Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated which are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for the intracellular growth defect, colony morphology on LB (X-p) media, and virulence in mice. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. Preliminary studies have also identified a convergently transcribed gene, tentatively designated mglC, immediately downstream of mglB. mglC null mutants are defective for intracellular growth and show the same phenotype on LB (X-p) agar as GB2. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Western blot analysis revealed the absence of MglA in an mglB null mutant, indicating MglB may influence MglA levels. Analysis of the regulation of mglA expression during growth in broth culture shows a decrease in expression upon entering late log-early stationary phase. mglA is also expressed during culture in macrophages. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida, most notably the absence of a 70 kDa secreted protein. A candidate clone for the gene encoding this 70 kDa protein has been isolated. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may control the expression of genes whose products contribute to survival and growth within macrophages. Roles for the putative MglC and possibly the 70 kDa secreted protein in this activity are also indicated. Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. The gene encoding AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella , was cloned and sequenced. The deduced amino acid sequence of AcpA showed limited similarity to phospholipase C proteins present in Pseudomonas aeruginosa and Mycobacterium tuberculosis. An F. novicida acpA null mutant was found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages as well as remaining virulent for mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida, and that any role it may play in virulence is subtle. / Graduate

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No description available.

No description available.

The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells. / Doctor of Philosophy (Ph.D.)

Concentration oscillations are a ubiquitous characteristic of intracellular dynamics. The period of these oscillations can vary from few seconds to many hours, well known examples being calcium oscillations (seconds to minutes), glycolytic oscillations (minutes) and circadian rhythms (1 day). Considerable advances into understanding the mechanisms and functionality of concentration oscillations have been made since glycolytic oscillations were observed in the late 1950s, and mathematical methods have been an essential part of this process. With increasing ability to experimentally measure oscillations in single cells as well as in cell ensembles, the gold standard of modelling is to provide tools that can elucidate how the system-wide dynamics in complex organisms emerge from a system of single cells. Both abstract and detailed mechanistic models are complementary in the insight they can bring, and for networks of coupled cells considerations such as intrinsic intracellular noise, cellular heterogeneity and coupling strength are all expected to play a part. Here, we investigate separately the potential roles played by intrinsic noise arising from finite numbers of interacting molecules and by coupling among cellular oscillators. Regarding the former, it is well known that internal or molecular noise induces concentration oscillations in chemical systems whose deterministic models exhibit damped oscillations. We show, using the linear-noise approximation of the chemical master equation, that noise can also induce oscillations in biochemical systems whose deterministic descriptions admit no damped oscillations, i.e., systems with a stable node. This non-intuitive phenomenon is remarkable since, unlike noise-induced oscillations in systems with damped deterministic oscillations, it cannot be explained by noise excitation of the deterministic resonant frequency of the system. We here prove the following general properties of stable-node noise-induced oscillations for systems with two species: (i) the upper bound of their frequency is given by the geometric mean of the real eigenvalues of the Jacobian of the system, (ii) the upper bound of the Q-factor of the oscillations is inversely proportional to the distance between the real eigenvalues of the Jacobian, and (iii) these oscillations are not necessarily exhibited by all interacting chemical species in the system. The existence and properties of stable-node oscillations are verified by stochastic simulations of the Brusselator, a cascade Brusselator reaction system, and two other simple chemical systems involving autocatalysis and trimerization. We also show that external noise induces stable node oscillations with different properties than those stimulated by internal noise. Having demonstrated and explored this non-intuitive effect of noise, we extend the work to investigate the phenomenon of noise induced oscillations in cellular reaction systems characterised by the ‘bursty’ production of one or more species. Experiments have shown that proteins are typically translated in sharp bursts and similar bursty phenomena have been observed for protein import into subcellular compartments. We investigate the effect of such burstiness on the stochastic properties of downstream pathways by considering two identical systems with equal mean input rates, except in one system molecules (e.g., proteins) are input one at a time and in the other molecules are input in bursts according to some probability distribution. We find that the stochastic behaviour falls in three categories: (i) both systems display or do not display noise-induced oscillations; (ii) the non-bursty input system displays noiseinduced oscillations whereas the bursty input system does not; (iii) the reverse of (ii). We derive necessary conditions for these three cases to classify pathways involving autocatalysis, trimerization and genetic feedback loops. Our results suggest that single cell rhythms can be controlled by regulation of burstiness in protein production. We go on to investigate roles played by intercellular coupling in whole organ-level oscillations with an experimental analysis of circadian rhythms in Arabidopsis thaliana †. Circadian clocks in animals are known to be tightly coupled among the cells of some tissues, and this coupling profoundly affects cellular rhythmicity. However, research on the clock in plant cells has largely ignored intercellular coupling. Our research group used luciferase reporter gene imaging to monitor circadian rhythms in leaves of Arabidopsis thaliana plants, with both a lower resolution, high throughput method and a high-resolution (cellular level), lower throughput method. Leaves were grown and imaged in a variety of light conditions to test the relative importance of intercellular coupling and cellular coupling to the environmental signal. We analysed the high throughput data and described the circadian phase by the timing of peak expression. Leaves grown for three weeks without entrainment reproducibly showed spatio-temporal waves of gene expression, consistent with intercellular coupling. A range of patterns was observed among the leaves, rather than a unique spatio-temporal pattern, although some patterns were more often observed. All of the measured leaves exposed to lightdark entrainment cycles had fully synchronised rhythms, which could desynchronise rather quickly when placed in a non-entraining environment (i.e., constant light conditions). After four days in constant light some of these leaves were as desynchronised as leaves grown without any rhythmic input, as described by the phase coherence across the leaf. The same fast transition was observed in the reverse experimental scenario, i.e., applying light-dark cycles to leaves grown in constant light resulted in full synchronisation within two to four days. From these results we conclude that single-cell circadian oscillators were coupled far more strongly to external light-dark cycles than to the other cellular oscillators. Leaves did not spontaneously completely desynchronise, which is consistent with a presence of intercellular coupling among heterogeneous clocks. We also developed a methodology, based on the notion of two functional spatial scales of expression across the leaf, to analyse the high-resolution microscope data and determine whether there is a difference in the phase of circadian expression between vein cells and mesophyll cells in the leaf. The result from a single leaf suggests that the global phase wave dominates the phase behaviour but that there are small delays in the veins compared to their nearby mesophyll cells. This result can be validated by applying the methodology developed here to new microscope leaf data which is currently being collected in the research group. † This work was performed as a collaboration between David Toner (DT) and Benedicte Wenden (BW). BW designed and carried out the experiments, DT performed the data analysis and led on data visualisation.

No description available.

Amino-functionalised polystyrene microspheres are promising candidates as delivery systems due to their unique features, tunable surface functionalities, and controllable release of the cargo. Herein several strategies for the conjugation of biologically relevant cargoes to these microspheres and their biological evaluation are described. Firstly, dispersion and suspension polymerisation methods were applied for the synthesis of these devices. Subsequently, these polymeric particles were employed in multistep solid phase synthesis to conjugate a broad range of cargoes. The capability of the resulting constructs to cross the cell membrane and deliver the desired cargo was evaluated by flow cytometry and confocal microscopy. Additionally, the effect of these particles on cell viability was determined. Moreover a chemical strategy for dual fuctionalisation allowed the production of microspheres capable of carrying two cargos simultaneously (e.g. a biologically relevant cargo and a tracking fluorophore). Several strategies were used to transport biomolecules such as peptides and oligonucleotides inside cells. Cell-impermeable peptides with neuroprotective activity were conjugated to microspheres to facilitate their internalisation and they were efficiently delivered into neuroblastom cells (SH-SY5Y) without affecting their therapeutic activity. In addition, several microsphere-mediated oligonucleotide delivery strategies were investigated. As a first approach, siRNA was successfully attached to microspheres via thiol linkage or via electrostatic interaction (by formation of polycationated microspheres-siRNA microplexes). Using both strategies EGFP expression was efficiently down-regulated in cervical cancer cells permanently expressing EGFP (HeLa-EGFP) following beadfection. Additionally embryonic stem (ES) cells were beadfected with siRNA linked to microspheres by amide formation and essential transcription factors implicated in cell renewal and differentiation were successfully silenced, exceeding the silencing capabilities of commercially available lipofection products. Furthermore, a novel approach for the intracellular delivery of plasmid DNA was designed. Following an easy protocol for the linearisation and functionalisation of the plasmid DNA, this was covalently coupled to beads and cells were homogeneously ‘beadfected’. Finally, the coupling of fluorogenic substrates for caspase-3 to microspheres allowed the in situ monitoring and quantification of apoptotic process within cells. In conclusion, these small particles are excellent devices for the efficient intracellular delivery of a broad range of cargoes.

Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE. Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3×Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

The intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV) where it abundantly releases its chaperonin HtpB, suggesting that HtpB may have virulence-related functions. To assess these functions, I attempted to construct an L. pneumophila ?htpBmutant but was unable to do so, likely because htpB is essential. In the absence of genetic deletion, functional tests were used to study the released HtpB. A small portion of the HtpB in L. pneumophila-infected cells was found in the cytoplasm of the infected cells, as judged by the CyaA reporter assay. To identify potential functions of the HtpB present in the eukaryotic cytoplasm, htpB was ectopically expressed in Saccharomyces cerevisiae. HtpB induced pseudohyphal growth (PHG) in yeast, suggesting it interacts with eukaryotic targets. A yeast two-hybrid screen showed that HtpB interacted with SAMDC, an essential yeast enzyme encoded by SPE2 that is required for polyamine biosynthesis. Overexpression of SPE2 induced PHG in S. cerevisiae, suggesting that HtpB induces PHG by activating polyamine synthesis, and that L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC reduced L. pneumophila replication in host cells, whereas exogenous polyamines enhanced intracellular growth. Bioinformatics revealed that most known enzymes required for polyamine biosynthesis in bacteria are absent in L. pneumophila, suggesting that L. pneumophila depends on exogenous polyamines transported from host cells. L. pneumophila possesses only one putative operon,potABCD, which encodes a polyamine transporter. Using GFP as a reporter of potABCD promoter (PpotA), we found that PpotA activity was turned on during exponential phase of growth in vitro. To test the potential function of this transporter in pathogenesis, potD was deleted.Although deletion of potD did not affect L. pneumophila growth in vitro, it reduced L. pneumophila attachment to phagocytic cells, intracellular growth, and the ability of the LCV to recruit vesicles. Collectively, these findings have contributed to a better understanding of the biology of L. pneumophila by suggesting that HtpB and PotD might collaborate to ensure a supply of polyamines required for the optimal intracellular growth of L. pneumophila.