Activator Protein-1 (AP-1) is an oncogenic transcription factor that is dysregulated in numerous human cancers, making it an attractive therapeutic target. AP-1 forms via interaction of cJun and cFos proteins, which intertwine to generate a ‘coiled coil’ (CC) structure. Thus, the cJun/cFos α-helical CC domains responsible for dimerisation are appealing targets for inhibiting AP-1 formation and activity. Helical peptide antagonists that sequester cJun can be derived from the cFos CC domain by selection of more optimal amino acids for increased binding affinity. Peptides can then be downsized and modified to improve therapeutic potential. Two approaches aimed to identify novel short peptides against cJun. The first was to covalently cyclise amino acid side chains in existing cFos-derived peptide “FosW”, with the aim of constraining FosW into a stable helix to allow downsizing without significant loss of binding structure and affinity. Using circular dichroism spectroscopy and isothermal titration calorimetry, a series of helix constrained peptides were characterised, from which a peptide was identified that retained 88 % of FosW binding affinity whilst being 22 % shorter, and which entered breast cancer cells in vitro, with preliminary data suggesting potential ability to inhibit AP-1 in cellulo. The second approach was to combine two existing high-throughput peptide selection systems, with the aim of benefitting from overlap in their strengths and weaknesses. Combination of in vitro CIS display and in cellulo Protein-fragment Complementation Assay successfully isolated a high affinity peptide from a hugely diverse library, and future refinements to further exploit this approach, particularly for short peptide selection, were formulated. Thus, molecules and techniques derived here may expedite the future development of therapies for cancers featuring AP-1 dysregulation.

Ph.D.

No / Coupling of peptide self-assembly to dynamic sequence exchange provides a useful approach for the discovery of self-assembling materials. In here, we demonstrate the discovery and optimization of aqueous, gel-phase nanostructures based on dynamically exchanging peptide sequences that self-select to maximize charge transfer of n-type semiconducting naphthalenediimide (NDI)-dipeptide bioconjugates with various π-electron-rich donors (dialkoxy/hydroxy/amino-naphthalene or pyrene derivatives). These gel-phase peptide libraries are characterized by spectroscopy (UV–vis and fluorescence), microscopy (TEM), HPLC, and oscillatory rheology and it is found that, of the various peptide sequences explored (tyrosine Y-NDI with tyrosine Y, phenylalanine F, leucine L, valine V, alanine A or glycine G-NH2), the optimum sequence is tyrosine-phenylalanine in each case; however, both its absolute and relative yield amplification is dictated by the properties of the donor component, indicating cooperativity of peptide sequence and donor/acceptor pairs in assembly. The methodology provides an in situ discovery tool for nanostructures that enable dynamic interfacing of supramolecular electronics with aqueous (biological) systems.

No description available.

Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Cyclic peptides provide a privileged scaffold when optimizing interactions with various biological targets. Their rigidified structure decreases the entropic cost of binding by preorganizing residues in a fixed conformation, which may enhance binding affinity. These molecules occupy a larger chemical space than typical small molecule drugs and may provide good candidates for inhibiting protein-protein interactions or being able to interact with previously undruggable targets. Given the benefits of these structures we aim to develop a one-bead-one-compound peptide library for screening against relevant biological targets. Herein we describe several routes to achieving cyclic peptides through side chain interactions and head-to tail amide bond linkages. Additional considerations for the development of the on resin library such as linker strategies and sequencing methods will be discussed. / Thesis (MS) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis.

Bikkavilli Rama Kamesh. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 212-223) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / "December 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Orientadores: Laura Sterian Ward, Luiz Ricardo Goulart Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T19:36:44Z (GMT). No. of bitstreams: 1 Reis_CarolinaFernandes_D.pdf: 11022078 bytes, checksum: 4506c2c2816405721c293e78c8fc5c46 (MD5) Previous issue date: 2013 / Resumo: A incidência do câncer de tireóide vem aumentando nas últimas décadas no Brasil, assim como em quase todos os países do mundo. Novos marcadores para uso no diagnóstico e prognóstico são essenciais para diminuir o número de cirurgias desnecessárias e melhorar a qualidade do tratamento dos pacientes, sendo que as técnicas utilizadas ainda não conseguiram obter marcadores de ampla utilidade. Os objetivos principais foram validar o peptídeo CaT12, um ligante de carcinoma papilífero de tireóide (CP) selecionado por Phage Display e avaliar em nódulos tireoidianos os possíveis alvos deste peptídeo. O CaT12 foi tumor específico quando testado em imunohistoquímica, foram usados um total de 775 tecidos de lesões não neoplásicas e neoplásicas (benignas e malignas), sendo 232 nódulos tireoidianos (15 sem neoplasia, 53 bócios, 54 adenomas, 69 carcinomas papilíferos e 41 carcinomas foliculares). O peptídeo CaT12 foi eficiente para identificar carcinomas papilíferos com 91,2% de sensibilidade e 85,1% de especificidade; este peptídeo também ajuda caracterizar lesões foliculares distinguindo carcinomas papilíferos de variante folicular (CPVF) de adenomas com 91,9% de acurácia. Tecidos benignos (nevos atípicos e nevos comuns) e não neoplásicos (vários tecidos sem neoplasia tais como mama, próstata, rim, cérebro, tireóide e outros) apresentaram apenas 13,74% de positividade, enquanto os carcinomas de mama, próstata e rim apresentaram 53,96%, 70,18% e 74,29% de positividade para o CaT12 respectivamente. Após verificar a eficiência do marcador CaT12 investigamos os possíveis ligantes que foram identificados como sendo queratinas (KRT), a expressão gênica de algumas dessas queratinas foram analisadas em tecidos micro dissecados de 114 nódulos tireoidianos, sendo 12 tecidos sem neoplasia, 38 com doença benigna (24 bócios e 14 adenomas) e 64 com carcinomas papilíferos (32 de variante folicular e 32 de variante clássica). As queratinas KRT5 (P=0,0002) e KRT14 (P<0,0001) estão mais expressas em pacientes com doença maligna, enquanto as queratinas KRT2 (P<0,0001), KRT6A (P=0,0014) e KRT10 (P<0,0001) foram expressas preferencialmente nos pacientes com doenças benignas. Ensaios de citotoxicidade e indução de inflamação avaliaram o possível uso não só como marcador, mas como carreador de drogas para uso terapêutico. O CaT12 não foi citotóxico para as células NPA e TPC1 (linhagens celulares de carcinoma papilífero de tireoide) e não demonstrou resposta imunológica quando testado em leucócitos de camundongos isogênicos. Visando futura aplicação deste peptídeo como carreador de drogas testamos a prostaglandina 15-dPGJ2 que foi tóxica para as células TPC1 (IC50=9,3 uM) e poderá ser utilizada conjugada ao CaT12 para entregar a droga apenas para as células cancerígenas. Outros peptídeos foram selecionados (CaT19, CaT42 e CaT66) para reconhecimento da superfície celular e poderão ser utilizados como marcadores auxiliares no diagnóstico durante a biópsia por agulha fina. Concluímos que o peptídeo CaT12 foi eficiente para distinguir malignidade em lesões tireoidianas, podendo ser usado para identificar carcinomas diferenciando as lesões malignas das benignas. O CaT12 também reconheceu malignidade em tumores de próstata, mama e rim. Além disso, foi demonstrado que a expressão gênica das queratinas são importantes na tumorigênese tireoidiana / Abstract: Incidence of thyroid cancer has increased in Brazil, as well as in almost countries of the world. New diagnostic and prognostic markers are essential to reduce numbers of unnecessary surgeries and improve the quality of patient's care. Unfortunately, recent markers used are not prove broad utility. Aim was validate CaT12 peptide selected by Phage Display as a marker to separate benign from malignant follicular lesions and evaluate in thyroid nodules possible targets for CaT12. The antibody-like peptide CaT12 was tumor-specific, which was further tested by immunohistochemistry against tissue comprised of 775 human benign and malignant tissues, including 232 thyroid nodular lesions: 15 normal thyroid tissues, 53 nodular goiters, 54 follicular adenomas; 69 papillary thyroid carcinomas (PTC); and 41 follicular carcinomas. CaT12 was able to identify PTC among thyroid nodular lesions with 91.2% sensitivity and 85.1% specificity; this peptide helped characterize follicular lesions distinguishing the follicular variant of PTC (FVPTC) from FA with 91.9% accuracy. Benign tissues (nevus atypical and nevus communes) and non neoplastic tissues (differents human non neoplastic tissues) showed only 13.74% positivity, while breast, prostate and kidney carcinomas showed respectively, 53.96%, 70.18% e 74.29% CaT12 positivity. After verifying the efficiency of marker CaT12 we investigated the possible ligands that have been identified as keratins (KRT), the mRNA expression these keratins were analyzed in microdissected tissues of 114 thyroid nodules: 12 tissues without cancer, 38 with benign disease (24 goiters and 14 adenomas) and 64 with papillary carcinomas (32 follicular variant and 32classic variant). The KRT5 (P = 0.0002) and KRT14 (P <0.0001) were most expressed in patients with malignant disease, while KRT2 (P <0.0001), KRT6A (P = 0.0014) and KRT10 (P <0.0001) were preferentially expressed in patients with benign diseases. Cytotoxicity and induction of inflammation assays evaluated the possible use as a biomarker and therapeutic by drug delivery. We showed CaT12 was not cytotoxic to NPA and TPC1 cells (papillary thyroid carcinoma cell lines) and no immune response demonstrated when tested in leukocytes of inbred mice. To future application of this peptide as a carrier of drugs we tested prostaglandin 15-dPGJ2 in TPC1 cells and was toxic to the cells (IC50 = 9.3 uM), this drug may be used conjugate with CaT12 for 15-dPGJ2 to be delivered only to cancer cells. Other peptides were selected (CaT19, CaT42 and CaT66) for recognition of cell surface and can be used as markers auxiliary to fine needle biopsy or drug deliveries. In conclusion, our CaT12 peptide was highly effective and resulted in a useful antibody-like biomarker that recognizes malignancy among thyroid nodules and may help distinguish follicular patterned lesions. CaT12 also recognized malignancy in prostate, breast and kidney tumors. Furthermore, it was demonstrated that the gene expression of keratins are important in thyroid tumorigenesis / Doutorado / Clinica Medica / Doutora em Clínica Médica