Itch lies second only to disturbance of body image as a reported symptom in dermatology. This study started by concentrating on improving the measurement of itch. Itch has a paired physical response, scratch. The pairing can be exploited: preliminary work by this unit had validated the use of wrist-worn movement-measuring machines called ‘accelerometers’ to measure itch-related movement (scratch and rub). The first part of this research developed use of these machines. Simple accelerometers (‘Actiwatch Plus’) were used to observe the pattern of variation of itch over clusters of nights and in different conditions. The accelerometer scores were able to identify controls’ scores from those with itchy disease. Considerable variation (56%) was discovered in objective score between subject and considerable variation was noted (46%) even within subject. More complex accelerometers, (‘DigiTrac’) which could potentially specifically identify itch-related movement on the basis of frequency of action derived from Fast Fourier Transform (FFT), were validated against the ‘gold standard’ measurement of itch-related movement, directly observed movement (via infra red video recording). It was necessary to characterise the ‘frequency of action’ of itch on video and, as an aside, the characteristics of human itch-related movement were compared to other mammals’ itch-related movement ‘frequency of action’. The ‘frequency of action’ and video data was used to enrich the DigiTrac readouts to improve specificity of itch-related movement detection. During the accelerometer studies, an unexpected finding came to light: objective score of itch was not related to subjective score. To try to explain the lack of relationship, a 42 day longitudinal study of atopic dermatitis patients’ subjective and objective scores was undertaken. The results demonstrated autocorrelation for subjective scores, but not for the objective scores but still did not fully explain the lack of relationship. In an effort to explain the disconnect between subjective and objective scores a second tranche of experiments and the second part of this research interrogated whether the methods with which we measure disease as a whole in dermatology are robust. One study investigated whether the way patients are asked about subjective symptoms in general was resistant to the effects of focusing and framing bias. The results were reassuring as they suggested that the commonly used and recommended symptom scoring systems were robust in the face of bias. In order to assess whether perspective or perception of disease explained the disconnect, a study was designed in collaboration with the Edinburgh College of Art. A series of computer-generated images of different psoriasis severities were created and used to assess how doctors and patients assessed disease-extent. This study showed that, whilst each group had a naturally divergent opinion of extent of disease, by scoring disease using the models it was possible to unify the perspective and perception of extent. Finally, an exploratory study to reduce recall bias to a minimum, in case this had caused the disconnect between objective and subjective, was undertaken. This employed a novel questionnaire, the Day Reconstruction Method.

No description available.

Spermatogenesis is an intricate process that strongly depends on the rapid turnover of short-lived proteins, both in the differentiating germ cells and in the supportive Sertoli cells. Recent evidence has demonstrated the importance of the ubiquitin-proteasome system for this turnover, with the final enzymatic E3 ligase providing the target specificity. One E3 ligase, Itch, has been well characterized in the immune system, but its role during spermatogenesis is not yet well understood. Mice lacking functional Itch protein display a late onset autoimmune disease characterized by severe inflammation, infiltration of immune cells into various organs, and most apparently chronic dermatitis, ultimately dying from pulmonary inflammation at 6 to 9 months of age. The work presented here evaluates the testes of itchy mice at two developmental time points, during the peri-pubertal period at postnatal day (PND) 28 and at adulthood, PND 56. Itchy mice are smaller in size and have lower spermatid head counts, most likely resulting from an increase in germ cell apoptosis rather than a decrease in Sertoli cell number. Litter sizes are reduced in the homozygous itchy colonies, with data suggesting a defect during fetal development and not in gamete production, although survival rates tend to be similar to that of wild type. At PND 28, itchy mice show a delay in spermatogenesis and an increase in meiotic figures, while PND 56 mice show alterations in germ cell layers, spermatid head formation, and irregular cell division. Examination of the previously identified targets of Itch revealed no significant increases in the testis, but led to discovery of immunoglobulin (IgG) deposits within the interstitial space. Changes in protein expression outside of the seminiferous epithelium suggest that cells of the immune system may be influencing proper development and functional spermatogenesis in the testis. While the previous studies using the itchy mice focused primarily on the late onset autoimmune dysfunction in these animals, increased spleen weights and changes in testicular protein are observed as early as PND 28, indicating that the loss of Itch impacts these animals much earlier during development. Taken together, these data indicate that Itch is required for functional spermatogenesis and that it may play different cellular roles depending on the developmental age of the animal. Future work is targeted at identifying the possible testis-specific targets of Itch and deciphering whether the observed phenotypes are the result of the primary loss of Itch or are a secondary effect from the overactive immune system. / text

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17822号 / 医博第3820号 / 新制||医||999(附属図書館) / 30637 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 鈴木 茂彦, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

臨床上止癢藥物的發展有限,故尋找具有廣泛止癢效果的藥物及探討癢覺傳遞的 神經受體機制是相當重要的。從過去研究中發現,bombesin 能在嚙齒類動物引發強 烈的搔抓行為。另外,bombesin 對 bombesin receptor family 中的 gastrin- releasing peptide receptor(GRPr)和 neuromedin B receptor(NMBr)也有高度親 和力。本研究主要目的為釐清 GRPr 和 NMBr 是否具有獨立調控癢覺搔抓行為的能 力。藉由側腦室於大白鼠中樞給藥並量化大白鼠的後肢搔抓行為,建立 GRP 與 NMB 這兩種胜肽在大白鼠上引發搔抓行為的基本藥理特性,並透過 GRPr 與 NMBr 這兩種受體的專一性拮抗劑探討受體間對於調控癢覺搔抓行為的獨立性。透過每天連 續施打 bombesin、GRP 與 NMB 這三種胜肽探討大白鼠搔抓行為的耐受性及三種胜 肽引發之搔抓行為在長時間觀測上的比較。最後,利用 GRPr 與 NMBr 這兩種受體 的專一性拮抗劑來探討 bombesin 引發搔抓行為的機制。利用側腦室給予 GRP (0.03 - 0.3 nmol)與 NMB(0.1-1 nmol)皆引發劑量相關性的搔抓行為反應。GRPr 的拮抗劑 RC-3095 (0.1-1 nmol)可以劑量相關性地阻斷 GRP 所引發的搔抓行 為,但不能阻斷 NMB 所引發的搔抓行為。相對而言,NMBr 的拮抗劑 PD168368 (0.3-3 nmol)可以劑量相關性地阻斷 NMB 引發的搔抓行為,但不能阻斷 GRP 所引 發的搔抓行為。藉由這些拮抗劑實驗,証實了 GRPr 與 NMBr 兩受體皆調控癢覺的訊 息傳遞並獨立不互相影響。Bombesin、GRP 與 NMB 都能引發搔抓行為,但程度與 持續時間上都有差異。但是每天施打這三種胜肽皆引發搔抓行為的耐受性。然而, RC-3095 與 PD168368 的有效劑量或兩個拮抗劑的組合並不能阻斷 bombesin 所引 發的搔抓行為,意指 bombesin 所引發的搔抓行為可能由其他受體調控。由本研究得 知,GRPr 與 NMBr 分別獨立調控中樞神經系統的癢覺訊息傳遞,並有程度與持續時 間上的差異。更重要的是,綜合先前文獻研究讓我們瞭解 bombesin-recognized neurons 中還有其他重要的受體機制調控於中樞神經系統的癢覺傳遞。 / Bombesin is a pruritogenic agent that causes intense itch-scratching activity in rodents. Bombesin has high affinity for the gastrin-releasing peptide receptor (GRPr) and the neuromedin B receptor (NMBr). The aim of this study was to investigate pharmacologically the ability of GRPr and NMBr to themselves elicit scratching behavior in rats. The intracerebroventricular (i.c.v.) route was selected for drug delivery because the study focused on supraspinal sites of action. The magnitude and duration of scratching produced by the naturally occurring peptides GRP and NMB were characterized. Antagonists selective for GRPr (RC-3095) and NMBr (PD168368) were used to define the role of GRPr and NMBr in the scratching response. After i.c.v. administration, GRP (0.03-0.3 nmol) and NMB (0.1-1 nmol) dose-dependently elicited marked scratching. There was a tolerance to scratching elicited by daily repeated administration of bombesin, GRP, or NMB. Presession administration of RC-3095 (0.1-1 nmol) and PD168368 (0.3-3 nmol) dose- dependently antagonized scratching elicited by GRP and NMB, respectively. More important, 1 nmol of RC-3095 failed to block NMB- elicited scratching and 3 nmol of PD168368 failed to block GRP-elicited scratching. In addition, pretreatment with effective doses of RC-3095 or PD168368 alone or in combination did not block bombesin-elicited scratching. Through the use of the selective antagonists RC-3095 and PD168368, this study demonstrates that central GRPr and NMBr may act independently to elicit scratching behavior. Although the receptor mechanisms underlying bombesin-elicited scratching elude us, this study provides a pharmacological basis for GRPr and NMBr antagonists as potential antipruritics.

No description available.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.

Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.