Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed March 3, 2009). Graduate School of Arts and Sciences, Dept. of Physics. Includes bibliographical references (p. 133-155).

Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 14 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Includes bibliographical references. Also available in print form.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

Thesis (M.S.)--Rochester Institute of Technology, 2006. / Typescript. Includes bibliographical references (leaves 47-48).

This thesis describes work done with the thyroid hormone receptor (TR), a nuclear protein which binds to specific DNA sequences and regulates transcription in response to thyroid hormone levels. The studies can be divided into two broad categories: structure/function studies of the TR protein, particularly with regards to DNA binding function; and, structure/function studies of the DNA sequences to which the thyroid hormone receptor binds in order to regulate gene transcription. In order to examine the DNA binding properties of the TR an electrophoretic mobility shift assay (EMSA) was utilized. Conditions of this assay were optimized for the use of in vitro translated TR. Mutant forms of the β-isoform of thyroid hormone receptor were generated using a PCR-based mutagenesis protocol. Each mutant substituted a different residue of the 12 amino acid-long α-recognition helix with alanine. The mutants were analyzed for their abilities to bind to thyroid hormone response elements (TREs), and to activate transcription in transfected eukaryotic cells. The DNA binding results were consistent with a conserved α-helix structure, with conserved function for many residues, that is similar to that of the related receptors for glucocorticoids and estrogen. Only the first of the three non-conserved residues lying in the P-box (EGG), a portion of the recognition α-helix that facilitate differential binding of distinct DNA sequences, disrupted binding when substituted with alanine. The third position of the P-box, when substituted with alanine exhibits an altered ability to bind to certain natural TREs. The mutant form of TR with alanine substituted for the second P-box position displayed only a modest decrease in DNA binding affinity compared to wild-type TR (roughly 3-fold), yet was completely deficient in trans-activation. The structure-function studies of TR binding sites on DNA applied a methylation interference protocol to examine the interactions of TR with direct repeats (DR) of the idealized hexameric sequence, spaced by three to five base-pairs. The interactions of both the TR/TR homodimer and the TR/RXR (9-cis-retinoic acid receptor) heterodimer with the DRs were examined. The methylation interference patterns for the TR/TR homodimer bound to the DR sequences are virtually identical for spacers of four and five base-pairs, but with three base-pairs, there is some evidence that at least one DNA binding domain is misaligned with the DNA to accomodate the unfavourable spacer length. The TR/RXR heterodimer methylation interference pattern is distinct on all three DRs, probably due to the fact that in the heterodimer cooperative intermolecular contacts are made between the DNA binding domains of the two receptors, but only when the spacer distance is four base-pairs. When a poorly conserved everted repeat (EvR) that overlaps the idealized DR is present. the homodimer, but not the heterodimer, binds this cryptic EvR in competition with the DR. The binding modality of the TR homodimer and TR/RXR heterodimer to DRs was reevalutated using point mutants and EMSA. The TR homodimer and TR/RXR heterodimer both bind to idealized direct repeats with DBDs aligned appropriately for a direct repeat; however, evidence is presented that there are certain poorly conserved sequences that are intermediate between DRs and EvRs that are differentially recognized by the TR homodimer and the TR/RXR heterodimer. That is, the homodimer binds with the DBDs aligned appropriately for a EvR, and the heterodimer DBDs are aligned appropriately for a DR. / Graduate

Transcription factors (TFs) are biologically important proteins that interact with transcription machinery and bind DNA regulatory sequences to regulate gene expression by modulating the synthesis of the messenger RNA. The regulatory sequences comprise of short conserved regions of a specific length called motifs . TFs have very diverse roles in different cells and play a very significant role in development. TFs have been associated with carcinogenesis in various tissue types, as well as developmental and hormone response disorders. They may be responsible for the regulation of oncogenes and can be oncogenic. Consequently, understanding TF binding and knowing the motifs to which they bind is worthy of attention and research focus. Various projects have made the study of TF binding their main focus; nevertheless, much about TF binding remains confounding. Chromatin immunoprecipitation in conjunction with deep sequencing (ChIP-seq) techniques are a popular method used to investigate DNA-TF interactions in vivo. This procedure is followed by motif discovery and motif enrichment analysis using relevant tools. Protein Binding Microarrays (PBMs) are an in vitro method for investigating DNA-TF interactions. We use a motif enrichment analysis tools (CentriMo and AME) and an empirical quality assessment tool (Area under the ROC curve) to investigate which method yields motifs that are a true representation of in vivo binding. Motif enrichment analysis: On average, ChIP-seq derived motifs from the JASPAR Core database outperformed PBM derived ones from the UniPROBE mouse database. However, the performance of motifs derived using these two methods is not much different from each other when using CentriMo and AME. The E-values from Motif enrichment analysis were not too different from each other or 0. CentriMo showed that in 35 cases JASPAR Core ChIP-seq derived motifs outperformed UniPROBE mouse PBM derived motifs, while it was only in 11 cases that PBM derived motifs outperformed ChIP-seq derived motifs. AME showed that in 18 cases JASPAR Core ChIP-seq derived motifs did better, while only it was only in 3 cases that UniPROBE motifs outperformed ChIP-seq derived motifs. We could not distinguish the performance in 25 cases. Empirical quality assessment: Area under the ROC curve values computations followed by a two-sided t-test showed that there is no significant difference in the average performances of the motifs from the two databases (with 95% confidence, mean of differences=0.0088125 p-value= 0.4874, DF=47) .

Bacteria live under constant assault by bacteriophages and have evolved a diverse array of defense strategies. CRISPR-Cas systems are prokaryotic adaptive immune systems that rely on RNA-guided binding for the recognition and degradation of invading nucleic acids. Intriguingly, some bacteria also encode divergent CRISPR-Cas systems that can bind to — but cannot degrade — target nucleic acids. In this dissertation, I describe the study of nuclease-deficient CRISPR-Cas systems alongside the evolutionary pressures that led to their persistence in bacterial genomes. I present experimental data for the existence of CRISPR-associated transposons (CASTs) that utilize the RNA-guided DNA binding ability of Type I-F CRISPR-Cas systems to direct transposition to new target sites in a heterologous Escherichia coli host. This RNA-guided DNA integration pathway can tolerate large cargos of up to 10 kilo-base pairs in size, and is highly specific for the programmed target site, as determined by deep sequencing experiments. We further reveal the physical link between the CRISPR-Cas and transposition machineries through biochemical experiments and by determining cryo-EM structures of the transposition protein TniQ in complex with the CRISPR-Cas effector. After bioinformatic analyses and experimental validation we established an array of twenty diverse CAST systems for which a subset works completely orthogonally. This dataset revealed the modular nature of CASTs by showcasing the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Further analysis of the transposon-encoded cargo genes uncovered the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Finally, we exploit high-throughput screening assays to determine the specific sequence and spacing requirements of the transposon ends, and use this knowledge to develop a CAST-mediated endogenous gene-tagging approach. Intriguingly, our experiments uncover the involvement of a previously unknown cellular protein, integration host factor (IHF), which is critical for transposition of VchCAST, but not other homologous systems. Collectively, the work presented in this dissertation describes the discovery of RNA-guided DNA integration employed by CASTs, substantially advances our biological understanding of these systems, and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

我們假設，將scleraxis (Scx)基因轉導入肌腱來源的幹細胞（TDSC），製成TDSC-Scx細胞系。TDSC-Scx會促進肌腱修復。本研究的目的在於探索Scx促進TDSC成肌腱分化的作用，以及TDSC-Scx對肌腱修復的促進作用。 / 使用慢病毒載體將Scx轉導入TDSCs，不含Scx的空載作為對照也轉導入TDSCs，用載體上帶有的抗性基因，殺稻瘟菌素對細胞進行篩選。分別建成TDSC-Scx和TDSC-Mock細胞系。Scx 的表達分別用定量PCR以及免疫螢光在mRNA和蛋白水準進行鑒定。TDSC-Scx成肌腱，成軟骨和成骨方向的分化能力用定量PCR檢驗。用大鼠臏腱視窗損傷模型進行了細胞移植試驗，測試TDSC-Scx對肌腱損傷的修復作用。實驗分為三組：（1）支架組，（2）空載體組，（3）Scx組。在細胞移植後的第二，四，八周，收集正在修復中的肌腱樣品，進行植入細胞的存留狀態，鈣化，組織學和生物力學測驗。 / TDSC-Scx比TDSC-Mock有更強的成肌腱分化能力。但是，在成軟骨-成骨分化方面，沒有結論。在動物試驗，植入的細胞在第二周仍然可見，但自第四周起，就不見了。在第八周，各組均有個別樣品輕微異位鈣化，但各組別間並無顯著差異。在早期，TDSC-Scx組比空載體組和支架組合得來更好的修復肌腱的能力。 / TDSC-Scx可能促進肌腱損傷的早期修復。 / We hypothesized that transduction of tendon-derived stem cell (TDSC) with scleraxis (Scx) might promote its tenogenic differentiation and promote better tendon repair compared to TDSC without Scx transduction. This study thus aimed to investigate the effect of Scx transduction on the tenogenic differentiation of TDSC and the effect of the resulting cell line in the promotion of tendon repair. / TDSCs were transduced with lentivirus-mediated Scx or empty vector and selected by blasticidin. The mRNA and protein expression of Scx were checked by qRT-PCR and Immuno fluorescence, respectively. The expression of different lineage markers were examined by qRT-PCR. A rat patellar tendon window injury model was used. The operated rats were divided into 3 groups: (1) scaffold-only group, (2) TDSC-Mock group and (3) TDSC-Scx group. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, or biomechanical test. / TDSC-Scx consistently showed higher expression of tendon-related markers compared to TDSC-Mock. However, the effect of Scx transduction on the expression of chondro-osteogenic markers was less conclusive. The transplanted TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic ossification was detected in some samples at week 8 but there was no difference among different groups. The TDSC-Scx group promoted early tendon repair histologically and biomechanically compared to the scaffold-only group and the TDSC-Mock group. / TDSC-Scx might be used for the promotion of early tendon repair. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Chunlai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-70). / Abstracts also in Chinese. / Chapter Thesis/Assessment Committee --- p.i / Acknowledgment --- p.ii / Publication --- p.vi / Abstract --- p.vii / 摘要 --- p.viii / Chapter Chapter 1 --- Tendon injury and tendon tissue engineering --- p.1 / Chapter 1.1 --- Anatomy of tendon --- p.1 / Chapter 1.2 --- Epidemiology of tendon injury --- p.3 / Chapter 1.3 --- Process and problems of tendon healing --- p.4 / Chapter 1.4 --- Current treatment and cell-based therapy for tendon repair --- p.5 / Chapter 1.5 --- Transcriptional factor Scleraxis and tendon --- p.8 / Chapter 1.5.1 --- Helix-loop-helix (HLH) and bHLH proteins --- p.8 / Chapter 1.5.2 --- Scleraxis --- p.9 / Chapter 1.6 --- Research focus and implications --- p.11 / Chapter 1.7 --- Hypotheses and objectives of this study --- p.12 / Chapter 1.8 --- Clinical significance --- p.13 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Study Design --- p.14 / Chapter 2.2 --- Establishment of TDSC-Scx cell line --- p.15 / Chapter 2.2.1 --- Isolation of TDSC and cell culture --- p.15 / Chapter 2.2.2 --- Establishment of TDSC-Scx cell line --- p.17 / Chapter 2.2.2.1 --- Construction of plasmid --- p.17 / Chapter 2.2.2.2 --- Transfection --- p.23 / Chapter 2.2.2.3 --- Infection --- p.24 / Chapter 2.2.2.4 --- Selection --- p.24 / Chapter 2.2.2.5 --- Characterization of TDSC-Scx and TDSC-Mock --- p.25 / Chapter 2.2.2.5.1 --- qRT-PCR --- p.25 / Chapter 2.2.2.5.2 --- Immune-fluorescent (IF) --- p.26 / Chapter 2.2.2.6 --- Lineage marker expression --- p.26 / Chapter 2.2.3 --- Data analysis --- p.29 / Chapter 2.3 --- The effect of TDSC-Scx on healing in a patellar tendon window injury model --- p.30 / Chapter 2.3.1 --- Animal surgery --- p.30 / Chapter 2.3.2 --- Ex Vivo Fluorescence Imaging --- p.32 / Chapter 2.3.3 --- vivaCT --- p.33 / Chapter 2.3.4 --- Histology --- p.33 / Chapter 2.3.5 --- Biomechanical test --- p.35 / Chapter 2.3.6 --- Data analysis --- p.37 / Chapter Chapter 3 --- Results --- p.38 / Chapter 3.1 --- Generation of TDSC-Scx and TDSC-Mock cell lines --- p.38 / Chapter 3.1.1 --- Plasmid --- p.38 / Chapter 3.1.2 --- Cell morphology --- p.38 / Chapter 3.1.3 --- Expression of Scx --- p.38 / Chapter 3.1.4 --- Expression of chondro-/osteo-/tenogenic markers --- p.39 / Chapter 3.2 --- The healing effect of TDSC-Scx on a patella tendon window injury model --- p.40 / Chapter 3.2.1 --- The fate of transplanted cells --- p.40 / Chapter 3.2.2 --- Ossification --- p.40 / Chapter 3.2.3 --- Histology --- p.40 / Chapter 3.2.3.1 --- Fiber arrangement --- p.41 / Chapter 3.2.3.2 --- Cellularity --- p.41 / Chapter 3.2.3.3 --- Cell alignment --- p.42 / Chapter 3.2.3.4 --- Cell rounding --- p.42 / Chapter 3.2.3.5 --- Vascularity --- p.43 / Chapter 3.2.3.6 --- Fiber structure --- p.43 / Chapter 3.2.3.7 --- Hyaline degeneration --- p.43 / Chapter 3.2.3.8 --- Inflammation --- p.44 / Chapter 3.2.3.9 --- Ossification --- p.44 / Chapter 3.2.4 --- Biomechanical properties --- p.44 / Chapter Chapter 4 --- Discussion --- p.55 / Chapter 4.1 --- In vitro --- p.55 / Chapter 4.1.1 --- Scx transduction did not lead to morphological change in TDSCs --- p.55 / Chapter 4.1.2 --- Scx transduction led to higher expression of Scx mRNA --- p.55 / Chapter 4.1.3 --- TDSC-Scx expressed higher levels of tenogenic markers --- p.56 / Chapter 4.2 --- In vivo --- p.58 / Chapter 4.2.1 --- The fate of transplanted cells --- p.58 / Chapter 4.2.2 --- Ossification was vague --- p.58 / Chapter 4.2.3 --- TDSC-Scx promoted tendon healing --- p.58 / Chapter 4.2.4 --- Biomechanical properties --- p.59 / Chapter 4.2.5 --- Clinical consideration --- p.60 / Chapter 4.3 --- Similar studies --- p.61 / Chapter Chapter 5 --- Limitations --- p.63 / Chapter 5.1 --- Direct Scx protein expression and function information unavailable --- p.63 / Chapter 5.2 --- Differentiation assay --- p.64 / Chapter Chapter 6 --- Conclusion --- p.65 / Reference --- p.66 / Appendix --- p.71