela PA. A intensidade da inflamação sistêmica devido à PA é semelhante em ratos jovens e idosos, porém, a duração da inflamação sistêmica parece ser muito maior nos idosos. A idade avançada é considerada um fator prognóstico independente para um pior prognóstico na PA, mas os mecanismos envolvidos não são totalmente compreendidos. Decidimos analisar a expressão gênica de diversas citocinas, quimiocinas e fatores de crescimento no coração de ratos idosos e jovens submetidos ao modelo animal de PA, com o objetivo de investigar os efeitos da inflamação sistêmica no coração e determinar se esses efeitos sofrem algum tipo de modificação durante a senilidade. Ratos jovens e idosos foram submetidos à PA utilizando-se ácido taurocólico 2,5% e, após 11 horas, o tecido cardíaco foi coletado para histologia e para a extração de RNA. A determinação do RNAm de citocinas e quimiocinas foi feita através do PCR Array. Como resultado, a expressão das interleucinas IL-6 E IL-10, e as quimiocinas CCL7 e CCL19 nos ratos idosos com PA foi menor do que nos ratos jovens com PA. Entretanto, os níveis de CXCL3 e CCL20 aumentaram nos idosos com PA. A expressão de CCL20 e CCL19 foi significativamente alta em ambos os grupos jovens e idosos com PA quando comparados com seus respectivos controles. Nos idosos com PA houve uma redução na expressão de CXCL1, CCL1, CCL11 e IL-10 comparados com seus controles, já entre os grupos saudáveis os idosos expressaram mais do que os jovens. Na análise histológica, a infiltração dos neutrófilos foi significativamente maior no tecido cardíaco dos ratos jovens e idosos com PA, comparado com seus controles. Esse estudo indica que a inflamação sistêmica pode mostrar características únicas para diferentes órgãos do corpo, e a diminuição da expressão gênica de algumas quimiocinas no tecido cardíaco de ratos mais velhos com PA pode sugerir um possível mecanismo cardioprotetor em animais mais velhos / Acute pancreatitis (AP) is a disease frequently associated with uncontrolled local and systemic inflammation that is responsible for the morbidity and mortality of this disease. Regardless of the initial mechanism, the inflammatory process is triggered by injury to the pancreatic acinar cells, leading to recruitment of inflammatory cell that produce cytokines and chemokines, which play an important role in the pathogenesis of this disease. Pancreatic inflammation induces profound disturbances in homeostasis, leading to tissue injury in other organs such as the intestine and lung. On the other hand, organs such as the brain have structural and cellular mechanisms of protection against inflammation. It has been described that patients with heart failure exhibit cardiac injury, leading to the production of pro-inflammatory cytokines, activation of T cells and activation of the complement system. In addition to inflammatory cascade during all stage of the PA, the cardiovascular system may suffer cardiac and histological changes compatible with inflammatory process triggered by the AP. The intensity of systemic inflammation associated with AP is similar in young and old rats, however, the duration of systemic inflammation is much longer in older animals. Advanced age is considered to be an independent prognostic factor for a poorer prognosis in AP, but the mechanisms involved are not fully understood. We analyzed the gene expression of several cytokines, chemokines and growth factors in young and aged rats hearts in an animal model of AP to investigate the effects of systemic inflammation on the heart, and to determine whether the effects are modified with age. Young and old rats were subjected to AP using taurocholic acid (2.5%) and after 11 hours, the cardiac tissue was collected to histology and RNA extraction. The determination of mRNA of cytokines and chemokines was performed by PCR array. The expression of interleukins IL-6 and IL-10 and the chemokines CCL7 and CCL19 in the aged AP rats was lower than young AP rats. However, CXCL3 and CCL20 levels showed opposite results, with increased levels in the aged AP group. The expression of CCL20 and CCL19 was greater in both young and aged AP rats compared with their respective controls. In aged AP rats there were a reduction in the expression of CXCL1, CCL1, CCL11 and IL-10 compared to their controls, and among healthy groups, the elderly rats expressed more than the young rats. In histological analysis, the neutrophils infiltration was significantly higher in the heart tissue of AP rats, both young and old, compared with their controls. This study indicates that systemic inflammation may show unique features for different organs in the body and decreased gene expression of some chemokines in the heart tissue of older AP may suggest a possible cardioprotective mechanism in older animals