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研究生：	蘇愉雅 Su, Yu-Ya
論文名稱：	TIAM2S基因對於大腦發育、大腦可塑性和行為特徵的影響 The effects of TIAM2S gene on brain development, plasticity and behavioral traits
指導教授：	孫孝芳 Sun, H. Sunny 朱俊憲 Chu, Chun-Hsien
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	45
中文關鍵詞：	T細胞淋巴癌侵略和轉移基因、大腦可塑性、過動、社交行為
外文關鍵詞：	TIAM gene, brain plasticity, hyperlocomotion, social interaction
相關次數：	點閱：137 下載：6
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跨膜結構域(transmembrane domain)缺失的短片段T細胞淋巴癌侵略和轉移2蛋白質(short form of T-cell lymphoma invasion and metastasis 2 ;TIAM2S)在結構上屬於鳥糞嘌呤核甘酸交換因子(guanine nucleotide exchange factor; GEF)的成員。在我們實驗室2012年的研究報告中，首次指出過度表現TIAM2S能促使肝癌細胞增生與轉移。有趣的是在正常成年人類的大腦內邊緣系統的神經細胞中，亦發現到TIAM2S蛋白的高度表達。然而此蛋白在人類大腦內的功能仍屬未知。已知Tiam1(TIAM2的同源基因)在胚胎發育時期的大腦形成(brain formation)扮演重要角色，同時也調控著軸突的生長 (neurite outgrowth)。然不同於Tiam1，我們先前的實驗結果指出TIAM2S蛋白並不具備GEF的酵素活性，而且其表現主要坐落在細胞核內。並且目前對於TIAM family所影響的行為特徵一無所知。在本研究中，我們假設TIAM2S參與在成年時期的大腦可塑性(brain plasticity)，並在行為塑造(behavioral shaping)上扮演關鍵的角色。為此，我們建立了過度表達人類TIAM2S基因轉殖鼠(transgenic mice overexpressing human TIAM2S; TIAM2S-TG mice)動物模型來進行相關的研究探討。結果顯示此TIAM2S-TG mice並未出現胚胎時期異常的大腦發育，其能正常地出生，並在外觀上與正常老鼠(wildtype mice)無顯著差異。進一步透過核磁共振的T2加權像(T2-weighted MRI)與神經細胞的染色，我們發現到成熟期乃至中年(mature and middle adulthood)的TIAM2S-TG mice具有與相同年齡的正常老鼠相似的神經結構與神經細胞數目。然而，利用擴散張量影像分析(DTI-MRI analysis)和Golgi染色指出過度表達人類TIAM2S會造成小鼠大腦內的神經細胞的樹突生長與分枝增加且改變腦內神經路徑。最重要的是TIAM2-TG mice顯示出過動與正向的社會互動的行為特徵。綜合上述結果，我們的研究顯示腦內的TIAM2S基因具有調控神經可塑性的全新功能，能去改變樹突可塑性且增加運動(hyperlocomotion)與社交能力(increased social interaction)。

Short form of T-cell lymphoma invasion and metastasis 2 (TIAM2S), structurally belonging to the TIAM subfamily of guanine nucleotide exchange factor (GEF) and lacking transmembrane domain, has been noted for its ability in prompting cancer proliferation and metastasis by a previous study of our laboratory in 2012. Unexpectedly, this protein also highly expresses in neuronal cells of limbic system of adult human brain at non-neoplastic, physiological conditions with unknown function. Tiam1, a homology of TIAM2, plays a critical role in developing brain formation at early embryonic stage and also participates in regulation of neurites outgrowth. Different to Tiam1, our previous results reveal that TIAM2S fails to show the GEF activity and mainly locates in nuclear. But little is known about behavioral influences of the TIAM subfamilies. Thus, we hypothesize that human TIAM2S is a key regulator for brain plasticity and behavioral shaping at adult stage. To address it, a transgenic mouse model that overexpresses human TIAM2S protein (TIAM2S-TG) is generated. Our results showed that TIAM2S-TG mice are born and grown normally into adolescence. Furthermore, TIAM2-TG mice at mature and middle adulthood exhibit normal structure and neurons viability in brain detected by T2-weighted magnetic resonance imaging (MRI) and chemical staining for neurons number. Moreover, through using DTI-MRI (diffusion tensor imaging-MRI) analysis and Golgi staining, we find that overexpressed human TIAM2S protein leads animals increase dendrite growth, branch and routing of brain. By contrast, knockdown of TIAM2S impairs neurite outgrowth of induced human NT2/N cells. Most importantly, the TIAM2S-TG mice display hyperlocomotion and positive social interaction behaviors. Taken together, our data demonstrate that TIAM2S functions as a novel neuroplasticity gene to modulate dendritic plasticity along with increased locomotion and social interaction behaviors.

摘要 I
Abstract III
誌謝 V
Introduction 1
1. TIAM subfamily 1
1.1 Conserved protein domain and its function in TIAM subfamily	1
1.2 T-Cell Lymphoma Invasion and Metastasis 1 (TIAM1) 1
1.3 Mouse T-cell lymphoma invasion and metastasis 2 (Tiam2/Stef) 2
1.4 Human T-cell lymphoma invasion and metastasis 2 (TIAM2) 3
2. TIAM2S protein expresses in neurons of restricted brain regions (our previous findings) 4
3. Objective of this study 5
Materials and Methods 6
1. Generation of TIAM2S transgenic mice 6
2. Magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) examination 7
3. Brain cryostat section 9
4. Immunofluorescence staining 9
5. Nissl and Golgi staining 10
6. Animal behavior tests 10
6.1 Open field test and locomotor activity 10
6.2 Social interaction test 10
6.3 Four-paw grip-strength test 11
6.4 Accelerating rotarod test 11
7. Cell culture 11
8. TIAM2S shRNA lentivirus infection 12
9. NT2/N cells induction and neurite length measurement 12
10. Gel electrophoresis and western blot 13
11. Antibody 14
12. Statistical analysis 14
Results 15
1. Transgenic mice for overexpression of human TIAM2S protein are born and grown normally into adolescence 15
2. Adult TIAM2-TG mice exhibit normal structure and neurons viability in brain 16
3. Overexpressed TIAM2S protein promotes dendritic plasticity 17
4. TIAM2S-TG mice display hyperactive behaviors	18
Discussion 19
Different to TIAM1, TIAM2S is critical for postnatal growth of brain 19
Behavioral phenotype of TIAM2S-TG mice 20
Possible mechanism of TIAM2S-mediated dendritic plasticity 21
Conclusion 23
Figures 24
Figure 1. Timeline for experiments 24
Figure 2. TIAM2S-TG mice are born and grown normally into adolescence 25
Figure 3. TIAM2S-TG mice possess normal neuroanatomy structure of brain at mature and middle adulthood 27
Figure 4. Overexpressed TIAM2S protein promotes dendritic plasticity 30
Figure 5. TIAM2S-TG mice display hyperlocomotion 32
Figure 6. TIAM2S-TG mice increase social interaction behaviors 34
Figure 7. TIAM2S-TG mice have not body weight loss and motor dysfunctions 35
Tables 36
Supplements 38
Supplementary Figure 1. Knockdown of TIAM2S protein decreases neurite outgrowth of NT2/N cells 39
Reference 41
                                    

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