The circadian input kinase gene (cikA) was first identified from a Tn5 mutant of Synechococcus elongatus PCC 7942. A cikA null strain shows a striking phenotype related to circadian gene regulation: all sampled loci show a shortened circadian period and reduced amplitude of oscillation and a failure to exhibit a wild-type resetting of the phase of the rhythm after an environmental signal. This global defect in response to the environment suggests a key role for CikA in the circadian input pathways. Bioinformatics results classify CikA as a divergent member of the bacteriophytochrome family, suggesting a role in light signal transduction. In vitro analysis previously showed that CikA is a bona fide histidine protein kinase (HPK), and its kinase activity is regulated by the presence of other domains. Its own pseudo-receiver (PsR) domain is not the cognate receiver domain of its kinase HPK domain, and its GAF domain does not likely bind a bilin chromophore as do photoreceptive phytochromes. Recent results suggested that CikA may function as a redox-sensor. In this study, we examined the function of each domain of CikA using different mutant cikA alleles, and determined their phenotypes with respect to complementation of a null mutant and overexpression in both wild type and cikA null strains. All domains except the featureless N-terminus were required for CikA function. Overexpression of all mutant alleles that encoded the PsR domain, whether or not the HPK was functional, caused a dominant arrhythmia phenotype. In the absence of PsR, overexpressed variants did not cause arrhythmia, but affected the amplitude and period of oscillation. The results suggest a model in which the PsR domain regulates kinase activity and mediates interaction with other input pathway components to allow CikA to reach the correct cellular position to fulfill its function. Cellular localization assays showed CikA can interact with a complex and showed a polar localization pattern, whereas its variant without PsR showed uniform distribution in the cell. In summary, CikA is an autoregulated kinase in which the PsR domain regulates activity of the HPK domain and also serves as an interaction module to lead the CikA to a specific cellular position.

The assisted living industry is predicted to experience considerable growth due to the simultaneous aging of the baby boom generation and their subsequent need for long-term care. This study investigates individuals’ pathways to administration in assisted living and the ways in which assisted living administrators define and carry out their roles. The study contributes to the understanding of the knowledge, skills, and personal traits needed by those who assume these vital leadership roles in an industry poised to care for the bourgeoning population of older adults. The sample for this study is 44 administrators who work in a random sample of 45 assisted living facilities in Georgia. Qualitative methods were used to analyze the data. The data revealed that multiple multi-level factors not only facilitate an administrator’s pathway to AL administration, but these factors also help to shape how an administrator experiences his or her role within an ALF.

Mammalian γ-glutamyltransferase (GGT) is a glycoprotein consisting of two subunits - a light chain and a heavy chain. The light chain contains the catalytic activity; the heavy chain anchors the protein to the membrane. GGT catalyzes the hydrolysis of the γ-glutamyl isopeptide bond of glutathione conjugates, releasing glutamic acid, or the transfer of the γ-glutamyl group to an acceptor substrate. The specificity of the enzyme for xenobiotic donor substrates has not been fully characterized. The transpeptidation activity of bovine kidney GGT was measured with glycylglycine as acceptor substrate and several glutathione conjugate donor substrates, representative of detoxication products of polycyclic aromatic xenobiotics. HPLC separation with UV detection was used for quantitation. The commonly-used chromogenic donor substrate γ-glutamyl-p-nitroanilide was also tested. Michaelis constants (Km) were obtained for γ-glutamyl-p-nitroanilide (0.74 mM), 4-nitrobenzyl glutathione (0.075 mM), 2,4-dinitrophenyl glutathione (0.30 mM), 4-methylbiphenylyl glutathione (0.12 mM), 1-menaphthyl glutathione (0.23 mM), and 9-methylanthracenyl glutathione (0.22 mM), indicating that enzyme activity is affected, but not strongly, by the nature of the S-substituent attached to glutathione, and there is a slight trend of higher Km values with bulkier aromatic S-substituents.

RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815

The activation of many pro-hormones occurs through cleavage at pairs of basic residues and is mediated by two serine proteases, PC2 and PC3. Like their substrates, they are also synthesised as inactive precurors (pro-PC2 and pro-PC3). Maturation is autocatalytic and requires removal of the N-terminal pro-peptide. Pro-PC3 matures within the endoplasmic reticulum (ER), whereas maturation of pro-PC2 proceeds within the later compartments of the Golgi network (TGN)/secretory vesicle (SV) and is thought to be regulated by 7B2. In this study the molecular basis for the differences in the maturation location and the interaction with 7B2 was examined by performing domain swap and site directed mutagenesis experiments. The mutant constructs were analysed within an <I>in vitro</I> cell free system. The results suggest that the oxyanion hole residue (Asp³¹⁰) of pro-PC2 restricts maturation to a late secretory compartment and is important for the interaction with 7B2. Mutation of this residue to resemble that of PC3 (Asp310Ans), altered pro-PC2 maturation from a TGN/SV like environment to an ER like environment. Maturation of pro-PC2, but not pro-PC3, was shown to be inhibited by 7B2. Residue Asp³¹⁰ of PC2 was necessary to mediate this interaction. When a similar mutant of PC3 was created to resemble PC2 (Asn309Asp), this residue was not sufficient to alter the maturation profile of pro-PC3 nor was it able to confer 7B2 sensitivity. The pro-region of PC2 was sufficient to alter maturation of PC3 from the ER-like compartment to a TGN/SV-like compartment, but was not able to confer 7B2 sensitivity onto PC3. This study also demonstrated that a basic cluster (HHKQQ⁸⁸) of pro-PC2 was important for delaying maturation to a late compartment and that the residue Phe¹⁰⁴, was important for efficient maturation. Mutational analysis of pro-7B2 revealed that a region within residues 1-151 was important for the interaction with pro-PC2.

No description available.

Most microbial activity in nature takes place at interfaces where redox discontinuities are present. Organic pollutants in groundwater encounter oxic/anoxic interfaces when they emerge to surface water bodies or volatilize above the plume. Such oxic/anoxic interfaces are key habitats for aerobic bacteria and are in turn created by the bacteria that degrade organic electron donors. In the absence of biodegradation, synthetic pollutants can migrate from the plume and impact a variety of receptors. The aims of our study were to determine whether microbes at oxic/anoxic interfaces can use synthetic chemicals as electron donors and protect the overlying vadose zone or surface water from groundwater pollutants. The approach was to design columns representing the interfaces and measure activities of the microbial communities responsible for the biodegradation of synthetic compounds.Taken together the above studies established clearly that contaminants recalcitrant under anaerobic conditions but degradable under aerobic conditions can be biodegraded at the narrow oxic/anoxic interface resulting in the protection of the overlying soil or water. The findings provide the basis for new approaches to natural attenuation that can serve to dramatically reduce the cost of bioremediation actions. Synthetic chemicals are widespread in the environment because of their extensive use in industry. These chemicals were recalcitrant until their microbial degradation pathways evolved. Currently the biodegradation pathways of many synthetic chemicals are known and serve as the basis for bioremediation strategies. The second part of the research described here involved discovery of the aerobic degradation pathway of a dye additive: 4-nitroaniline (4NA). Annotation of the whole genome sequence coupled with assays and supported with cloned enzymes revealed that the 4NA biodegradation pathway contains two monooxygenase steps prior to ring cleavage. Because nitroaniline degradation was not previously understood our work advanced the understanding of metabolic diversity in degradation of amino and nitro compounds by providing enzymes with unique activities.

The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease of unclear aetiology, although the general consensus is of a multifactorial disease. The kynurenine pathway (KP), activated during neuroinflammation, is emerging as a possible contributory factor in ALS. The KP is the major route for tryptophan (TRP) catabolism. The intermediates generated can be either neurotoxic, such as quinolinic acid (QUIN), or neuroprotective, such as picolinic acid (PIC), an important endogenous metal chelator. The first and inducible enzyme is indoleamine 2,3-dioxygenase (IDO). As the extent of the involvement of the KP in ALS is unknown, the main aim of this thesis was to attempt to answer that question. The techniques used in this work include HPLC, GC/MS, RT-PCR, immunohistochemistry and immunocyctochemsitry. The main findings of this project are: (1) the complete KP is present in the mouse motor neuron cell line, NSC-34; (2) QUIN toxicity on NSC-34 cells may be ameliorated through the administration of NMDA antagonists, neuroprotective kynurenines, kynurenine inhibitor and QUIN monoclonal antibody; (3) in ALS patients, QUIN CSF and serum levels are significantly elevated, while PIC serum levels are significantly reduced; (4) ALS brain and spinal cord tissue show extensive microglia activation and positive immunoreactivity IDO and QUIN in spinal motor neurons and Betz cells in the motor cortex; and (5) kynurenine pathway inhibitor and analogue, R061-8048 and tranilast, are able to prolong the survival in the G93A SOD1 ALS transgenic mouse model. In conclusion, this study provide the first strong evidence for the involvement of the KP in ALS, and these data point to an inflammation-driven excitotoxic-chelation defective mechanism in ALS, which is amenable to KP analogue and inhibitor in ALS transgenic mice.

In this thesis, the transmission of spike trains in a neuron model is studied in order to obtain a better understanding of the role played by stochastic activity, i.e. uncorrelated spikes, in the auditory pathway. Fluctuations of the neuron membrane potential are given by a first-order stochastic differential equation, using a leaky integrate-and-fire model. In contrast to most previous studies the model has a finite number of synapses, and the usual diffusion approximation does not hold. / The input signal is modeled by spike trains with spiking times described by inhomogenous Poisson processes. The membrane potential is a shot noise process for which statistical properties are derived with a Gaussian approximation. The statistics of the output spike train are obtained by using the property that a pool of a large number of output spike trains can be modeled by an inhomogeneous Poisson process. It is shown that, under certain conditions, the addition of uncorrelated input spikes, i.e. noise, can enhance the transmission of periodic temporal information. This phenomenon, called stochastic resonance, is demonstrated analytically and supported by computer simulations. / Results are compared with those obtained from the traditional leaky integrate-and- fire neuron receiving a continuous waveform input. The shot-noise property of the membrane potential, which implies that its variance is de facto modulated by the input stimulus, is shown to enhance the phenomenon of stochastic resonance. Indeed, for a given average noise level, a modulated noise gives a higher output signal-to-noise ratio than an unmodulated noise with the same average amplitude. / The derivation is then extended to certain polyperiodic stimuli mimicking vowel sounds. The fact that the addition of uncorrelated input spikes can enhance the transmission of information is discussed in the context of cochlear implants. The results provide supportive evidence to the postulate that a cochlear implant speech coding strategy that elicits stochastic firing neural activity might benefit the user.