Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

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The goal of this study was to show that quantifiable metabolic changes may be used to screen for cancer. NIH III nude mice (n=22) were injected with human GBM cells, with daily urine samples collected pre and post-injection. 14 mice were injected with saline to serve as controls. The measurement of metabolite concentrations took place on an 800 MHz NMR spectrometer. 34 metabolites were identified and quantified, through targeted profiling, with Chenomx Suite 5.1. Univariate statistical analysis showed that 3 metabolites (2-oxoglutarate, glucose and trimethylamine n-oxide) were significantly altered in the presence of tumour, while PCA and PLS-DA models found the maximum variance between the healthy and tumour-bearing groups. Receiver operating characteristic (ROC) curve analysis was applied to the data set to provide a measure of clinical utility. ROC statistics were as high as 0.85 for the analysis of individual metabolites, 0.939 for the analysis of metabolite pairs and 0.996 for PLS-DA models. These results show that metabolomics has potential as a screening tool for cancer. / Medical Physics

MeCP2 was identified by its ability to bind DNA in a methylation-specific manner. Yet, how it interprets the DNA methylome remains unclear. Several mechanisms have been proposed, including a role in transcriptional repression. MeCP2 is highly abundant in the brain, and loss-of-function mutations result in a neurological disorder called Rett syndrome (RTT). Strikingly, RTT-causing missense mutations are almost all located in either the methyl-CpG-binding domain (MBD) or a region that has been shown to bind the NCoR/SMRT co-repressor complex (NID). This suggests that the MBD and the NID are the key functional domains in MeCP2, and that the role of MeCP2 is to form a ‘bridge’ between chromatin and the co-repressor complex to regulate gene expression. To test this ‘bridge’ hypothesis, I have made an allelic series of knock-in mice with truncated forms of MeCP2 to determine whether the other regions are dispensable for protein function. The three other regions of MeCP2 (the N-terminus before the MBD, the Intervening region between the MBD and the NID, and the C-terminus after the NID) were deleted in a step-wise manner to produce progressively smaller truncated proteins. Knock-in mice which lack just the N-terminus or both the N- and C-termini are phenotypically normal. Therefore, these regions, which together make up 46% of the protein sequence, are dispensable for MeCP2 function in vivo. Additional deletion of the Intervening region, retaining only 34% of the original sequence, results in mild RTT-like symptoms in the knock in mice. This is likely to be caused by this protein’s decreased stability and reduced ability to bind the NCoR/SMRT complex in the brain. The most severely truncated protein is nevertheless able to reverse the Mecp2-null phenotype when reactivated after the onset of symptoms. Together, these findings strongly support the ‘bridge’ hypothesis.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantrone can improve the outcome of RVFV infection in our mouse model. The underlying mechanism of mitoxantrone to inhibit RVFV replication remains to be investigated. Our studies build the foundation for identification and development of antivirals against RVFV in a BSL-2 environment.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

<p>  </p> <p>Recent advancements in genetics have revealed that <em>SCN2A</em> is one of the leading genes associated with neurodevelopmental disorders including autism spectrum disorder and epilepsy. In particular, loss-of-function and truncation variants account for a majority of cases. As there are no current treatments specific for <em>SCN2A</em>, the neuropharmacogenomics field has strived to further elucidate the role of <em>SCN2A</em> in neurodevelopment to identify intervention targets. Rodent models offer <em>in vivo</em>, pre-clinical insight into the effects of genetic variation on behavior, biochemistry, and electrophysiology as well as the mechanisms on molecular, cellular, and circuitry levels. Due to <em>SCN2A</em>’s critical involvement in the initiation and propagation of action potential neuronal firing early in neurological development, full null homozygous knockout of <em>Scn2a</em> in mice is perinatal lethal. Furthermore, canonical heterozygous knockout of <em>Scn2a </em>in mice does not render phenotypes that recapitulate <em>SCN2A</em> deficiency in humans. Therefore my dissertation aims at developing a mouse model that better parallels the human condition, then using that pre-clinical platform to explore precision medicine.</p> <p>  </p> <p>Using the unconventional strategy of gene trapping, we generated mice with a severe reduction in <em>Scn2a</em> expression, resulting in significant behavioral and electrophysiological differences from neurotypical wild-type mice with full <em>Scn2a</em> expression, but enough residual expression that the <em>Scn2a</em>-deficient mice survived into adulthood. The severely decreased sociability accompanied by increased high and low order repetitive behaviors observed with the <em>Scn2a</em>-deficient mice suggest autism-like phenotypes. In addition, <em>Scn2a</em>-deficient mice also displayed other co-morbidities of neurodevelopmental disorders including atypical innate behavior, increased anxiety, increased sensitivity to stimuli, motor discoordination, and impaired learning and memory. On the electrophysiological level, these mice displayed enhanced intrinsic excitabilities of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in seizures and social behavior. This increased excitability was autonomous and reversible by the genetic restoration of <em>Scn2a</em> expression in adult mice. Further, RNA-sequencing revealed a downregulation of multiple potassium channels as well as differential expression of glutamate excitatory and GABA inhibitory signaling, which led to the pursuit of targeting these pathways. Indeed, the use of potassium channel openers alleviated the hyperexcitability of <em>Scn2a</em>-deficient neurons, thus supporting the pursuit of these targets.  </p> <p>Since characterization of the <em>Scn2a</em>-deficient mouse model revealed disruption in excitatory and inhibitory pathways, excitatory/inhibitory balance was examined further as a precision medicine target. Increasing <em>Scn2a</em> expression throughout the whole brain by excising the gene trap, as well as specific targeting of the striatum and the neurons that project to it using a retrograde viral vector, rescued social deficits. However the striatum-specific injection did not lead to a social rescue. This shifted the focus to the neurons that project to the striatum such as the medial prefrontal cortex. Using chemogenetics to reduce excitatory signaling in the prelimbic region of the medial prefrontal cortex, we were able to increase the social behavior in <em>Scn2a</em>-deficient mice. Synthesizing the results from the retrograde striatum and prelimbic-specific rescue, the next hypothesis tested was a circuity-level manipulation of the medial prefrontal cortex projections to the striatum. Retrograde control (striatum) of chemogenetics (medial prefrontal cortex) decreased the excitatory signaling in the medial prefrontal cortex neurons that project to the striatum, which also led to improved sociability. On the other side of the excitatory/inhibitory balance, increasing inhibitory signaling through acute exposure to small-molecule GABA receptor positive allosteric modulators, clonazepam and AZD7325, rescued sociability.</p> <p>This dissertation opens up new avenues of research by supporting the use of a pre-clinical mouse model of <em>Scn2a</em> deficiency to advance the study of underlying mechanisms behind <em>SCN2A</em>-related neurodevelopmental disorders. Although the results of this dissertation need additional validation such as cellular support, the data and results in this dissertation can serve as a guide to further explore excitatory/inhibitory balance as a neuropharmacogenomics precision medicine target to treat <em>SCN2A</em>-related neurodevelopmental disorders. </p> <p><br></p> <p><br></p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24504号 / 医博第4946号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 溝脇 尚志, 教授 浅野 雅秀, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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