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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

In Vivo Analysis of Cruciform Extrusion and Resolution of DNA Palindromes in Eukaryotes

Cote, Atina 23 September 2009 (has links)
DNA palindromes are implicated in several examples of gross chromosomal aberrations in the human genome, however, the molecular mechanism(s) that govern palindrome instability are largely under-investigated. Because of their propensity for intrastrand base pairing, it is suspected that the acquisition of a secondary structure, such as a hairpin or cruciform, instigates the rearrangement process. A significant hurdle in defining palindrome-provoked instability lies in the fact that reliable methods for examining in vivo cruciform extrusion remain underdeveloped. A challenge is to provide straightforward evidence for cruciform extrusion in eukaryotic cells. Here, I present a plasmid system for use in Saccharomyces cerevisiae that enables for the detection of cruciforms in vivo. Cruciform extrusion, of either an in vitro-prepared palindrome or a near-palindrome from the human genome, is monitored by scoring for the product of cruciform resolution, being a dually hairpin-capped linear DNA molecule. These results not only provide evidence for the occurrence of cruciform extrusion in eukaryotic chromatin, they also identify a novel source of endogenous double strand break formation. A screen for candidate genes that are required for resolution revealed that the Mus81 Endonuclease, a candidate Holliday junction resolvase, provides the majority of cruciform resolution activity in mitotic cells, validating the notion that cellular HJ resolvases can misrecognize a cruciform for a Holliday junction. A second screen identified a requirement for the Sgs1-Top3-Rmi1 complex in the prevention of double strand break formation, including cruciform resolution, of DNA palindromes. These results uncover a new role for the RecQ helicase in prevention of palindrome-provoked instability, possibly through the intrusion of cruciform structures. Together, this work contributes significantly to our understanding of cruciform metabolism in eukaryotes and supports suggestions that cruciform extrusion instigates instability in the human genome.
2

Regulation of MDM2 and the p53 Family by the NEDD8 Pathway

Watson, Ian 17 February 2011 (has links)
NEDD8 is an ubiquitin-like protein sharing approximately 60% amino acid identity with ubiquitin and has biological roles in cell cycle progression, viability and development. Recently, a number of oncoproteins and tumor suppressors have been identified as NEDD8 substrates, including MDM2 and p53. MDM2 is an oncogenic E3 ligase that promotes NEDD8 modification and ubiquitin-mediated degradation of the tumor suppressor transcription factor, p53. Cellular stresses such as DNA damage lead to p53 activation due, in part, to MDM2 destabilization by mechanisms that are not completely understood. Studies in mice demonstrate the biological role of MDM2 is to negatively regulate p53 function, however, when overexpressed or amplified, MDM2 has p53-independent oncogenic functions presumably due to the regulation of additional substrates. One such substrate may be the p53 family member, p73. p73 exists as multiple isoforms and accumulating evidence suggests that the N-terminal isoforms dictate its role in tumorigenesis. The full-length pro-apoptotic TAp73 isoforms are induced by chemotherapies and are able to transactivate p53-target genes to initiate cell cycle arrest and apoptosis. Conversely, the N-terminally truncated ΔNp73 isoforms lack the transactivation domain (TAD) and consequently act as dominant-negative inhibitors for all TA isoforms of the p53 family, and are overexpressed in human tumors. Here, we report that TAp73, but not ΔNp73, is covalently modified by NEDD8 in an MDM2-dependent manner, attenuating its transactivation function and promoting cytoplasmic localization of neddylated TAp73. These results provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73, and identify the MDM2-TAp73 interaction as a promising therapeutic target. We also demonstrate that the stability of MDM2 is regulated by the NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization, concomitant with p53 activation. This study identifies a novel p53 activating mechanism in response to chemotherapy. In conclusion, the work presented herein has helped characterize the function of NEDD8 modification of MDM2 and the p53 family, and identify mechanisms by which MDM2 and the NEDD8 pathway may be targeted in the development of anti-cancer therapeutics.
3

In Vivo Analysis of Cruciform Extrusion and Resolution of DNA Palindromes in Eukaryotes

Cote, Atina 23 September 2009 (has links)
DNA palindromes are implicated in several examples of gross chromosomal aberrations in the human genome, however, the molecular mechanism(s) that govern palindrome instability are largely under-investigated. Because of their propensity for intrastrand base pairing, it is suspected that the acquisition of a secondary structure, such as a hairpin or cruciform, instigates the rearrangement process. A significant hurdle in defining palindrome-provoked instability lies in the fact that reliable methods for examining in vivo cruciform extrusion remain underdeveloped. A challenge is to provide straightforward evidence for cruciform extrusion in eukaryotic cells. Here, I present a plasmid system for use in Saccharomyces cerevisiae that enables for the detection of cruciforms in vivo. Cruciform extrusion, of either an in vitro-prepared palindrome or a near-palindrome from the human genome, is monitored by scoring for the product of cruciform resolution, being a dually hairpin-capped linear DNA molecule. These results not only provide evidence for the occurrence of cruciform extrusion in eukaryotic chromatin, they also identify a novel source of endogenous double strand break formation. A screen for candidate genes that are required for resolution revealed that the Mus81 Endonuclease, a candidate Holliday junction resolvase, provides the majority of cruciform resolution activity in mitotic cells, validating the notion that cellular HJ resolvases can misrecognize a cruciform for a Holliday junction. A second screen identified a requirement for the Sgs1-Top3-Rmi1 complex in the prevention of double strand break formation, including cruciform resolution, of DNA palindromes. These results uncover a new role for the RecQ helicase in prevention of palindrome-provoked instability, possibly through the intrusion of cruciform structures. Together, this work contributes significantly to our understanding of cruciform metabolism in eukaryotes and supports suggestions that cruciform extrusion instigates instability in the human genome.
4

Regulation of MDM2 and the p53 Family by the NEDD8 Pathway

Watson, Ian 17 February 2011 (has links)
NEDD8 is an ubiquitin-like protein sharing approximately 60% amino acid identity with ubiquitin and has biological roles in cell cycle progression, viability and development. Recently, a number of oncoproteins and tumor suppressors have been identified as NEDD8 substrates, including MDM2 and p53. MDM2 is an oncogenic E3 ligase that promotes NEDD8 modification and ubiquitin-mediated degradation of the tumor suppressor transcription factor, p53. Cellular stresses such as DNA damage lead to p53 activation due, in part, to MDM2 destabilization by mechanisms that are not completely understood. Studies in mice demonstrate the biological role of MDM2 is to negatively regulate p53 function, however, when overexpressed or amplified, MDM2 has p53-independent oncogenic functions presumably due to the regulation of additional substrates. One such substrate may be the p53 family member, p73. p73 exists as multiple isoforms and accumulating evidence suggests that the N-terminal isoforms dictate its role in tumorigenesis. The full-length pro-apoptotic TAp73 isoforms are induced by chemotherapies and are able to transactivate p53-target genes to initiate cell cycle arrest and apoptosis. Conversely, the N-terminally truncated ΔNp73 isoforms lack the transactivation domain (TAD) and consequently act as dominant-negative inhibitors for all TA isoforms of the p53 family, and are overexpressed in human tumors. Here, we report that TAp73, but not ΔNp73, is covalently modified by NEDD8 in an MDM2-dependent manner, attenuating its transactivation function and promoting cytoplasmic localization of neddylated TAp73. These results provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73, and identify the MDM2-TAp73 interaction as a promising therapeutic target. We also demonstrate that the stability of MDM2 is regulated by the NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization, concomitant with p53 activation. This study identifies a novel p53 activating mechanism in response to chemotherapy. In conclusion, the work presented herein has helped characterize the function of NEDD8 modification of MDM2 and the p53 family, and identify mechanisms by which MDM2 and the NEDD8 pathway may be targeted in the development of anti-cancer therapeutics.
5

Generation of Anti-CD133 Human Synthetic Antibodies as Tools for Exploring CD133 Function

Williams, Rashida 11 December 2013 (has links)
Two synthetic human antibody fragments against the human pentaspan membrane protein CD133 were isolated using a novel selection method involving direct selections on cells coupled with Illumina deep sequencing. The antibody fragments were isolated through a PCR-based recovery strategy developed in the lab and subsequently converted to full length IgGs. Termed RW01 and RW03, the antibodies bind separate epitopes on CD133 and are able to detect the protein using various molecular techniques. Finally, experiments have shown that RW01 and RW03 treatment affect the stability of CD133 on live cells. Additional experiments are required to reveal the specific epitope recognized by each antibody, which organelles they are targeted to when internalized and whether they have an effect on cellular differentiation or cellular viability. In addition to the therapeutic potential of these antibodies, they will have many applications towards expanding our knowledge concerning the CD133 protein and its role in cancer.
6

Generation of Anti-CD133 Human Synthetic Antibodies as Tools for Exploring CD133 Function

Williams, Rashida 11 December 2013 (has links)
Two synthetic human antibody fragments against the human pentaspan membrane protein CD133 were isolated using a novel selection method involving direct selections on cells coupled with Illumina deep sequencing. The antibody fragments were isolated through a PCR-based recovery strategy developed in the lab and subsequently converted to full length IgGs. Termed RW01 and RW03, the antibodies bind separate epitopes on CD133 and are able to detect the protein using various molecular techniques. Finally, experiments have shown that RW01 and RW03 treatment affect the stability of CD133 on live cells. Additional experiments are required to reveal the specific epitope recognized by each antibody, which organelles they are targeted to when internalized and whether they have an effect on cellular differentiation or cellular viability. In addition to the therapeutic potential of these antibodies, they will have many applications towards expanding our knowledge concerning the CD133 protein and its role in cancer.
7

Exploration of Peptide Recognition using Directed Evolution of the PDZ Domain Fold

McLaughlin, Megan 15 July 2013 (has links)
The PDZ domain family is one of the most abundant peptide recognition modules in metazoan proteomes. Characterization of natural PDZ domains has provided insight into the structural basis and diversity of peptide recognition by this fold. In order to test the limits of the current model, I evolved synthetic PDZ domains. Based on the Erbin PDZ domain and selected for binding to pep- tides with different position-2 residues, synthetic variants were characterized using high throughput peptide profiling. This approach generated insight into subclass specificities in the most common natural specificity classes (I [ST]-2 and II Φ-2), demonstrated an alternative basis for a rare specific- ity (Class III D-2) and predicted that some natural domains may exhibit an easily-evolved novel specificity (Class IV R-2). These results also emphasize that some non-contact residues may have a disproportionate effect on position-2 specificity, contrary to the predictions of the original model.
8

Exploration of Peptide Recognition using Directed Evolution of the PDZ Domain Fold

McLaughlin, Megan 15 July 2013 (has links)
The PDZ domain family is one of the most abundant peptide recognition modules in metazoan proteomes. Characterization of natural PDZ domains has provided insight into the structural basis and diversity of peptide recognition by this fold. In order to test the limits of the current model, I evolved synthetic PDZ domains. Based on the Erbin PDZ domain and selected for binding to pep- tides with different position-2 residues, synthetic variants were characterized using high throughput peptide profiling. This approach generated insight into subclass specificities in the most common natural specificity classes (I [ST]-2 and II Φ-2), demonstrated an alternative basis for a rare specific- ity (Class III D-2) and predicted that some natural domains may exhibit an easily-evolved novel specificity (Class IV R-2). These results also emphasize that some non-contact residues may have a disproportionate effect on position-2 specificity, contrary to the predictions of the original model.
9

Differential Protein Interactions of NMDA Receptor NR2 Subunits

Sam, Kevin 11 January 2011 (has links)
NMDA-type glutamate receptors (NMDAR) regulate neurotransmission and excitotoxicity. NMDAR signaling is believed to be dependent on NR2 subunit (A-D) composition and interactions with intracellular proteins. To determine the role of individual NR2 subunits in NMDAR signaling, I examined the biochemical interactions and colocalization of NR2A and NR2B NMDAR subunits with PSD-95 and CaMKII. Immunofluorescent colocalization revealed that by perturbing PSD-95 PDZ interactions using a targeted peptide (TAT-NR2B9c) increased association of PSD-95 with NR2A and CaMKII with NR2B; furthermore, decreases in association of CaMKII with NR2A and PSD-95 with NR2B were observed. The effects of TAT-NR2B9c were dependent upon NMDAR stimulation with 10μM NMDA and were not observed in untreated cells or at toxic doses of NMDA (40 μM). Thus, disrupting PSD-95 PDZ interactions produced activity-dependent differences in the co-localization of NR2A and NR2B with key signaling proteins, providing evidence that individual NR2 subunits may confer differential signaling to NMDARs.
10

Differential Protein Interactions of NMDA Receptor NR2 Subunits

Sam, Kevin 11 January 2011 (has links)
NMDA-type glutamate receptors (NMDAR) regulate neurotransmission and excitotoxicity. NMDAR signaling is believed to be dependent on NR2 subunit (A-D) composition and interactions with intracellular proteins. To determine the role of individual NR2 subunits in NMDAR signaling, I examined the biochemical interactions and colocalization of NR2A and NR2B NMDAR subunits with PSD-95 and CaMKII. Immunofluorescent colocalization revealed that by perturbing PSD-95 PDZ interactions using a targeted peptide (TAT-NR2B9c) increased association of PSD-95 with NR2A and CaMKII with NR2B; furthermore, decreases in association of CaMKII with NR2A and PSD-95 with NR2B were observed. The effects of TAT-NR2B9c were dependent upon NMDAR stimulation with 10μM NMDA and were not observed in untreated cells or at toxic doses of NMDA (40 μM). Thus, disrupting PSD-95 PDZ interactions produced activity-dependent differences in the co-localization of NR2A and NR2B with key signaling proteins, providing evidence that individual NR2 subunits may confer differential signaling to NMDARs.

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