Abstract not available

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 191-209).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 161-184).

Les facteurs de transcription de la famille Rel/NF-κB régulent l’expression d’un grand nombre de gènes impliqués dans les réponses immunitaires et inflammatoires ainsi que dans la régulation de la prolifération et de la survie cellulaire. Le caractère transitoire de l’activation de NF-κB est donc crucial pour poterger les cellules de l’autoxicité due à une trop forte expression des gènes cibles de ce facteur de transcription. Dans le cadre de notre thèse de doctorat, nous avons étudié les mécanismes moléculaires régulant la cinétique d’activation de NF-κB, en accordant une attention toute particulière au complexe kinase IKK, qui semble être le regulateur clef de l’activation de NF-κB. Nos résultats suggèrent que p300 pourrait réguler la durée d’activation des IKKs d’une part par acétylation directe, et d’autre part, indépendamment de son activité HAT, en stabilisant les IKKs et donc en prolongeant leur demie-vie et par conséquent leur activation.<p>Certains virus utilisent la voie de signalisation NF-κB afin de promouvoir leur propre réplication. C’est le cas du virus HIV-1 (Human Immunodeficiency Virus type 1), qui contient dans son promoteur deux sites de liaison pour NF-κB. Notre laboratoire a précédemment montré que l’utilisation du TNFα en combinaison avec la TSA, active l’expression virale de manière synergique. L’administration combinée d’un activateur du facteur NF-κB et d’un inhibiteur de désacétylases pourrait, en présence d’une thérapie anti-HIV-1 efficace, être envisagée dans le but d’éliminer les cellules réservoirs infectées de manière latente. L’utilisation thérapeutique du TNFα ou de la TSA étant inenvisageable en raison de leur toxicité, nous avons étudié l’effet d’autres substances ayant un plus grand potentiel thérapeutique et nous avons apporté une preuve de principe du potentiel thérapeutique de la coadministration de plusieurs activateurs viraux (inhibiteurs de HDACs[HDACIs]+inducteurs de la voie NF-κB) pour réduire le pool des réservoirs cellulaires infectés de manière latente.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Watson-Crick paired B-form DNA is the genetic material in most of the biological systems. Integrity of DNA is of utmost importance for the normal functioning of any organism. Various environmental factors, chemicals and endogenous agents constantly challenge integrity of the genome resulting in mutagenesis. Over the past few decades multiple reports suggest that DNA can adopt alternative conformations other than the right handed double helix. Such structures occur within the context of B-DNA as sequence dependent structural variations and are facilitated by free energy derived from negative supercoiling, which may be generated during physiological processes like transcription, replication, etc. or binding of proteins. Multiple groups have shown that these structures render fragility to the genome owing to single-strandedness (presence of unpaired bases). This conformational polymorphism of the DNA is due to the presence of several repetitive elements across the genome. Some of the common non-B DNA structures include Z-DNA, H-DNA (triplex DNA), cruciform DNA, G-quadruplexes and RNA: DNA hybrid (R-loops). Over the past few decades G-quadruplex structures have gained tremendous importance owing to its role in physiology and pathology. Recently it has been shown that novel sequence motifs, called GNG or bulges can fold into G-quadruplexes, thus increasing the propensity of such structures genome-wide. Neurological diseases, psychiatric diseases and genomic disorders (due to deletions, translocations, duplications and inversions) are some of the consequences of non-B DNA structures in the human genome. Inadvertent genomic rearrangements in human can lead to different diseases including cancer. Immediate consequence of genomic rearrangement includes structural alteration of genome through joining of distant sequences. t(8;14) translocation is the hallmark of Burkitt’s lymphoma, which results in deregulation of c-MYC gene that may contribute to oncogenic transformation. In the present study, we delineate the causes of fragility within the c-MYC gene. In order to do this, breakpoints at the c-MYC locus from Burkitt’s lymphoma patient sequences reported in database were plotted and analysed. Interestingly, unlike many other translocations, breakpoints at c-MYC locus were widespread, except for a cluster of breakpoints downstream to promoter 2 (P2). Previous studies indicate that the translocation breakpoint clusters often correlate with formation of non-B DNA structures. The entire breakpoint cluster downstream of P2 was divided into Region 1, Region 2 and Region 3. Interestingly, in silico analysis of the breakpoint clusters revealed no evidence for predictive classic non-B DNA motifs in Region 2; whereas Region 1 harboured a G-quadruplex motif on the template strand and Region 3 had two short inverted repeats. Intriguingly, as the nontemplate strand of Region 2 was G skewed with a good number of AID binding motifs, we tested the MYC breakpoint Region 2 for its potential to form R-loop due to binding of nascent RNA to template DNA. Our results showed that MYC Region 2 can form RNA-DNA hybrid in a transcription dependent manner in physiological orientation. Observed structure was sensitive to RNase H. We showed Region 2 hindered action of Dpn I upon transcription confirming formation of R-loop structure. Owing to single strandedness, Region 2 R-loop was shown to be sensitive to P1 nuclease as opposed to the untranscribed control. The single strandedness of the Region 2 R-loop was characterized at a single molecule level through bisulfite modification assay. The assay corroborated formation of R-loop along with providing snapshots of various length R-loops formed upon Region 2 transcription. Besides, various biophysical and biochemical assays showed the complementary region (template strand) to be single-stranded in stretches, upon transcription. Length of RNA within the R-loop was within a range of 75 to 250 nt. To delineate the mechanism of R-loop formation we tested the sensitivity of R-loop formation to RNase A during and post transcription; and found that R-loop formation was abrogated in presence of RNase A during transcription suggesting that R-loop formation followed a “thread back model”. Intriguingly we observed that two short regions of the template strand exhibited high degree of single strandedness. To investigate the reason for such unusual single strandedness, oligonucleotides spanning the region was designed and subjected for CD and EMSA studies. EMSA showed robust intramolecular G-quadruplex structure formation in presence of KCl, whereas CD confirmed that both regions formed parallel G-quadruplexes. We also showed the precise involvement of guanines in structure formation through DMS protection assay. Further, the region of interest was cloned into appropriate vectors and primer extension assays were performed in presence of G-quadruplex stabilizing agents like TMPyP4 and KCl. Increasing concentration of these stabilizing agents enhanced the formation of G-quadruplexes in a double stranded context, which hindered polymerase progression. Since these G-quadruplex structures utilized sequences which are deviant to the consensus of G-quadruplex motifs, non-B DNA predicting tools were unable to score them. On closer analysis of the sequences we found that, these G-quadruplexes involve duplex hairpin and GNG motifs during structure formation. Besides, both the G-quadruplexes were highly thermostable and were able to fold back upon renaturation. Till recently, it has been believed that G-quadruplex structures are formed using a minimum of four, 3 guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In the present study, guanines along with GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the core elements of a G-quadruplex are not equally important in structure formation when flanked by GNG motifs. Importantly, the positioning and number of GNG/GNGNG can dictate the formation of G-quadruplexes. In addition to HOX11 fragile region, GNG motifs of HIF1-alpha can fold into intramolecular G-quartet. However, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Interestingly, intra molecular GNG G-quadruplexes were able to fold back after complete denaturation of the oligonucleotides. Besides one of the intra molecular GNG G-quadruplexes was purified and confirmed for parallel conformation. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody BG4 to SHOX G-quadruplex through EMSA studies. Thus, the study provides novel insights into the role of GNG motifs in G-quadruplex structure formation, which may have both physiological and pathological implications. In conclusion, we show formation of transcription dependent R-loop and G-quadruplex structures at the c-MYC gene locus in a mutually exclusive manner. The data presented here, in conjunction with studies from other laboratories suggests that these structures could impart fragility within the c-MYC gene locus during t(8;14) translocation. Besides, we characterised unusual G-quadruplexes harbouring GNG motifs. We find that positioning and number of GNG can dictate the formation of G-quadruplexes and is context dependent.

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.

No description available.

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. The nuclear factor κB (NF-κB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including CRC. Therefore, understanding the regulation of NF-κB is of ultimate importance for cancer therapy. Using a novel validation-based insertional mutagenesis (VBIM) strategy, our lab has identified the novel adenomatous polyposis coli (APC) like protein (ALP) gene as a negative regulator of NF-κB. Preliminary studies from our lab demonstrated that overexpression of ALP led to decreased NF-κB activity by κB reporter assay and electrophoresis mobility gel shift assay (EMSA). The current project aims to further evaluate the role of ALP in the regulation of NF-κB signaling in CRC cells. We found that overexpression of ALP in human CRC HT29 cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay. ALP overexpression also decreased the cell growth rate and cell migration ability, while shRNA mediated knockdown of ALP showed opposite effects, confirming that ALP is a tumor suppressor in CRC HT29 cells. Overexpression of ALP led to decreased NF-κB activity by κB reporter assay and condition media assay in CRC HT29 cells. Furthermore, immunohistochemical analysis with human colon vii tissues revealed that there is a gradual loss of ALP protein with tumor progression. We also found that ALP predominantly localizes in the cytoplasm, and binds to the p65 subunit of NF-κB, and might be functioning downstream of IκB kinase (IKK). In summary, in this study, we provide evidence regarding the tumor suppressor role of ALP in CRC by functioning as novel negative regulator of NF-κB. This discovery could lead to the establishment of ALP as a potential biomarker and therapeutic target in CRC.

The majority of structural studies on DNA have been carried out using fibre diffraction, while studies of its dynamics and thermal behaviour have been mainly performed in solution. When the DNA double helix is heated, it exhibits local separation of the two strands that grow in size with temperature and lead to their complete separation. This work has investigated various aspects of this phenomenon. The experiments reported in this thesis were carried out on films of oriented fibres of DNA prepared with the Wet Spinning Apparatus. Thus, sample preparation and characterisation are essential parts of the research. The structures of two forms of DNA, A and B, have been explored as a function of relative humidity at fixed ionic conditions. A method to eliminate traces of ever-present B-form contamination in A-form samples was established. The high orientation of the DNA molecules within the samples allowed us to investigate dynamical fluctuations and the melting transition of DNA using neutron scattering, which can provide the spatial information crucial to understand a phase transition, probing the static correlation length along the molecule as a function of temperature. The transition has been investigated for A and B-forms in order to understand its dependence on molecular configuration.Furthermore, after the first melting, denatured DNA films show typical glass behaviour. Their thermal relaxation has been explored using calorimetry.Neutron and X-ray inelastic scattering (INS and IXS) were used in the past to measure longitudinal phonons in fibre DNA, and the results shown disagreement. Recent INS measurements supported with phonon simulations have been crucial to understand the different dispersion curves reported to date. Experiments using INS and IXS have been carried out to continue with this investigation. Attempts to observe the transverse fluctuations associated to the thermal denaturing of DNA, never experimentally investigated before, have been made.

La majorité des études structurales sur l’ADN avaient été réalisées par diffraction sur des fibres tandis que ses propriétés dynamiques thermiques avaient été étudiées en solution. Lorsque la double hélice d’ADN est chauffée elle présente des séparations locales des deux brins, dont la taille augmente avec la température jusqu’à la séparation complète des brins. Ce travail étudie différents aspects de ce phénomène. Les expériences présentées dans cette thèse ont été réalisées sur des films formés de fibres orientées d’ADN préparés par la méthode du ”filage humide“. La préparation et la caractérisation des échantillons en deux formes A et B de l’ADN ont constitué une partie importante de la recherche. Une méthode pour éliminer la contamination résiduelle de la forme B dans les échantillons de forme A a été mise au point. La bonne orientation des molécules d’ADN dans les échantillons nous a permis d’étudier les fluctuations dynamiques et la transition de dénaturation thermique de l’ADN par diffraction de neutrons, sensibles à la longueur de corrélation statique le long de la molécule en fonction de la température. La transition a été étudiée pour les formes A et B pour déterminer comment elle dépend de la conformation. De plus, après la première dénaturation thermique, les films d’ADN présentent un comportement typique d’un verre. Leur relaxation thermique a été étudiée par calorimétrie. La diffusion inélastique de neutrons et de rayons X (INS et IXS) avaient été utilisées antérieurement pour mesurer les phonons longitudinaux dans des fibres d’ADN, avec des désaccords entre les résultats. Des mesures INS récentes, complétées par des simulations, avaient été cruciales pour comprendre les différentes courbes de dispersion observées. Nous avons mené des expériences INS et IXS pour poursuivre cette analyse. Des tentatives pour observer les mouvements transversaux associés à la dénaturation thermique de l’ADN, jamais observés expérimentalement, ont également été faites. / The majority of structural studies on DNA have been carried out using fibre diffraction, while studies of its dynamics and thermal behaviour have been mainly performed in solution. When the DNA double helix is heated, it exhibits local separation of the two strands that grow in size with temperature and lead to their complete separation. This work has investigated various aspects of this phenomenon. The experiments reported in this thesis were carried out on films of oriented fibres of DNA prepared with the Wet Spinning Apparatus. Thus, sample preparation and characterisation are essential parts of the research. The structures of two forms of DNA, A and B, have been explored as a function of relative humidity at fixed ionic conditions. A method to eliminate traces of ever-present B-form contamination in A-form samples was established. The high orientation of the DNA molecules within the samples allowed us to investigate dynamical fluctuations and the melting transition of DNA using neutron scattering, which can provide the spatial information crucial to understand a phase transition, probing the static correlation length along the molecule as a function of temperature. The transition has been investigated for A and B-forms in order to understand its dependence on molecular configuration.Furthermore, after the first melting, denatured DNA films show typical glass behaviour. Their thermal relaxation has been explored using calorimetry.Neutron and X-ray inelastic scattering (INS and IXS) were used in the past to measure longitudinal phonons in fibre DNA, and the results shown disagreement. Recent INS measurements supported with phonon simulations have been crucial to understand the different dispersion curves reported to date. Experiments using INS and IXS have been carried out to continue with this investigation. Attempts to observe the transverse fluctuations associated to the thermal denaturing of DNA, never experimentally investigated before, have been made.