59 research outputs found

    生化学反応による計算能力の研究

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    早大学位記番号:新6514早稲田大

    FLOW INJECTION AND MULTIVARIATE CALIBRATION TECHNIQUES FOR PROCESS ANALYSIS

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    The role of process analytical chemistry is summarised in chapter one with particular emphasis on a multidisciplinary approach and the instrumental requirements for on-plant analysis. These concepts are extended to process FIA, highlighting its potential for simultaneous multicomponent determinations. The development of an automated FIA monitor for the on-line determination of sulphite in potassium chloride brine is covered in the second chapter. Reaction stability is demonstrated and the results of on-plant validation and on-line trials are presented. The next chapter deals with the concepts of multivariate calibration. Direct multicomponent analysis, principal components regression and partial least squares regression are critically examined in practical spectroscopic terms and statistical terms. The relative predictive abilities of these techniques are compared in chapter four for the resolution of a multicomponent UV-visible spectrophotometric data set. Chapter five describes the development of an automated FIA-diode array system for the simultaneous determination of phosphate and chlorine. The implications of combining reaction chemistries and the influence of a number of calibration parameters are considered in detail. Finally, the jackknife is presented as a means of dimensionality estimation' and bias correction in PLS modelling. Data sets from the literature are analysed and the results compared with those obtaining using commercial software.ICI Chemicals & Polymer

    T cells in solid tumors : investigating the immunomodulation in the tumor microenvironment

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    The immune system protects human from cancer through an immunosurveillance mechanism. However, the progressive nature of tumor cells to differentiate and the complexity of the tumor microenvironment may result in the immunomodulation of immune cells. In this thesis, we aim to explore the T cell immunomodulation inside the intricate solid tumor microenvironment in patients. First, we investigated suppressive regulatory T cells (Tregs) in urinary bladder cancer (UBC). Our group previously demonstrated a contradictory finding that a high FOXP3+ tumor infiltrating lymphocyte (TIL) number correlates positively to survival. In here, we answered that FOXP3+ CD4+ T cells in the tumor were real Tregs which protectively regulated tumor invasiveness by suppressing MMP2 expression in tumor-associated macrophages (TAMs) and tumor cells. Next, we explored the subset of tissue-resident memory CD8+ T (TRM) cells from UBC tumor. It is less known whether TRM cells are effective killers of tumor cells. We revealed that tumor TRM cells were epigenetically committed to express perforin. Although TRM cells expressed exhaustion marker PD-1, they were not terminally exhausted. As a result, we found that an increased number of TRM cells in the tumor correlated with a lower tumor stage. Furthermore, we looked into the cytotoxic CD8+ T cells in the sentinel nodes (SNs) of UBC patients. Surprisingly, we discovered that SN CD8+ T cells displayed a deficiency of their cytotoxic constituent perforin, whereas granzyme B was still expressed. Thereafter, we revealed that muscle invasive UBC suppressed perforin expression using an ICAM-1/TGFβ2 – mediated pathway as an immune escape mechanism. In the next study, we focused on the effect of standard neoadjuvant chemotherapy (NAC) and T cell responses in the SNs. We found that NAC reinforced the anti-tumor T cell activities by reducing the exhaustion in CD8+ and CD4+ effector T cells, which consequently increased their cytotoxicity and clonal expansion, respectively. Additionally, NAC also reduced the frequency and activation of the suppressive Tregs. Lastly, as a result of escaping the immune destruction, tumor can grow and metastasize. In this study, we revealed that micrometastases in lymph nodes of renal tumors could be reliably detected by flow cytometry. This method is more sensitive, objective, time- and cost-effective compared to the gold standard histopathological examination. In conclusion, T cells are modulated in the solid tumor microenvironment. By understanding the molecular and cellular aspects of T cells in this microenvironment, we may unveil new strategies for designing cancer immunotherapies in the future

    Acupuncture in Modern Medicine

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    This book contains four integrated sections: 1) Acupuncture Research; 2) New Developments in Acupuncture; 3) Acupuncture Therapy for Clinical Conditions and 4) Assessment and Accessibility in Acupuncture Therapy. Section 1 provides updates on acupuncture research. From acupuncture effects in modulation of immune system to the role of nitric oxide in acupuncture mechanisms, chapters in this section offer readers the newest trends in acupuncture research. Section 2 summarizes new developments in acupuncture. The included chapters discuss new tools and methods in acupuncture such as laser acupuncture, sham needles, and new technologies. Section 3 discusses acupuncture therapy for clinical conditions. The chapters in this section provide comprehensive and critical views of acupuncture therapy and its application in common clinical practice. Section 4 takes a new look at the issues related to assessment and accessibility in acupuncture therapy. These issues are central to developing new standards for outcome assessment and policies that will increase the accessibility to acupuncture therapy

    Epigenetic and Proteomic Signatures for Chronic Pain Patients after Total Knee Replacement

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    A proteomic investigation of \u3cem\u3ePhytophthora\u3c/em\u3e species using mass spectrometry and reverse genetics

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    Organisms in the genus Phytophthora are important plant pathogens, although understudied. Phytophthora was first brought into human awareness with the identification of P. infestans as the culprit for the Irish potato famine in the mid 1800s. Since then, over 80 Phytophthora species have been identified, many of which infect a wide variety of crops worldwide with devastating results. Traditionally, much of the work aimed at controlling Phytophthora diseases involved applied research. In recent years there has been a marked increase in molecular work on Phytophthora. This increase is evident not only from increased funding by agencies such as the National Science Foundation (NSF), but also from the type of research applied to Phytophthora for the first time. The first Phytophthora species to have their genomes sequenced were P. sojae and P. ramorum at 2004. Since then the genomes of two more Phytophthora species- P. capsici and P. infestans, were also sequenced. Availability of Phytophthora genome sequences provided us with the basis necessary for a proteomic investigation of these organisms. The study presented here represents the first large scale proteomic study of any Phytophthora species. Using mass spectrometry and available or newly developed bioinformatic tools we measured the proteomes of different asexual Phytophthora life stages. We also measured the protein complement of P. capsici infected tomato plants, the so called “interactome”, in order to gain an insight into the biological processes occurring in the pathogen during infection, and in the plant in response to the pathogen. We also used data from these proteomic experiments as a part of a novel approach aimed at improving the genome annotation of those Phytophthora species. Finally, we used different molecular techniques, including a reverse genetic technique called Targeted Induced Local Lesions in Genome (TILLING), to begin characterization of a few protein targets identified in those experiments. The accumulated data from all our experiments identified certain molecular processes, metabolic and others, that may explain the success of Phytophthora as a plant pathogen. The data from these experiments provides a platform on which future experiments can be based on to further characterize these interesting organisms

    The aetiology of pain in chronic midportion Achilles tendinopathy

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    Background Achilles tendinopathy (AT) is a common injury in athletes and sedentary individuals, which presents as pain and loss of function in the lower limb. Tendon pathology can exist without pain, but the hallmark of the condition is pain, which is classically of insidious onset, related to loading activity and often resistant to treatment. While the biology of pain in general is well described, the mechanisms of pain in AT are not fully understood. Most commonly, the nociceptive driver associated with AT is thought to be a result of the structural changes that occur in the tendon or the inflammatory cascades that occur in the pathological tendon and/or reflective of altered central pain mechanisms. Evidence from other chronic pain conditions also shows that genetic variation explains, at least in part, some of the heterogeneity observed in chronic pain conditions. The presentation of chronic Achilles tendon pain is variable and therefore it is reasonable to propose that this variability may be influenced by a genetic component. The absence of a definitive cause or mechanism of pain in AT is reflected in the plethora of treatment strategies available to manage it, most of which are not universally effective. In order to improve the management of pain in chronic AT, it is imperative that its mechanisms be better understood. Aims of the thesis The aims of this thesis were therefore to characterise Achilles tendon pain using other pain questionnaires, to investigate the relationship between structural changes and central pain mechanisms with self-reported tendon pain. Additionally, the thesis sought to evaluate the relationship between selected gene variants and pain in a cohort of recreational athletes with chronic Achilles tendinopathy using a candidate gene approach. Candidate genes: COMT rs4818 (C/G), COMT rs4633 (C/T), TAC1rs2072100 (C/T), TACR1 rs3771829 (C/G) and SCN9A rs6746030 (G/A) were selected based on the biological function of their encoded proteins within the pain pathways. The objectives of the specific chapters which addressed these aims were: • Describe Achilles tendon pain using multidimensional pain scales; the short forms of the McGill pain questionnaire (sf-MPQ) and Brief Pain Inventory (sf-BPI), as well as the Victorian Institute of Sports Assessment – Achilles questionnaire (VISA-A) (Chapter 2). • Evaluate the relationship between self- reported tendon pain, the grey scale ultrasound and colour Doppler characteristics in chronic AT (Chapter 3). • Evaluate the relationship between conditioned pain modulation and chronic AT (Chapter 4). • Explore and evaluate if variants in genes [COMT rs4818 (C/G), COMT rs4633 (C/T), TAC1 rs2072100 (C/T), TACR1 rs3771829 (C/G) and SCN9A rs6746030 (G/A)] involved in the pain pathways are associated with either self-reported tendon pain and/or conditioned pain modulation (Chapter 5). Methods Two hundred and eighty-two (282) recreational athletes with at least one year's experience in their main sport were recruited for the studies in this thesis but fifty-two (52) were excluded for not meeting the inclusion criteria of the studies. Hence, 103 recreational athletes without a history of chronic AT (CON) and 127 participants clinically diagnosed with chronic AT (TEN) were included in the study. All participants completed demographic questionnaires on their medical, sporting, training, and injury history. Participants with AT (TEN) also completed the self-administered eight question VISA-A questionnaire, the sf-MPQ and the sf-BPI. Additionally, all participants had grey scale ultrasound (US) and colour Doppler (CD) assessments of both their tendons performed and had conditioned pain modulation (CPM) assessed using pressure and cold pain. Lastly, participants were genotyped for variants in COMT rs4818 (C/G), COMT rs4633 (C/T), TAC1 rs2072100 (C/T), TACR1 rs3771829 (C/G) and SCN9A rs6746030 (G/A) using standard PCR methods. Data were analysed using Statistica Version 13.2.50. Normality of data was assessed using the Shapiro-Wilks test. Evaluations of differences between normally distributed quantitative data were conducted with the independent students t-test or one-way ANOVA, while Mann-Whitney-U and Kruskall-Wallis tests were used for non-normally distributed data. The Fisher's exact and χ2 tests were used for categorical data. For post-hoc analyses, the Kruskal-Wallis associated multiple comparisons test with Bonferroni adjustment was used for quantitative data. For the genotyping data, Hardy– Weinberg equilibrium (HWE) was calculated using ‘HardyWeinberg' version 1.6.3. package. The overall level of significance was set at p0.3; p0.05). However, the median interference index scores of the VISA-A questionnaire of participants with US abnormalities [median (IQR)] [35.5 (30.0 - 41.0), n=36] was significantly higher than those without US abnormalities [32.5 (26.0 - 37.0), n=39, p=0.046]. Additionally, participants from the TEN group who reported no stabbing pain, those who reported mild, moderate or severe stabbing pain on the sf-MPQ had significantly thicker tendons [median (IQR)] [6.0mm (5.2 - 7.6) vs 7.0mm (5.9 - 8.9), 7.7mm (6.2 - 9.1) and 6.3mm (4.9 - 7.4), p=0.037]. From the CPM analysis, participants with tendinopathy had a lower pressure pain threshold (PPT) before [median (IQR)] [TEN: 417kPa (364 - 516) vs CON 601kPa (459 - 724), p<0.001] and during [TEN: 458kPa (358 - 550) vs CON 633kPa (506 - 753), p<0.001] the cold pressor test. However, there was no difference in the CPM effect between the two groups [median (IQR)] [TEN: 34kPa (-2 - 79) vs CON: 45kPa (4 - 94), p=0.490]. From the sf-BPI, PPT before the cold pressor test were significantly lower in individuals who reported mild to severe interferences in mood (p=0.023), general activity (p=0.038) and walking ability (p=0.004) when compared to those who reported no interferences. Pressure pain thresholds before the cold pressor test were also significantly lower in those participants who reported mild to severe pain at the time of testing (p=0.024) or reported moderate to severe pain on average (p=0.014) on the sf- BPI. Additionally, from the sf-BPI, a low CPM effect was significantly associated with mild to severe interference with sleep (p=0.043). The genotype analysis showed that the median total scores of self-reported tendon pain from the sf-MPQ were significantly different (p=0.019) among the three COMT rs4818 (G/C) genotype groups [median (IQR)] [CC: 9.1 (4.0 - 13.0) n=61; CG: 7.3 (4.0 - 0.0) n=50; GG: 4.0 (1.0 - 5.0) n=7], with the CC genotype having a significantly higher pain score (p=0.018) than the GG genotype. No other associations were observed between genotype distributions of COMT rs4633, TAC1 rs2072100, TACR1 rs3771829, SCN9A rs746030 and the median self-reported total tendon pain scores for the sf-MPQ, sf-BPI, VISA-A, or their subscales. Conclusion The novel findings of this thesis suggest that the language of chronic AT pain ought to be further investigated as it may help extend our knowledge of the underlying mechanisms in chronic AT pain. In addition, that AT pain interferes with more than physical and sporting ability should be considered in the overall management of this condition in athletes. While no associations were observed between imaging findings and tendon pain, the relationship between imaging findings and physical limitations suggests that using pain as a primary outcome measure in rehabilitation may be insufficient and highlights the need to further study the relationship between tendon structure, imaging and pain. Furthermore, impaired CPM was associated with interferences with sleep which suggests that, though not quite clear, some central mechanisms are at play in chronic AT pain. This finding also reaffirms the need to consider factors other than physical function in AT management. Another novel finding of this thesis was the association between COMT rs4818 (C/G) and chronic tendon pain. This finding suggests that the catecholaminergic pathway is involved in the chronic AT pain pathway. COMT variants are associated with maladaptive coping mechanisms which may be important to consider in managing chronic pain conditions such as AT. In future, larger studies are required in order to replicate these findings and large, prospective cohort studies are required to confirm the role of genetic variation in chronic AT pain. Overall, the mechanisms of pain in tendinopathy are complex and not yet well described, emphasising the further need for multi-sectorial research

    Phytophthora parasitica and lupin (Lupinus angustifolius) interactions: changes in gene expression during infection and after phosphite treatment

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    Phytophthora species are Oomycete pathogens that cause highly destructive diseases in a variety of agricultural and horticultural crops, and natural ecosystems. An understanding of the key biological processes that occur during development and infection of hosts is important for the development of effective Phytophthora control mechanisms. An infection assay model system was developed for P. parasitica based on lupin (Lupinus angustifolius) seedlings. The progress of lesion development and colonisation of P. parasitica in inoculated root tissues was assessed macroscopically and using light microscopy of sectioned material. At 24 hours post inoculation (hpi), a few hyphae were observed in the epidermal and outer cortical cells in the region of the root that had been at the surface of the zoospore suspension during the inoculation period. As root infection progressed, the hyphae grew both towards the vascular tissue at the centre of the root and longitudinally along the root. At 42 hpi, P. parasitica hyphae developed haustoria within root cortical cells. No evidence of callose deposition, a typical plant defence response, by the lupin root cells was observed after infected roots stained with aniline blue. Development of the model lupin-P. parasitica infection assay system facilitated ensuing studies of this plant-pathogen interaction, including the cellular and molecular basis of plant infection. The model assay system was used to examine levels of resistance of different lupin cultivars following inoculation with P. parasitica and to analyse temporal patterns of P. parasitica gene expression using quantitative real-time PCR (qPCR) during lupin root infection. One crucial component of Phytophthora pathogenicity is the digestion of the plant cell wall to allow penetration of the plant surface and colonisation within the plant tissues. Plant cell walls are complicated structures that are composed of a wide range of complex polysaccharides (i.e. cellulose, hemicelluloses and pectins) and proteins and they constitute an effective barrier that impedes the entry of many potential pathogens. In order to penetrate the plant cell wall, pathogens secrete a diverse array of cell wall degrading enzymes (CWDEs). The identity and timing of the expression of genes encoding P. parasitica CWDEs was analysed using qPCR. It is believed that pathogens secrete cascades of CWDEs during the infection process and evidence supporting this hypothesis was obtained from the lupin-P. parasitica data. One management strategy used in the control of Phytophthora diseases is the application of the chemical phosphite. Our understanding of the mechanism(s) underlying phosphite inhibition of Phytophthora diseases in plants is limited. Phosphite is known to have effects on both host plants and Phytophthora pathogens. In the present study, RNA-Seq was used to investigate the effects of phosphite on P. parasitica gene expression in vitro and in planta. Phosphite treatment was found to induce extensive changes in the expression of many pathogen genes both in vitro and in planta. One of the exciting results was the discovery that there was a general tendency for phosphite to up-regulate the expression of genes that are normally expressed early in lupin infection (30-36 hpi) and to down-regulate the expression of genes that are normally expressed during late infection (54-60 hpi). This was exemplified in particular by P. parasitica genes encoding pectinase and cellulase CWDEs and RxLR effectors. In conclusion, the research described in this thesis has developed a new and robust model infection assay for use in studies of plant infection by P. parasitica and, potentially, by other Phytophthora species. The research also presents the results of using this assay in transcriptomic studies of pathogen gene expression during plant infection. The results that have been obtained provide a better understanding of Phytophthora pathogenicity mechanisms and should aid the future development of improved methods of controlling Phytophthora diseases
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