Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome

Matthew J. Nicholson, Michael K. Theodorou and Jayne L. Brookman. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome. Microbiology, 151 (1), 121-133. Sponsorship: BBSRC RAE2008

Alterações metabólicas no diabético

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Inibidores do co-transportador renal de sódio-glicose 2

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Molecular characterisation of the mechanisms of compatible solute accumulation in Listeria monocytogenes

The ability of the Gram-positive foodborne pathogen Listeria monocytogenes to survive and grow in environments of elevated osmolarity can be attributed, at least in part, to the accumulation of a restricted range of low molecular mass solutes compatible with cellular function. Accumulated to high internal concentrations in hyper-saline environments, compatible solutes, either transported into the cell or synthesized de novo, play a dual role: helping to stabilize protein structure and function while also counterbalancing external osmotic strength, thus preventing water loss from the cell and plasmolysis. While previous physiological investigations identified glycine betaine, carnitine, and proline as the principal compatible solutes in the listerial osmostress response, genetic alanysis of the uptake/synthesis systems governing the accumulation of these compounds has, until now, remained largely unexplored. Representing the first genetic analysis of compatible solute accumulation in L. monocytogenes, this thesis describes the molecular characterization of BetL; a highly specific secondary glycine betaine transport system, OpuC; a multicomponent carnitine/glycine betaine transporter, and finally proBA; a two-gene operon encoding the first two enzymes of the listerial proline piosynthesis pathway. In addition to their role in osmotolerance, the potential of each system in contributing to listerial pathogenesis was investigated. While mutations in each gene cluster exhibited dramatic reductions in listerial osmotolerance, OpuC- mutants were additionally shown to exhibit reduced virulence when admisistered via the oral route. This represents the first direct link between the salt stress response and virulence in L. monocytogenes.

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Novel roles for the GABAB receptor in anxiety and cocaine addiction

The GABAB receptor has been postulated as a possible drug target in the treatment of anxiety disorders and cocaine addiction. Indeed, a wealth of preclinical data is emerging that has shown that mice lacking functional GABAB receptors display a highly anxious behaviour across a range of behavioural models of anxiety. Additionally, novel compounds that act by altering the allosteric conformation of the GABAB receptor to a more active state; the GABAB receptor positive modulators, have been repeatedly demonstrated to have anxiolytic effects in animals. In addition to being a putative anxiolytic drug target, the GABAB receptor has been identified as a novel target for antiaddictive therapies. Indeed GABAB receptor positive modulators have been demonstrated to have anti-addictive properties across a broad variety of behavioural paradigms. Despite these findings, several gaps in our knowledge of the role played by the GABAB receptor in both anxiety and drug abuse disorder exist. The aim of this thesis was to use preclinical animal models in an effort to further probe the role played by the GABAB receptor in anxiety and addiction. Our studies initially examined the role played by the GABAB receptor in the neurodevelopmental processes underpinning of anxiety. Our studies demonstrated that treating mouse pups in early life with the GABAB receptor agonist baclofen produced an anxious phenotype in adult life, whereas treatment with the GABAB receptor antagonist CGP52432 produced no effects on adult behaviour. Further to this, we examined whether the anxious behaviour induced by early life blockade of the serotonin reuptake transporter was dependant on alterations in GABAB receptor function. Our studies however revealed no effect of early life selective serotonin reuptake inhibitor treatment on adult life baclofen sensitivity. The next issue addressed in this thesis is the characterization of the effects of a GABAB receptor positive modulator and a GABAB receptor antagonist in a behavioural model of conditioned fear behaviour. These novel classes of GABAB receptor ligands have been considerably less well characterized in this facet of preclinical anxiety behaviour than in terms of innate anxiety behaviour. Our study however revealed that the GABAB receptor positive modulator GS39783 and the GABAB receptor antagonist CGP52432 were without effect on the acquisition, expression or extinction of conditioned fear in our model. The next element of this thesis dealt with the characterization of a novel mouse model, the GABAB(2)- S892A mouse. This mouse has been engineered to express a form of the GABAB(2) receptor subunit wherein the function determining serine phosphorylation site cannot be phosphorylated. We initially tested this mouse in terms of its GABAB receptor function in adult life, followed by testing it in a battery of tests of unconditioned and learned anxiety behaviour. We also examined the behavioural and molecular responses of the GABAB(2)-S892A mouse to cocaine. All of our studies appear to show that the GABAB(2)-S892A mouse is indistinguishable from wildtype controls. The final aim of the thesis was to investigate the behavioural and molecular sensitivity of the GABAB(1) subunit isoform null mice, the GABAB(1a) -/- and GABAB(1b) -/- mice to cocaine. Our studies revealed that these mice display differing behavioural responses to cocaine, with the GABAB(1a) -/- mouse displaying a hypersensitivity to the acute locomotor effects of cocaine, while the GABAB(1b) -/- displayed blunted locomotor sensitisation to cocaine.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.

Thuricin CD: a potential therapeutic targeted against Clostridium difficile-associated diarrhea (CDAD)

Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibioticassociated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. The main etiological agent of C. difficile-associated diarrhea (CDAD) is perturbations to the gut microbiota by broad-spectrum antibiotics. Recently, thuricin CD, a two-peptide narrow spectrum sactibiotic bacteriocin with potent activity against C. difficile has been discovered. It is produced by Bacillus thuringiensis DPC6431. The efficacy of thuricin CD against a range of C. difficile clinical isolates has been determined in the form of minimum inhibitory concentration (MIC) values and compared to metronidazole, vancomycin, ramoplanin and actagardine in this thesis. Furthermore, by assessing paired combinations of the above-mentioned antimicrobials, it was determined that ramoplanin and actagardine function in a synergistic manner against the majority of C. difficile isolates. The functions of the genes in the thuricin CD gene cluster have also been elucidated by cloning the cluster and expressing thuricin CD in a heterologous Bacillus subtilis host and are described herein. In addition, the immunity mechanisms employed by the B. thuringiensis DPC6431 producer to protect itself from the antimicrobial actions of thuricin CD have also been elucidated. It has been shown that a small immunity peptide, TrnI, is involved in thuricin CD immunity, most likely by intercepting the thuricin CD peptides and/or blocking their access to the thuricin CD receptor. This immunity peptide and also the ABC-transporter system TrnFG serve to protect the B. thuringiensis host against thuricin CD.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.

Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.

A longitudinal study of epigenetic variation in twins.

DNA methylation is a key epigenetic mechanism involved in the developmental regulation of gene expression. Alterations in DNA methylation are established contributors to inter-individual phenotypic variation and have been associated with disease susceptibility. The degree to which changes in loci-specific DNA methylation are under the influence of heritable and environmental factors is largely unknown. In this study, we quantitatively measured DNA methylation across the promoter regions of the dopamine receptor 4 gene (DRD4), the serotonin transporter gene (SLC6A4/SERT) and the X-linked monoamine oxidase A gene (MAOA) using DNA sampled at both ages 5 and 10 years in 46 MZ twin-pairs and 45 DZ twin-pairs (total n=182). Our data suggest that DNA methylation differences are apparent already in early childhood, even between genetically identical individuals, and that individual differences in methylation are not stable over time. Our longitudinal-developmental study suggests that environmental influences are important factors accounting for interindividual DNA methylation differences, and that these influences differ across the genome. The observation of dynamic changes in DNA methylation over time highlights the importance of longitudinal research designs for epigenetic research.

Serotonin synthesis, release and reuptake in terminals: a mathematical model.

BACKGROUND: Serotonin is a neurotransmitter that has been linked to a wide variety of behaviors including feeding and body-weight regulation, social hierarchies, aggression and suicidality, obsessive compulsive disorder, alcoholism, anxiety, and affective disorders. Full understanding of serotonergic systems in the central nervous system involves genomics, neurochemistry, electrophysiology, and behavior. Though associations have been found between functions at these different levels, in most cases the causal mechanisms are unknown. The scientific issues are daunting but important for human health because of the use of selective serotonin reuptake inhibitors and other pharmacological agents to treat disorders in the serotonergic signaling system. METHODS: We construct a mathematical model of serotonin synthesis, release, and reuptake in a single serotonergic neuron terminal. The model includes the effects of autoreceptors, the transport of tryptophan into the terminal, and the metabolism of serotonin, as well as the dependence of release on the firing rate. The model is based on real physiology determined experimentally and is compared to experimental data. RESULTS: We compare the variations in serotonin and dopamine synthesis due to meals and find that dopamine synthesis is insensitive to the availability of tyrosine but serotonin synthesis is sensitive to the availability of tryptophan. We conduct in silico experiments on the clearance of extracellular serotonin, normally and in the presence of fluoxetine, and compare to experimental data. We study the effects of various polymorphisms in the genes for the serotonin transporter and for tryptophan hydroxylase on synthesis, release, and reuptake. We find that, because of the homeostatic feedback mechanisms of the autoreceptors, the polymorphisms have smaller effects than one expects. We compute the expected steady concentrations of serotonin transporter knockout mice and compare to experimental data. Finally, we study how the properties of the the serotonin transporter and the autoreceptors give rise to the time courses of extracellular serotonin in various projection regions after a dose of fluoxetine. CONCLUSIONS: Serotonergic systems must respond robustly to important biological signals, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of the serotonin transporters and the serotonin autoreceptors. Many difficult questions remain in order to fully understand how serotonin biochemistry affects serotonin electrophysiology and vice versa, and how both are changed in the presence of selective serotonin reuptake inhibitors. Mathematical models are useful tools for investigating some of these questions.

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex.

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.