The Role of the Small G Protein Rap1 in T cell Activation

Rap1會受到T細胞抗原受體 (TCR) 的活化，並在細胞中扮演調控黏著分子integrin的角色。然而Rap1在T細胞活化上所扮演的角色及影響黏著分子integrin時受到的調控機制，並無明確的結論。在本論文中，我們將分三個方面探討Rap1的角色。第一，探討CD28的刺激對於Rap1的活化。第二，利用表現抑制型Rap1N17及持續活化型Rap1V12的DO11.10細胞株，與建立Rap1V12轉殖基因小鼠來探討Rap1在T細胞活化時的影響。第三，了解p38 MAPK是否參與Rap1的訊息傳遞。 從我們的研究中發現，T細胞株EL4受到CD28的刺激會增加TCR對於Rap1的活化。而在表現Rap1V12的轉殖小鼠內，不影響胸腺T細胞與脾臟T細胞之發育(包含細胞數目、胸腺T細胞的正向選擇、CD4與CD8細胞之比例、TCR的表現)。而胸腺T細胞與脾臟T細胞受到活化後之細胞增生及IL-2的產量則較對照組高，顯示Rap1在T細胞內為正向調節分子。但在T 細胞株DO11.10內表現持續活化型Rap1V12及抑制型Rap1N17，其活化所產生的IL-2則未受到影響，也不影響DO11.10與抗原呈現細胞在有抗原存在下之結合比率。 在探討TCR活化Rap1的訊息傳遞上，我們發現T細胞株EL4以p38 MAPK特異抑制劑SB203580的預先處理後，會促進Rap1受到TCR刺激後的活性。但小鼠脾臟T細胞以SB203580的預先處理，增加了未活化T細胞內Rap1的活性，但經TCR及CD28刺激後則Rap1之活性並未明顯增加。因此，p38 MAPK調控Rap1的活化因不同細胞而異。 總結來說，我們的研究支持Rap1在正常細胞內扮演正向調節的角色。CD28的訊息會促進Rap1在T細胞內的活化。此外，p38 MAPK可能參與在TCR活化Rap1的訊息傳遞路徑中。我們同時也發現在不同的細胞中有相異的實驗結果，顯示調控Rap1的活化會因為在不同的細胞、細胞內的分布及活化量上的不同而有相當之複雜性。所以Rap1在T細胞活化及訊息傳導上所扮演的真正角色，需要更多研究來進一步釐清。Rap1 is activated by TCR signaling and is participated in integrin activation. The exact role of Rap1 in T cell activation and the mechanism on how Rap1 regulates integrin activation have not been precisely defined. In this study, we explored the role of Rap1 in three different aspects. First, we examined the effect of CD28 co-stimulation on Rap1 activation. Second, we elucidated the role of Rap1 in T cell activation by expressing the dominant negative Rap1N17 and constitutive active Rap1V12 in DO11.10 hybridoma and Rap1V12 in transgenic mice. Third, we examined the possible involvement of p38 MAPK in Rap1 activation. Results from our study illustrated CD28 co-stimulation increased Rap1 activation both in EL4 T lymphoma and splenic T cell, supporting a positive role of CD28. The T cell-specific transgenic expression of Rap1V12 did not affect T cell development, including cell number, CD4/CD8 ratio, positive selection and TCR levels. T cell proliferation and IL-2 production were increased in Rap1V12-transgenic mice, suggesting a positive role of Rap1 in T cell activation. In contrast, expression of Rap1V12 and Rap1N17 in DO11.10 T hybridoma did not affect CD3 or antigen peptide induced IL-2 production nor was antigen peptide-mediated T cell-antigen presenting cell conjugation. In the study of the TCR signals that activate Rap1, we found that pretreatment with p38 MAPK specific inhibitor SB203580 increased TCR-stimulated Rap1 activation in EL4 cell. Pretreatment of splenic T cell with SB203580 resulted in increase Rap1 basal level not TCR-stimulated Rap1 activation. Therefore, the involvement of p38 MAPK in Rap1 activation is cell-type dependent. In summary, our results clearly support a positive role of Rap1 in the activation of normal T cells. CD28 co-stimulation promoted the Rap1 activation. In addition, p38 MAPK may couple TCR activation to Rap1 activation. However, we also found exceptional results in different types of T cells, suggesting a complicated regulation of Rap1 which may depend on cell type, distribution, and quantity of Rap1. Further studies are required to delineate the exact role of Rap1 in T cell activation and signal coupling.中文摘要 i 英文摘要 iii 縮寫對照表 viii 第一章 簡介 一、Rap1的發現 1 二、Rap1在細胞內之弁? 1.Rap1與Ras的交互作用 2 2.Rap1影響細胞型態與integrin的活化 4 3.Rap1與p38 MAPK的交互作用 6 研究目的 7 第二章 材料與方法 壹、細胞株與細胞培養 一、細胞株 8 二、小鼠胸腺與脾臟細胞 8 三、細胞培養 9 貳、藥品與試劑 9 參、抗體 9 肆、質體構築 一、pcDNA3-myc-Rap1 N17及pcDNA3-myc-Rap1 V12 質體 10 二、pGC-myc-Rap1 N17-YFP及pGC-myc-Rap1V12-YFP 質體 11 三、CD2-myc-Rap1 V12質體 11 伍、在DO11.10細胞表現Rap1突變株 一、以calcium phosphate轉染法將質體送入Phoenix細胞 11 二、以離心法感染DO11.10細胞 12 陸、基因轉殖鼠之建立 一、Genomic DNA測試 12 二、mRNA測試 1.以TRIzol試劑抽取細胞全RNA 13 2. RNA反轉錄反應 14 3. 反轉錄聚

Insight into One-Electron Oxidation of the {Fe(NO)₂}⁹ Dinitrosyl Iron Complex (DNIC): Aminyl Radical Stabilized by [Fe(NO)₂] Motif

A reversible redox reaction ({Fe­(NO)₂}⁹ DNIC [(NO)₂Fe­(N­(Mes)­(TMS))₂]⁻ (<b>4</b>) ⇄ oxidized-form DNIC [(NO)₂Fe­(N­(Mes)­(TMS))₂] (<b>5</b>) (Mes = mesityl, TMS = trimethylsilane)), characterized by IR, UV–vis, ¹H/¹⁵N NMR, SQUID, XAS, single-crystal X-ray structure, and DFT calculation, was demonstrated. The electronic structure of the oxidized-form DNIC <b>5</b> (<i>S</i>_total = 0) may be best described as the delocalized aminyl radical [(N­(Mes)­(TMS))₂]₂^–• stabilized by the electron-deficient {Fe^III(NO^–)₂}⁹ motif, that is, substantial spin is delocalized onto the [(N­(Mes)­(TMS))₂]₂^–• such that the highly covalent dinitrosyl iron core (DNIC) is preserved. In addition to IR, EPR (<i>g</i> ≈ 2.03 for {Fe­(NO)₂}⁹), single-crystal X-ray structure (Fe–N­(O) and N–O bond distances), and Fe K-edge pre-edge energy (7113.1–7113.3 eV for {Fe­(NO)₂}¹⁰ vs 7113.4–7113.9 eV for {Fe­(NO)₂}⁹), the ¹⁵N NMR spectrum of [Fe­(¹⁵NO)₂] was also explored to serve as an efficient tool to characterize and discriminate {Fe­(NO)₂}⁹ (δ 23.1–76.1 ppm) and {Fe­(NO)₂}¹⁰ (δ −7.8–25.0 ppm) DNICs. To the best of our knowledge, DNIC <b>5</b> is the first structurally characterized tetrahedral DNIC formulated as covalent–delocalized [{Fe^III(NO^–)₂}⁹–[N­(Mes)­(TMS)]₂^–•]. This result may explain why all tetrahedral DNICs containing monodentate-coordinate ligands isolated and characterized nowadays are confined in the {Fe­(NO)₂}⁹ and {Fe­(NO)₂}¹⁰ DNICs in chemistry and biology

Insight into One-Electron Oxidation of the {Fe(NO)₂}⁹ Dinitrosyl Iron Complex (DNIC): Aminyl Radical Stabilized by [Fe(NO)₂] Motif

A reversible redox reaction ({Fe­(NO)₂}⁹ DNIC [(NO)₂Fe­(N­(Mes)­(TMS))₂]⁻ (<b>4</b>) ⇄ oxidized-form DNIC [(NO)₂Fe­(N­(Mes)­(TMS))₂] (<b>5</b>) (Mes = mesityl, TMS = trimethylsilane)), characterized by IR, UV–vis, ¹H/¹⁵N NMR, SQUID, XAS, single-crystal X-ray structure, and DFT calculation, was demonstrated. The electronic structure of the oxidized-form DNIC <b>5</b> (<i>S</i>_total = 0) may be best described as the delocalized aminyl radical [(N­(Mes)­(TMS))₂]₂^–• stabilized by the electron-deficient {Fe^III(NO^–)₂}⁹ motif, that is, substantial spin is delocalized onto the [(N­(Mes)­(TMS))₂]₂^–• such that the highly covalent dinitrosyl iron core (DNIC) is preserved. In addition to IR, EPR (<i>g</i> ≈ 2.03 for {Fe­(NO)₂}⁹), single-crystal X-ray structure (Fe–N­(O) and N–O bond distances), and Fe K-edge pre-edge energy (7113.1–7113.3 eV for {Fe­(NO)₂}¹⁰ vs 7113.4–7113.9 eV for {Fe­(NO)₂}⁹), the ¹⁵N NMR spectrum of [Fe­(¹⁵NO)₂] was also explored to serve as an efficient tool to characterize and discriminate {Fe­(NO)₂}⁹ (δ 23.1–76.1 ppm) and {Fe­(NO)₂}¹⁰ (δ −7.8–25.0 ppm) DNICs. To the best of our knowledge, DNIC <b>5</b> is the first structurally characterized tetrahedral DNIC formulated as covalent–delocalized [{Fe^III(NO^–)₂}⁹–[N­(Mes)­(TMS)]₂^–•]. This result may explain why all tetrahedral DNICs containing monodentate-coordinate ligands isolated and characterized nowadays are confined in the {Fe­(NO)₂}⁹ and {Fe­(NO)₂}¹⁰ DNICs in chemistry and biology

Patient–machine interactions of intravenous patient-controlled analgesia in bilateral versus unilateral total knee arthroplasty: A retrospective study

Background: Continuous passive motion after a major knee surgery optimizes functional prognosis, but causes severe pain. In this study, we assessed the effect of intravenous patient-controlled analgesia (IVPCA) on postoperative pain management in unilateral and bilateral total knee arthroplasty (TKA). Methods: Data were collected retrospectively from a single medical center from March 2003 to October 2007. All patients who had undergone TKA were given general anesthesia, and the type of surgery that each patient received was planned according to individual needs. A total of 223 patients qualified for this study, with 174 patients in the unilateral TKA group. Data on patient demography, pain scores, and side effect scores were collected. Total dose consumption, demand, delivery doses, demand-to-delivery ratio, and infusion rate were collected from PCA machines and analyzed. Results: The patient pain score and patient satisfaction showed no significant difference between the unilateral and bilateral TKA groups. The incidence of sedation (p < 0.001), nausea (p = 0.013), and vomiting (p = 0.044) during the postoperative 24–48-hour period was higher in the bilateral TKA group. Compared with the patients in the unilateral group, those in the bilateral group showed significantly greater dose consumption during the postoperative 6–12-, 12–18-, and 18–24-hour periods. They also showed more demand for medication during the postoperative 12–18- and 18–24-hour periods and received more bolus doses during the postoperative 12–18-, 18–24-, and 30–36-hour periods. In addition, there was also a significantly higher demand-to-delivery ratio for patients in the bilateral group during the postoperative 6–12-, 12–18-hour periods. Conclusion: In this study, we successfully demonstrated that our IVPCA protocol can provide adequate analgesia for patients after both bilateral and unilateral TKA. However, sedation, nausea, and vomiting occurred more frequently during the postoperative 24–48-hour period in patients who underwent bilateral than unilateral TKA. This may due of the increased number of bolus doses administered to the patients in the bilateral TKA group during the postoperative 12–18, 18–24, and 30–36-hour periods. Therefore, the initial infusion rates for patients undergoing bilateral TKA could be set at a lower threshold in order to reduce the incidence of these side effects

Visceral Adiposity, Pro-Inflammatory Signaling and Vasculopathy in Metabolically Unhealthy Non-Obesity Phenotype

The debate regarding the actual cardiovascular burden in metabolically healthy obese or metabolically unhealthy non-obesity individuals is ongoing. Accumulating data have suggested a unique pathophysiological role of pro-inflammatory cytokines in mediating metabolic and cardiovascular disorders by dysregulated visceral adiposity. To compare the burden of visceral adiposity, the inflammatory marker high-sensitivity C-reactive protein (hs-CRP) and the prevalent atherosclerotic burden in metabolically healthy obese (MHO) or metabolically unhealthy (MU) populations, were compared to those of metabolically healthy non-obesity subjects (MHNO). Coronary artery calcification score (CACS) and visceral fat, including pericardial fat (PCF)/thoracic peri-aortic fat (TAT), were quantified in 2846 asymptomatic subjects using a CT dataset. A cross-sectional analysis comparing CACS, inflammatory marker hs-CRP, and visceral fat burden among four obesity phenotypes (MHNO, metabolically unhealthy non-obesity (MUNO), MHO, and metabolically unhealthy obese (MUO)) was performed. Both MUNO and MUO demonstrated significantly higher hs-CRP and greater CACS than MHNO/MHO (adjusted coefficient: 25.46, 95% confidence interval (CI): 5.29&ndash;45.63; 43.55, 95% CI: 23.38&ndash;63.73 for MUNO and MUO (MHNO as reference); both p &lt; 0.05). Visceral fat (PCF/TAT) was an independent determinant of MU and was similarly higher in the MUNO/MHO groups than in the MHNO group, with the MUO group having the largest amount. PCF/TAT, obesity, and MU remained significantly associated with higher CACS even after adjustment, with larger PCF/TAT modified effects for MU and diabetes in CACS (both pinteraction &lt; 0.05). MU tightly linked to excessive visceral adiposity was a strong and independent risk factor for coronary atherosclerosis even in lean individuals, which could be partially explained by its coalignment with pathological pro-inflammatory signaling

Investigation of Bone Growth in Additive-Manufactured Pedicle Screw Implant by Using Ti-6Al-4V and Bioactive Glass Powder Composite

In this study, we optimized the geometry and composition of additive-manufactured pedicle screws. Metal powders of titanium-aluminum-vanadium (Ti-6Al-4V) were mixed with reactive glass-ceramic biomaterials of bioactive glass (BG) powders. To optimize the geometry of pedicle screws, we applied a novel numerical approach to proposing the optimal shape of the healing chamber to promote biological healing. We examined the geometry and composition effects of pedicle screw implants on the interfacial autologous bone attachment and bone graft incorporation through in vivo studies. The addition of an optimal amount of BG to Ti-6Al-4V leads to a lower elastic modulus of the ceramic-metal composite material, effectively reducing the stress-shielding effects. Pedicle screw implants with optimal shape design and made of the composite material of Ti-6Al-4V doped with BG fabricated through additive manufacturing exhibit greater osseointegration and a more rapid bone volume fraction during the fracture healing process 120 days after implantation, per in vivo studies

Additional file 1: of Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase

List of Primers used in this study. (PDF 180 kb