Nitric oxide (NO) is a neurotransmitter which is synthesized on demand from L-arginine by the enzyme nitric-oxide-oxidase, and is implicated in a variety of physiological functions, including release and uptake of dopamine. Amphetamine induces stereotyped behavior via release of dopamine from dopaminergic neurons in the striatum and related structures, while apomorphine induces such behavior via activation of central dopaminergic receptors. Recently we have demonstrated that a NO donors and a NO-synthase inhibitor modify the response of some central dopaminergic receptors to their agonists and antagonists. In the present study we examined the effect of two NO donors and one NO-synthase inhibitor on stereotyped behavior induced in rats by amphetamine and apomorphine, and the sex-selectivity of this effect. A highly significant dose-dependent sexual differentiation was recorded in the stereotyped behavior of amphetamine, as the duration and intensity of this effect was shortened by L-NAME but not by L-arginine and Molsidomine. Differences in the stereotyped behavior between female and male rats administered apomorphine were dose-dependent, but were not affected by any of the three drugs tested. It is concluded that while nitric oxide is involved in the reactivity of central dopamine receptors, the intensity and duration of this effect is drug- and sex-dependent.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 μg/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with β-tubulin III. However, 21-day treatment (50 μg/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

In the aftermath of cancer surgery, Natural killer (NK) cells are severely suppressed. NK cells are critical for anti-tumour surveillance and their postoperative dysfunction creates an opportunity for metastases. I hypothesized that NK cell suppression is mediated by multiple suppressive mechanisms of surgery-induced Myeloid Derived Suppressor Cells (Sx-MDSCs). In this thesis, I first show that NK cell dysfunction is far worse than previously described. In a cohort of colorectal cancer (CRC) surgery patients (n=42), the ability of NK cells to secrete IFN-gamma in response to stimulation was suppressed for up to 2 months after surgery. Secondly, since Sx-MDSCs have been poorly characterized in humans, I thoroughly phenotyped Sx-MDSCs from cancer surgery patients using flow cytometry (n=32 patient samples) and single-cell RNA sequencing (n=6 patient samples). Additionally, upon screening a library of 150 compounds, I showed that Sx-MDSC rely on PI3K signaling for their suppression of NK cells in ex vivo NK cell suppression assays. The third part of this thesis explores the contribution of Sx-MDSCs to the rapid reduction in postoperative arginine, the perioperative importance of arginine for NK cells, and the therapeutic effects of a perioperative arginine enriched supplement (AES) on metastases in murine models of surgical stress. Here, I showed that perioperative AES attenuates postoperative metastases by accelerating NK cell recovery after surgery. These promising preclinical data combined with evidence from the scientific literature led us to initiate a Phase II randomized-controlled clinical trial assessing the ability of perioperative AES to improve NK cell function after surgery in CRC patients (n=12/arm). In the last part of this thesis, I present the results from our clinical trial, which showed only a transient and, at best, modest improvement in NK cell function. Importantly, this may have been heavily influenced by poor postoperative patient compliance in taking the AES. In conclusion, this body of work describes the multifactorial role that Sx-MDSCs play in mediating postoperative NK cell suppression, and that safe, effective, and targeted perioperative interventions should be further investigated as a strategy to attenuate metastatic disease recurrence after surgery.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20661号 / 医博第4271号 / 新制||医||1024(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 川口 義弥, 教授 柳田 素子, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The aim of this study was to compare the effects of arginine vasotocin (AVT) administration in the endangered boreal toad (Bufo boreas boreas) and common Fowler’s toad (Bufo fowleri). The objectives of this study were to determine if AVT could elicit: 1) calling, and 2) amplexus behaviors. Toads were paired into single male:female groups and administered AVT at varying concentrations: 0.1, 1.0, 5.0, 10.0 and 25.0 μg/g and in different combinations: 1) only male was treated; 2) only female was treated, and 3) both male and female treated. AVT failed to stimulate any breeding behavior in the boreal toad, but the administration of AVT to both B. Fowleri genders significantly affected the duration of amplexus (p<0.0347). Also, the concentration of AVT significantly affected the length of amplexus (p<0.0429) and call frequency (p<0.0294). These results will be valuable for breeding programs where animals are failing to show natural reproductive behavior.

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This dissertation is a compilation of published work and a manuscript that seeks to understand the role of polyamine metabolism in the regulation of pneumococcal physiology. Streptococcus pneumoniae (pneumococcus) is the major cause of community-acquired pneumonia, and otitis media worldwide. Genetic diversity and serotype replacement, and antibiotics resistance to confound existing therapeutic strategies and limit the effectiveness of the available capsule polysaccharide (CPS) based vaccines. Polyamines such as putrescine, spermidine and cadaverine are ubiquitous polycationic hydrocarbons that interact with negatively charged molecules and modulate important cellular processes. Intracellular polyamine concentrations are regulated by biosynthesis, degradation, and transport. This work investigated the impact of the deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine/arginine decarboxylase covered in the second, third and fourth chapters), on growth, Gram staining characteristics, capsule production, proteome and stress responses of virulent pneumococcal serotype 4 (TIGR4). We identified loss of capsular polysaccharide (CPS) in DELTA SP_0916 strain. Our proteome results showed a shift in metabolism towards the pentose phosphate pathway (PPP) that could reduce the availability of precursors for CPS and could explain the un-encapsulated phenotype of DELTA SP_0916. Since a shift towards the PPP is usually in response to stress, we compared the stress responses of DELTA SP_0916 to that of TIGR4. Our results show that the mutant was more susceptible to oxidative, nitrosative, and acid stress compared to the wild type. In the fifth chapter we compared the transcriptome, metabolome, stress responses and stress susceptibility of the polyamine transport deficient strain (DELTA potABCD) and S. pneumoniae TIGR4. Results in this chapter show that polyamine transport is essential for pneumococcal stress responses, and capsule biosynthesis. The impact of impaired polyamine synthesis (DELTA SP_0916), and transport (DELTA potABCD) on pneumococcal capsule is due to altered expression of Leloir pathway, reduced glycolysis, and increased PPP, possibly in response to impaired stress responses. These results demonstrate that alteration of polyamine pathways affects pneumococcal stress responses which in turn could limit the availability of precursors for capsule synthesis, and thus have an impact on virulence. Thus, polyamine metabolism is an attractive avenue for developing novel interventions for limiting the spread of S. pneumoniae, a versatile human pathogen.

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