Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Nov 29

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Roger Bradley. Includes bibliographical references (leaves 122-140).

Radiotherapy is second only to surgery in the curative management of patients with cancer, and yet the molecular mechanisms that determine the sensitivity of tumours to radiation remain largely unclear. A high-throughput radiosensitivity screening method based on clonogenicity was developed and a siRNA library against kinase targets was screened. The gold standard colony formation endpoint was chosen for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitisation. TPK1 knockdown caused significant radiosensitisation in cancer but not normal tissue cell lines. Other means of blocking this pathway such as knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumour specific radiosensitisation. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitisation target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumour specific radiosensitisation. Three additional genes, signal recognition particle-72 kDa (SRP72), glycogen synthase 3-beta (GSK3β) and MAP/Microtubule Affinity-Regulating Kinase 2 (MARK2) were also investigated. Knockdown of these genes radiosensitised both tumour and normal tissue cell lines and expression of two of them, GSK3β and SRP72 were found to be associated with poor recurrence-free survival in early breast cancer patients.

Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy Maia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

M.Tech. / RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged.

No description available.

Made available in DSpace on 2016-06-24T04:00:22Z (GMT). No. of bitstreams: 1 Sergio Araujo Figueiredo.pdf: 5051471 bytes, checksum: 845523d46a114f99e92e4c1f07b80e2a (MD5) Previous issue date: 2011-06-03 / ß-galactosidases are a class of glycosyl-hydrolases that act on the plant cell primary walls, hydrolyzing ß-D-galactose at the nonreducing ends of ß-D-galactosides present in several biological molecules. Initially a characterization of the monosaccharides present in the primary wall of the pericarp and endosperm of Coffea arabica fruits at different ripening stages was performed, identifying the polysaccharides present in these regions, along with the putative carbohydrate target for the ß-galactosidase. In parallel, a molecular and a biochemical characterization of ß-galactosidase was performed. A partial characterization of ß-galactosidase genomic DNA structure, along with a transcription analysis and an in vitro and in situ biochemical activity were performed, identifying peaks of expression in the early stages of growth and in fully ripe fruit. Finally, in order to evaluate the ß-galactosidase effects on coffee fruit ripening, C. arabica calli were transformed by biolistic using RNA interference approach, in order to obtain genetically modified coffee plants with a silenced ß-galactosidase expression. Three transgenic calli growing on selective medium containing ammonium glufosinate were obtained, two of which contained the ß-galactosidase gene fragment. These calli are under embryogenic regeneration and the resulting seedlings will be further analyzed in order to confirm the presence of the transgenes and to assess of the effects of ß-galactosidase gene silencing on coffee fruit ripening. / As ß-galactosidases sao uma classe de glicosil-hidrolases que atuam na parede primaria das celulas vegetais, hidrolisando residuos ß-D-galactosis de extremidades nao redutoras de ß-D galactosideos presentes em diversas moleculas biologicas. Inicialmente foi feita uma caracterizacao dos monossacarideos presentes na parede primaria do pericarpo e do endosperma de frutos de Coffea arabica em distintas fases de maturacao, identificando os polissacarideos presentes nesta regiao celular, juntamente com os provaveis carboidratos-alvo para as ß-galactosidases. Em paralelo, foi realizada uma caracterizacao molecular e bioquimica das ß-galactosidases. Foi realizada uma caracterização parcial da estrutura do seu DNA genomico, juntamente com uma analise do nivel de transcricao e da atividade bioquimica in vitro e in situ foram realizadas, identificando picos de expressao nas fases iniciais de crescimento e nos frutos completamente maduros. Por fim, visando avaliar os efeitos das ß-galactosidases na maturacao de frutos de cafe, calos embriogenicos de C. arabica foram transformados por biobalistica, utilizando a tecnica do RNA interferente, com a finalidade de obtencao de plantas geneticamente modificadas de cafeeiro para o silenciamento da expressao do gene das ß-galactosidases. Foram obtidos tres calos transgenicos crescendo em meio seletivo com glufosinato de amonio, dentre os quais dois continham o fragmento deste gene. Estes calos encontram-se em fase de formacao de embrioes somaticos e as plantulas resultantes desta regeneracao serao analisadas, a posteriori, para confirmacao da presenca dos transgenes e avaliacao dos efeitos do silenciamento do gene das ß-galactosidases sobre a maturacao de frutos de cafe.

成人骨量的更新与维持通过骨重塑来实现。骨重塑包括骨吸收与骨形成两个偶联的过程，其中成骨细胞介导骨形成，破骨细胞介导骨吸收，当偶联的骨吸收超过骨吸收就会导致骨量丢失，进而导致发生骨质疏松症的发生。目前，临床治疗骨质疏松的药物如阿仑膦酸盐、雌激素受体调节剂、活性维生素D、雌激素替代治疗、降钙素、骨化三醇等都是基于针对破骨细胞的调控来抑制骨吸收，但是对于已经丢失的骨量无法恢复。唯一被美国FDA批准用来通过刺激新骨形成来恢复丢失的骨量的治疗药物就是甲状旁腺激素。然而，这种药物在刺激新骨形成的同时也刺激了骨吸收，即：在使用18个月后有明显促进骨吸收的副作用。 / 酪蛋白激酶相互作用蛋白-1（CKIP-1）基因是一个新发现的骨形成的负调控基因，CKIP-1基因敲除小鼠在骨发育和正常骨代谢过程中均未发现激活骨吸收。CKIP-1敲除导致小鼠胫骨近端骨量与胫骨皮质骨形成速率显著高于野生型，且这一差异随着小鼠的增龄而显著，而骨外器官没有发现异常表型，提示CKIP-1是潜在相对安全的治疗骨质疏松的靶向基因。特别是我们最近研发的一种天门冬氨酸－丝氨酸－丝氨酸重复三肽修饰的脂质体递送（(Asp-Ser-Ser)₆-liposome）系统能够实现靶向骨形成表面的小干扰核酸的递送，并明显减少了小干扰核酸在非骨组织的分布。因此，提出本课题的研究假设：特异性静默骨内CKIP-1可以促进骨形成而不刺激骨吸收，从而为骨质疏松的临床治疗提供安全有效的治疗手段。 / 为了确定CKIP-1基因表达在老年绝经后妇女骨骼中与骨形成内在联系，首先，我们通过对发生骨折的老年绝经后妇女的骨痂标本中CKIP-1 mRNA和蛋白表达的测定，发现CKIP-1基因mRNA和蛋白表达水平与骨形成能力负相关。并且，这种相关性在骨质疏松动物模型中进一步得到证实。其次，针对我们研究假设，从一组针对大鼠、小鼠、猴和人类的成骨样细胞的CKIP-1 mRNA的跨种属siRNA序列中筛选出体外静默效率最高CKIP-1小干扰核酸序列si-3。接着，体内外实验证实si-3序列在健康动物体内的静默效率和促进成骨的功能。同时，确定尾静脉注射（Asp-Ser-Ser）₆-liposome 包裹的CKIP-1小干扰核酸在 大鼠和小鼠为的最佳剂量分别为3.75mg/kg和7.5mg/kg以及注射周期为每两周一次。最后，为了检验CKIP-1 小干扰核酸是否可通过促进骨形成从而逆转绝经后骨质疏松症中的骨丢失，我们分别以绝经后骨质疏松大鼠和小鼠为实验动物模型，通过测定骨形态计量学参数、骨量和骨结构等来评价骨靶向递送系统（(Asp-Ser-Ser)₆-liposome）递送的CKIP-1 siRNA对老年绝经后骨质疏松症的治疗效果。动态活体CT分析结果表明，与0周未治疗的基础值相比，经6周治疗骨密度（BMD）, 相对骨体积分数（BV/TV）和骨小梁厚度（Tb.Th）在小干扰核酸治疗组显著增加。此外，在治疗6周后小干扰核酸治疗组骨密度，相对骨体积和骨小梁厚度显示较高于模型对照组。0周与其它检测时间点之间的对比分析较显示，小干扰核酸治疗组的新生骨显著高于模型组或假手术组。组织形态学分析结果表明在治疗6周后，无论是股骨远端或中段的矿化沉积率（MAR）、骨形成速率（BFR） 和组的骨形成表面（Ob.S/ BS）在OVX组和siRNA组均显著高于模型对照组，而模型对照组和小干扰核酸治疗组的骨吸收表面（Oc.S/ BS）之间无显著性差异。 / 结论：CKIP-1基因小核酸干扰治疗在老年绝经后骨质疏松中能够显著促进骨形成并不会加剧骨吸收，该药物具有显著逆转骨丢失的作用。 / Osteoporosis is characterized by an imbalance between bone formation and bone resorption. Therefore, promoting bone formation and inhibiting bone resorption are the two major therapeutic strategies in the treatment of osteoporosis. Currently, the only Food and Drug Administration (FDA)-approved anabolic agent capable of stimulating bone formation is parathyroid hormone (PTH). However, dominant bone resorption after 18-month treatment with PTH is a great concern (Rubin and Bilezikian 2003). Thus, development of alternative bone anabolic agents is highly desirable. / Casein kinase-2 interacting protein-1 (CKIP-1), which is encoded by Plekho1, and thus also known as Plekho1, is a newly discovered negative regulator of bone formation during bone development and subsequent bone maintenance that does not activate bone resorption (Lu, Yin et al. 2008). Specifically, CKIP-1 protein functions as the auxiliary factor of ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1) to interrupt the bone anabolic BMP-signalling pathway, which has been demonstrated to be a specific suppressor of bone formation (Yamashita, Ying et al. 2005). In a previous study, we found that CKIP-1 expression in female rat bone increases with aging, whereas bone formation decreases with aging (Guo, Zhang et al. 2010). Systemic examination of the tissue distribution of CKIP-1 expression has revealed that is abundantly expressed in the musculoskeletal system but sparingly expressed in the liver, lungs, kidneys, pancreas, and other organs (Zhang, Tang et al. 2007). In addition, an abnormal tissue phenotype in heart, liver, spleen, lung, and kidney tissue has not been observed in CKIP-1 gene knockout mice (KO), even at an advanced age (Lu, Yin et al. 2008). Thus, CKIP-1 gene silencing might be a potential strategy for promoting bone anabolic action in reversing bone loss. / RNA interference (RNAi), a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNA and then blocking translation of the specific gene, has been employed for gene silencing in vivo (Frank-Kamenetsky, Grefhorst et al. 2008). Accordingly, RNAi should be an appropriate target for CKIP-1 gene silencing in vivo. / We raised the hypothesis that therapeutic RNAi targeting of CKIP-1 might promote bone formation for reversing postmenopausal bone loss. To test the hypothesis, we performed several studies to achieve the following specific aims: (1) To explore the relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis; (2) To Identify a cross-species CKIP-1 siRNA sequence with high knockdown efficiency; (3) To validate of the identified CKIP-1 siRNA in healthy rodents in vivo; (4) To examine the anabolic effect of the identified CKIP-1 siRNA on bone in osteoporotic animal models. / The relationship between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women: To explore the association between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women, the gene expression of CKIP-1 and ALP in the bone specimens from aged female patients were examined. We found the protein expression of CKIP-1 increased during aging and negatively correlate to bone formation as indicated by the mRNA expression of ALP (Guo., Zhang. et al. 2011). Further, we also found the decreased bone formation during aging was partly rescued in Ckip-1 KO mice during aging. / A cross-species CKIP-1 siRNA sequence: Recently, we identified a specific CKIP-1 siRNA sequence (CKIP-1 siRNA si-3) with high knockdown efficiency across rat, mouse, rhesus, and human osteoblast-like cells that does not induce immunostimulatory activity and promotes osteoblast differentiation across the species in vitro and bone formation in rats in vivo (Guo, Zheng et al. 2012). / Validation of the CKIP-1 siRNA si-3 capsulated by bone-targeted siRNA delivery system in healthy rodents in vivo: We developed a bone-targeting siRNA delivery system (tripeptide aspartate-serine-serine linked with liposome, i.e. (Asp-Ser-Ser)₆-liposome) that can remarkably reduce the exposure of non-bone tissue to CKIP-1 siRNA (Zhang, Guo et al. 2012). To validate the identified CKIP-1 siRNA in healthy rodents in vivo, the established continuous CKIP-1 gene silencing protocol is optimized in adult rats and mice in vivo by hydrodynamic tail vein injection of 3.75mg/kg for rats and 7.5 mg/kg for mice every 2 weeks (Guo, Zhang et al. 2010). The osteogenic effects of CKIP-1 siRNA in both rats and mice were further validated in vivo. / Anabolic effect of CKIP-1 siRNA si-3 on bone in aged postmenopausal osteoporosis: For evaluation of the anabolic effect of CKIP-1 siRNA si-3 on reversing bone loss due to osteoporosis in an animal model, we intravenously injected ovariectomized (OVX) rats and mice with CKIP-1 siRNA delivered by the (Asp-Ser-Ser)₆-liposome, a liposome linked with six repeated aspartate-serine-serine moiety, every 2 weeks for 6 weeks. In vivo and ex vivo microCT analysis demonstrated a change over time in the variables examined and different change patterns over time among the groups examined after administration. We found that the siRNA group had experienced a significant increase in bone mineral density (BMD), relative bone volume (BV/TV), and trabecular thickness (Tb.Th) between weeks 0 and 6; had a higher BMD, BV/TV, and Tb.Th compared to the OVX group at week 6; and had a similar Tb.Th to that of the SHAM group at week 6. Registration analysis between week 0 and other time points revealed that the siRNA had a greater number of newly formed bone than the OVX and SHAM groups. Histomorphometric analysis showed that the siRNA group had a significantly higher mineralization rate (MAR), a significantly higher bone-formation rate (BFR), a significantly larger osteoblast surface (Ob.S/BS) at both the distal and mid-shaft femur compared to the OVX group after 6 weeks of treatment but not a significantly different Oc.S/BS. / Significance: Confirmation of our hypothesis by our results helps establish CKIP-1’s role as a pivotal negative regulator of bone formation in the aging skeleton and provides evidence that inhibiting CKIP-1 is a novel anabolic treatment for osteoporosis, indicating great potential for the use of therapeutic RNAi in orthopaedics and traumatology. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Baosheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves [132-150]). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 论文摘要 --- p.vii / Table of Content --- p.ix / Abbreviations --- p.xvii / List of Figures --- p.xix / List of Tables --- p.xxii / Chapter CHAPTER 1 --- Review of recent anabolic therapy for osteoporosis --- p.1 / Chapter 1.1. --- Epidemiology of postmenopausal osteoporosis --- p.1 / Chapter 1.1.1. --- Definition of osteoporosis --- p.1 / Chapter 1.1.2. --- Epidemiology and health challenge of postmenopausal osteoporosis --- p.2 / Chapter 1.2. --- General pathophysiological understanding of osteoporosis and current challenge for osteoporosis treatment --- p.3 / Chapter 1.2.1. --- Bone modeling and remodeling --- p.3 / Chapter 1.2.2. --- Pathophysiological process of osteoporosis --- p.4 / Chapter 1.2.3. --- Systemic risk factors in the pathophysiology of osteoporosis --- p.5 / Chapter 1.2.4. --- Local risk factors in the osteoporosis pathophysiology --- p.6 / Chapter 1.2.5. --- Two therapeutic strategies for osteoporosis treatment --- p.7 / Chapter 1.3. --- Current and potential anabolic agents for osteoporosis treatment --- p.8 / Chapter 1.3.1. --- PTH analogues --- p.8 / Chapter 1.3.2. --- Potential concerns regarding PTH administration --- p.9 / Chapter 1.3.3. --- Potential PTH alternatives --- p.10 / Chapter 1.3.4. --- Modulation of Wnt/β-cateinin pathway --- p.10 / Chapter 1.3.5. --- Aptamer-based technology in osteoporosis treatment --- p.14 / Chapter 1.4. --- CKIP-1: A novel negative regulator of bone formation --- p.15 / Chapter 1.4.1. --- TGF-β/BMP signaling pathways involved in regulating bone formation --- p.15 / Chapter 1.4.2. --- CKIP-1 interrupts BMP signaling pathway --- p.16 / Chapter 1.4.3. --- CKIP-1 negatively regulates bone formation without activating bone resorption --- p.17 / Chapter 1.5. --- RNA interference strategy in anabolic therapy of osteoporosis --- p.18 / Chapter 1.5.1. --- siRNA-mediated gene silencing in osteoporosis treatment --- p.18 / Chapter 1.5.2. --- MicroRNAs as potential therapeutic targets in the anabolic treatment of osteoporosis --- p.20 / Chapter 1.5.3. --- Bone targeted RNAi-based anabolic-agents delivery --- p.23 / Chapter 1.6. --- Summary --- p.24 / Chapter CHAPTER 2 --- The relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- Bone specimen collection from aged postmenopausal women --- p.28 / Chapter 2.2.2 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.28 / Chapter 2.2.3 --- Total protein extraction and western blot analysis --- p.30 / Chapter 2.2.4 --- CKIP-1 expression in bone and other tissues --- p.31 / Chapter 2.2.5 --- Relationship between CKIP-1 expression and bone formation in aged ovariectomized rats --- p.31 / Chapter 2.2.6 --- Role of CKIP-1 in regulating bone formation in aged ovariectomized mice --- p.32 / Chapter 2.2.7 --- Statistics --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Correlation analysis between CKIP-1 expression and bone formation-related gene expression in bone specimens from agedd postmenopausal women across age --- p.33 / Chapter 2.3.2 --- CKIP-1 gene expression pattern in bone and other tissues --- p.37 / Chapter 2.3.3 --- Correlation between CKIP-1 expression and bone formation in rat bone --- p.38 / Chapter 2.3.4 --- CKIP-1 negatively regulates bone formation in aged ovariectomized mice by using CKIP-1 knockout mice --- p.39 / Chapter 2.4 --- Summary --- p.41 / Chapter CHAPTER 3 --- Identification of a cross-species CKIP-1 siRNA sequence --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Materials and methods --- p.44 / Chapter 3.2.1 --- Design rationale and modification for cross-species CKIP-1 siRNA --- p.44 / Chapter 3.2.2 --- In vitro screening for cross-species CKIP-1 siRNA sequences --- p.45 / Chapter 3.2.3 --- Investigation of the effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.47 / Chapter 3.2.4 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.47 / Chapter 3.2.5 --- Western blot analysis --- p.51 / Chapter 3.2.6 --- Evaluation of calcium deposition --- p.51 / Chapter 3.2.7 --- BMP-2 reporter activity assay in MC3T3-E1 cells --- p.52 / Chapter 3.2.8 --- Isolation of the primary human blood monocytes and IFN-α and TNF-α measurement --- p.53 / Chapter 3.2.9 --- Statistics --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- Bio-informatic analysis of the designed CKIP-1 siRNA sequences --- p.54 / Chapter 3.3.2 --- Identified the cross-species CKIP-1 siRNA sequences by In vitro screening --- p.56 / Chapter 3.3.3 --- Effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.60 / Chapter 3.3.4 --- Effects of the identified CKIP-1 siRNA on matrix mineralization --- p.65 / Chapter 3.3.5 --- Effect of the identified CKIP-1 siRNA on BMP signaling --- p.67 / Chapter 3.3.6 --- Effects of the identified CKIP-1 siRNA on the ratio of RANKL/OPG --- p.67 / Chapter 3.3.7 --- Effects of the identified CKIP-1 siRNA on immunostimulatory activity --- p.68 / Chapter 3.4 --- Summary --- p.71 / Chapter 3.4.1 --- CKIP-1 siRNA si-3 as the identified sequence --- p.71 / Chapter 3.4.2 --- CKIP-1 siRNA si-3 promoted osteoblast differentiation in vitro --- p.72 / Chapter CHAPTER 4 --- Validation of the identified CKIP-1 siRNA in healthy rodents in vivo --- p.74 / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and methods --- p.74 / Chapter 4.2.1 --- Localization of intraosseous siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.75 / Chapter 4.2.2 --- Cell-selective delivery in vivo of CKIP-1 siRNA --- p.76 / Chapter 4.2.3 --- Dose-response study of CKIP-1 siRNA --- p.77 / Chapter 4.2.4 --- Time-course study of CKIP-1 siRNA --- p.77 / Chapter 4.2.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.78 / Chapter 4.2.6 --- Measurement for serum PINP and urinary DPD --- p.80 / Chapter 4.2.7 --- 5’-RACE Analysis --- p.81 / Chapter 4.2.8 --- Laser captured micro-dissection (LCM) --- p.82 / Chapter 4.2.9 --- Evaluation the anabolic effect of the identified siRNA on healthy rat bone --- p.82 / Chapter 4.2.10 --- Evaluation the anabolic effect of the identified siRNA on healthy mouse bone --- p.84 / Chapter 4.2.11 --- Micro CT analysis --- p.84 / Chapter 4.2.12 --- Dynamic bone histomorphometric analysis --- p.85 / Chapter 4.2.13 --- Statistics --- p.86 / Chapter 4.3 --- Results --- p.87 / Chapter 4.3.1 --- Rationale of bone targeted delivery of CKIP-1 siRNA by (Asp-Ser-Ser)₆-liposome --- p.87 / Chapter 4.3.2 --- Intraosseous distribution of siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.89 / Chapter 4.3.3 --- Optimal dosage and duration for CKIP-1 siRNA administration in vivo --- p.92 / Chapter 4.3.4 --- Knockdown efficiency of CKIP-1 siRNA in osteoblasts by LCM in combination with Q-PCR --- p.94 / Chapter 4.3.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.96 / Chapter 4.3.6 --- RNAi mechanism of CKIP-1 siRNA action in vivo --- p.99 / Chapter 4.3.7 --- Anabolic effect of the identified siRNA on healthy rat bone --- p.101 / Chapter 4.3.8 --- Anabolic effect of the identified siRNA on healthy mouse bone . --- p.104 / Chapter 4.4 --- Summary --- p.107 / Chapter 4.4.1 --- Intraosseous localization of CKIP-1 siRNA after systemic administration --- p.107 / Chapter 4.4.2 --- Evidence of RNAi in bone tissue from systemic administration of CKIP-I siRNA --- p.107 / Chapter 4.4.3 --- CKIP-1 siRNA si-3 promots bone formation in rats and mice in vivo --- p.108 / Chapter CHAPTER 5 --- Anabolic effect of the identified CKIP-1 siRNA on bone in postmenopausal osteoporostic animal models --- p.110 / Chapter 5.1. --- Introduction --- p.110 / Chapter 5.2. --- Materials and Methods --- p.110 / Chapter 5.2.1. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic mouse bone --- p.111 / Chapter 5.2.2. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic rat bone --- p.112 / Chapter 5.2.3. --- In vivo micro-CT analysis and registration of proximal tibia from osteoporotic rats --- p.112 / Chapter 5.2.4. --- Ex vivo micro-CT analysis of the distal femur and 5th lumbar vertebrae body of osteoporotic rats --- p.115 / Chapter 5.2.5. --- Ex vivo micro-CT analysis of distal femur from osteoporotic mice --- p.115 / Chapter 5.2.6. --- Bone histomorphometric analysis --- p.116 / Chapter 5.2.7. --- Mechanical testing --- p.117 / Chapter 5.2.8. --- Statistics --- p.118 / Chapter 5.3. --- Results --- p.116 / Chapter 5.3.1. --- Anabolic effect of CKIP-1 siRNA si-3 on osteoporotic mouse bone --- p.118 / Chapter 5.3.2. --- In vivo microCT data of proximal tibia from aged osteoporotic rats --- p.121 / Chapter 5.3.3. --- Ex vivo microCT data of distal femur from aged osteoporotic rats --- p.124 / Chapter 5.3.4. --- Ex vivo microCT data of 5th LV body from aged osteoporotic rats --- p.126 / Chapter 5.3.5. --- Bone histomorphometric analysis of aged osteoporotic rats --- p.129 / Chapter 5.3.6. --- Mechanical testing of the mid-shaft femur of aged osteoporotic rats --- p.132 / Chapter 5.4. --- Summary --- p.134 / Chapter CHAPTER 6 --- Discussions --- p.134 / Chapter 6.1 --- CKIP-1 siRNA design rationale and further modification --- p.135 / Chapter 6.1.1 --- Specificity design rationale of the CKIP-1 siRNA --- p.135 / Chapter 6.1.2 --- Stability enhancing modification of CKIP-1 siRNA --- p.136 / Chapter 6.1.3 --- Safety concerns with CKIP-1 siRNA therapy --- p.136 / Chapter 6.2 --- Development of bone-targeted siRNA delivery --- p.136 / Chapter 6.3 --- Prospects for and limitation of application of study findings to clinical therapeutics --- p.137 / References --- p.139 / Publications --- p.159

By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line. / Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs. / In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation. / MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition. / Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs. / This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis. / To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays. / Lee, Sharon Ka-wai. / "December 2009." / Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 216-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.