In order to maintain genome stability, DNA damage needs to be detected and repaired in a timely fashion. To cope with damaged DNA, cells have evolved mechanisms termed "checkpoints", where, upon damage, cells initiate a signal transduction cascade that results in the slowing or halting of the cell cycle, allowing efficient DNA repair. Defects in the DNA damage checkpoint result in an overall increase in genomic instability and are thought to fuel cancer progression. To facilitate our understanding of how DNA damage leads to cancer progression, it is crucial to fully comprehend how these signal transduction mechanisms function. In this work, we have characterized in great detail the mechanisms of regulation of Rad53 (a central regulator of the DNA damage response in Saccharomyces cerevisiae) at the genetic, biochemical and structural level. Firstly, we describe a complex biochemical two-step mode of activation of Rad53 by protein-protein interaction and multi-step phosphorylation. We also shed light onto the mechanisms by which Rad53 is turned off to allow the cell cycle to resume, a process termed DNA damage recovery and adaptation. We found that during adaptation, the polo-like kinase Cdc5 is required to attenuate Rad53 catalytic activity. Finally, the study of Rad53 at the molecular and atomic level revealed that in addition to being regulated through a complex network of protein-protein interactions, Rad53 autophosphorylation is orchestrated by a mechanism of dimerization, activation segment phosphorylation via A-loop exchange, as well as through an autoinhibition mechanism regulated by a specific alpha-helical region at the C-terminal extremity of its kinase domain. Such work is important in understanding the function of different proteins in DNA damage signaling. This knowledge will enhance our understanding of the progression of DNA damage related diseases such as cancer, and could eventually help in the long term the development of novel therapeutics as treatments against these conditions.

Neural precursor cells self-renew and differentiate throughout development and in response to neural injury in the adult brain. The DNA damage response in NPCs has yet to be characterized. Patients with defective nucleotide excision repair (NER) demonstrate neurodegeneration dismyelination, and microcephaly, suggesting a potential link to defective NPC function with accumulating DNA damage. We observed reduced self-renewal in Csbm/m and Xpam/m NPCs in response to UV damage. Serial passaging resulted in exhaustion of Csbm/m NPCs in the absence of exogenous DNA damage. In vitro neuronal differentiation resulted in abnormal neuritigenesis after UV DNA damage in Csbm/m NPCs, suggesting defects in the terminal differentiation process. Taken together, the results indicate that DNA damage can modulate the apoptotic, self-renewal and differentiation fates of NPCs.

Neural precursor cells self-renew and differentiate throughout development and in response to neural injury in the adult brain. The DNA damage response in NPCs has yet to be characterized. Patients with defective nucleotide excision repair (NER) demonstrate neurodegeneration dismyelination, and microcephaly, suggesting a potential link to defective NPC function with accumulating DNA damage. We observed reduced self-renewal in Csbm/m and Xpam/m NPCs in response to UV damage. Serial passaging resulted in exhaustion of Csbm/m NPCs in the absence of exogenous DNA damage. In vitro neuronal differentiation resulted in abnormal neuritigenesis after UV DNA damage in Csbm/m NPCs, suggesting defects in the terminal differentiation process. Taken together, the results indicate that DNA damage can modulate the apoptotic, self-renewal and differentiation fates of NPCs.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

A crucial factor in determining the accuracy of DNA replication is maintenance of a balanced supply of deoxyribonucleoside triphosphates (dNTPs) at replication forks. Perturbation of dNTP biosynthesis can induce dNTP pool imbalance with deleterious genetic consequences, including increased mutagenesis, recombination, chromosomal abnormalities and cell death. Using the T4 bacteriophage system, I investigated the molecular basis of mutations induced by imbalanced dNTP pools in vivo. Two approaches were adopted to disturb dNTP biosynthesis: 1) using mutations which affect the deoxyribonucleotide biosynthesis pathway; 2) exogenously supplying mutagenic deoxyribonucleoside analogs which are then taken up by cells and are metabolized to dNTPs. The levels of dNTPs under different conditions were measured in crude extracts of phage-infected cells, while mutagenic effects were quantitated by analysis of certain rII mutations, thought to revert to wild type along either GC-to-AT or AT-to-GC transition pathways. The mutation pathways stimulated by dNTP pool perturbations were confirmed by direct DNA sequencing after amplification of template by the polymerase chain reaction (PCR). By replacing phage ribonucleotide (rNDP) reductase with the host, Escherichia coli, rNDP reductase, in phage-infected cells, I examined the mechanism of mutation induced by the thymidine analog 5- bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to- AT transition mutations were stimulated many hundred-fold when cells were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions were stimulated predominantly when T4 reductase was active, while ATto- GC transitions were stimulated more when E. coli reductase was active. By examining the control by dNTPs on CDP reduction, I found that the T4 rNDP reductase is substantially inhibited by either BrdUTP or dTTP in crude enzyme extracts. These experimental results are consistent with the hypothesis that mutagenic effects of BrdUrd are based on dNTP perturbations, supporting the model that rNDP reductase is a major determinant of BrdUrd mutagenesis. I also studied the mutator phenotype of one temperature-sensitive conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4 deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions induced by ts LB3 at a semipermissive temperature (34° C). Sequence analysis of revertants from the most sensitive gene marker, rII SN103, showed that either cytosine within the mutated triplet can undergo change to either thymidine or adenine, supporting a model in which mutagenesis induced by ts LB3 at a semipermissive temperature is based on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine triphosphate (hm-dCTP) caused by the temperature-labile dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations. However, no significant dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected cells even when cells were grown at the semi-permissive temperature, suggesting that imbalanced dNTP pools occurred only locally, close to replication forks. These results support a model of dNTP "functional compartmentation", in which DNA replication is fed by a small and rapidly depleted pool, with the bulk of measurable dNTP in a cell representing a replication-inactive pool. To further characterize the mutagenic specificity and DNA site specificity induced by T4 ts LB3, I developed a fast forward mutation approach using thymidine kinase as a marker gene. The studies confirmed that the principal mutagenic effect induced by ts LB3 is C-to- T transition, while C-to-A transversion mutagenesis also occurs. Analysis of DNA sequences around each mutation also suggests that local DNA context influences mutation frequency. / Graduation date: 1991

In order to maintain genome stability, DNA damage needs to be detected and repaired in a timely fashion. To cope with damaged DNA, cells have evolved mechanisms termed "checkpoints", where, upon damage, cells initiate a signal transduction cascade that results in the slowing or halting of the cell cycle, allowing efficient DNA repair. Defects in the DNA damage checkpoint result in an overall increase in genomic instability and are thought to fuel cancer progression. To facilitate our understanding of how DNA damage leads to cancer progression, it is crucial to fully comprehend how these signal transduction mechanisms function. In this work, we have characterized in great detail the mechanisms of regulation of Rad53 (a central regulator of the DNA damage response in Saccharomyces cerevisiae) at the genetic, biochemical and structural level. Firstly, we describe a complex biochemical two-step mode of activation of Rad53 by protein-protein interaction and multi-step phosphorylation. We also shed light onto the mechanisms by which Rad53 is turned off to allow the cell cycle to resume, a process termed DNA damage recovery and adaptation. We found that during adaptation, the polo-like kinase Cdc5 is required to attenuate Rad53 catalytic activity. Finally, the study of Rad53 at the molecular and atomic level revealed that in addition to being regulated through a complex network of protein-protein interactions, Rad53 autophosphorylation is orchestrated by a mechanism of dimerization, activation segment phosphorylation via A-loop exchange, as well as through an autoinhibition mechanism regulated by a specific alpha-helical region at the C-terminal extremity of its kinase domain. Such work is important in understanding the function of different proteins in DNA damage signaling. This knowledge will enhance our understanding of the progression of DNA damage related diseases such as cancer, and could eventually help in the long term the development of novel therapeutics as treatments against these conditions.

S'han estudiat en profunditat els mecanismes apoptòtics, constituint un factor substancial en la mort neuronal després de diferents tipus de danys en el SNC. No obstant això, molt poc es sap sobre l'activació dels mecanismes apoptòtics en les cèl·lules glials i la seva rellevància en la terminació de la resposta glial. La caspasa-3 ha estat definida clàssicament com la principal executora de la mort cel·lular programada i li han estat atribuïts recentment altres papers no apoptòtics de participació en esdeveniments cel·lulars, incloent-hi la proliferació cel·lular, la regulació del cicle cel·lular, i la participació en vies cel·lulars apoptòtiques i antiapoptòtiques després d'un dany excito-tòxic en el cervell immadur de rata. Per aquest propòsit, s'ha utilitzat un model de excitotoxicitat ben caracteritzat, que ha consistit en la inducció d'un dany cerebral mitjançant una injecció intracortical de N-methyl-D-asparate (NMDA) en rates postnatals en el 9è dia de vida (P9). La mort cel·lular, les proteïnes apoptòtiques i antiapoptòtiques van ser analitzades a diferents terminis de supervivència després de la lesió per assaigs enzimàtics i anàlisis de doble fluorescència amb microscopia confocal. Els primers resultats com la caspase-3 clivada es presentava en neurones danyades amb els nuclis TUNEL+, encara que va ser observada majoritàriament en el nucli d'astròcits activats en l'hemisferi lesionat sense associació amb fragmentació del ADN, des de les 4 hores post-lesió (hpl) fins a la formació de la cicatriu glial als 7-14 dies, sense trobar-se associació amb marcatge amb TUNEL. L'activitat enzimàtica de la caspasa-3 va ser detectada a les primeres hores post-lesió i coincidint amb els fragments clivados per caspasas de la proteïna acídica fibrilar (CCP-GFAP) en astrocitos. No obstant això, sobre llargs terminis de supervivència, quan sorgia la hipertròfia astroglial, la caspasa-3 astroglial generalment no es va correlacionar amb el clivage de CCP-GFAP, però en canvi es va relacionar amb l'expressió de novo de vimentina. Per altra banda, la caspasa-3 clivada no s'ha vinculat a proliferació cel·lular. Aquests resultats inicials van mostrar l'evidència d'un paper gens tradicional de les caspases en la funció astroglial, suggerint que la seva activació pot ser important per a la reestructuració del citoesquelet després d'un dany. Secundàriament, al avaluar les vies d'activació de la caspasa-3 en neurones i cèl·lules glials, l'activació espai-temporal de les vies apoptòtiques intrínseca i extrínseca va ser analitzada. Es va expressar principalment en les neurones danyades corticals i hipocampals, una important contribució de la via intrínseca a través de la gran proporció de caspasa-9 activa amb l'activació neuronal de la caspase-3 i mort cel·lular. Per altra banda, la caspasa-8 va presentar una reduïda correlació amb la caspasa-3, i característicament es va presentar amb especificitat en les capes corticals. En les cèl·lules glials, l'activació de la caspasa-9, caspasa-8, i també p53, altra molècula que activa la caspasa-3 indirectament, va ser significant tant sols en la capa cortical VI i en el corpus callosum, suggerint que la caspasa-3 astroglial no es correlaciona generalment amb les clàssiques vies d'activació. La tercera part d'aquesta tesi es va focalitzar en l'estudi de les molècules antiapoptòtiques, com diversos inhibidors de l'apoptosis (IAPs) i les proteïnes heat shock (HSPs), conegudes com inhibidors de la caspasa-3 clivada en altres tipus cel·lulars. Les neurones van presentar una apreciable expressió de HSC70/HSP70 i cIAP-2, encara que aquestes es correlacionaven pobrament amb caspasa-3 en aquest tipus cel·lular. En astròcits , la survivina i la chaperona HSP25/27 van mostrar una forta correlació amb caspasa-3 en diverses regions cerebrals i a diversos terminis de supervivència, sent aquestes proteïnes destacades candidates per al bloqueig de l'activitat enzimàtica de la caspasa-3, la qual podria acumular-se en absència de mort astroglial. En conclusió, aquesta tesi ha descobert nous rols no apoptòtics de la caspasa-3 en cèl·lules astroglials i suggereix mecanismes de bloqueig de la caspasa-3, ajudant al coneixement dels mecanismes emprats pels astròcits per enfrontar-se al dany en el cervell immadur. / Los mecanismos apoptóticos han sido estudiados en profundidad, constituyendo un papel substancial en la muerte neuronal tras diferentes tipos de daño en el SNC. No obstante, muy poco se sabe acerca de la activación de los mecanismos apoptóticos en las células gliales y su relevancia en la terminación de la respuesta glial. La caspasa-3 ha sido definida clásicamente como al principal ejecutora de la muerte celular programada, le han sido recientemente atribuidos otros papeles no-apoptóticos, participando en acontecimientos celulares, incluyendo proliferación celular, regulación del ciclo celular, y vías celulares apoptóticas y antiapoptóticas después de un daño excitotóxico en el cerebro inmaduro de rata. Para este propósito, se ha utilizado un modelo de excitotoxicidad bien caracterizado, que consiste en la inducción de un daño cerebral mediante una inyección intracortical de N-methyl-D-asparate (NMDA) en ratas postnatales en el día 9 de vida (P9). La muerte celular, las proteínas apoptóticas y antiapoptóticas fueron analizadas a distintos tiempos de supervivencia después de la lesión por ensayos enzimáticos y análisis de doble fluorescencia con microscopía confocal. Los primeros resultados como la caspasa-3 clivada se presentaba en neuronas dañadas con los núcleos TUNEL+, aunque fue observada mayoritariamente en el núcleo de astrocitos activados en el hemisferio lesionado sin asociación con fragmentación del ADN, desde tiempos tempranos las 4 horas post-lesión (hpl) hasta la formación de la cicatriz glial a los 7-14 días, sin encontrarse asociación con marcaje con TUNEL. La actividad enzimática de la caspasa-3 fue detectada a las primeras horas post-lesión y coincidiendo con los fragmentos clivados por caspasas de la proteína acídica fibrilar (CCP-GFAP) en astrocitos. Sin embargo, a largos tiempos de supervivencia, cuando surge la hipertrofia astroglial, la caspasa-3 astroglial generalmente no correlacionó con el clivage de CCP-GFAP, pero en cambio se relacionó con la expresión de novo de vimentina. Por otra parte, la caspasa-3 clivada no se ha vinculado a proliferación celular. Estes resultados iniciales mostraron la evidencia de un no-tradicional papel de las caspasas en la función astroglial, sugeriendo que su activación puede ser importante para la reestructuración del citoesqueleto después de un daño.Secundariamente, al evaluar las vías de activación de la caspasa-3 en neuronas y células gliales, la activación espacio-temporal de las vías apoptóticas intrínseca y extrínseca fueron analizadas. Se expresó principalmente en las neuronas dañadas corticales e hipocampales, una importante contribución de la vía intrínseca através de la gran proporción de caspasa-9 activa con la activación neuronal de la caspase-3 y muerte celular. Por otra banda, la caspasa-8 presentó una reducida correlación con la caspasa-3, y característicamente presentó con especificidad en las capas corticales. En las células gliales, la activación de la caspasa-9, caspasa-8, y también p53, otra molécula que activa la caspasa-3 indirectamente, fue solamente significante en la capa cortical VI y en el corpus callosum, sugeriendo que la caspasa-3 astroglial no se correlaciona generalmente con las clásicas vías de activación. La tercera parte de esta tesis se focalizó en el estudio de las moléculas antiapoptóticas, como varios inhibitores de apoptosis (IAPs) y las heat shock proteins (HSPs), conocidas como inhibidoras de la caspasa-3 clivada en otros tipos celulares. Las neuronas presentaron una apreciable expresión de HSC70/HSP70 y cIAP-2, aunque estas pobremente se correlacionan con caspasa-3 en este tipo celular. En astrocitos , la survivina y la chaperona HSP25/27 mostraron una fuerte correlación con caspasa-3 en varias regiones cerebrales y a varios tiempos de supervivencia, siendo estas proteínas destacadas candidatas para el bloqueo de la actividad enzimática de la caspasa-3, la cual podría acumularse en ausencia de muerte astroglial. En conclusión esta tesis ha destapado nuevos roles no apoptóticos de la caspasa-3 en células astrogliales, sugeriendo mecanismos de bloqueo de la caspasa-3, ayudando a la mejor comprensión de los mecanismos empleados por los astrocitos para enfrentarse al daño en el cerebro inmaduro. / Os mecanismos apoptóticos estudiáronse en profundidade, constituíndo un papel substancial na morte neuronal despois de diferentes tipos de dano no SNC. Nembergantes, moi pouco se sabe acerca da activación dos mecanismos apoptóticos nas células gliais e a sua relevancia na terminación da resposta glial. A caspasa-3 definiuse clásicamente coma principal executura da morte celular programada, fóronlle recentemente atribuídos outros papeis non-apoptóticos, participando en acontecementos celulais, incluíndo proliferación, regulación do ciclo celular, e vías apoptóticas e antiapoptóticas despois dun dano excitotóxico no cerebro inmaduro de rata. Para este propósito, utilizáronse un modelo de excitotoxicidade ben caracterizado, que consiste na inducción dun dano cerebral mediante unha inxección intracortical de N-methyl-D-asparate (NMDA) en ratas postnatais no día 9 de vida (P9). A morte celular, as proteínas apoptóticas e antiapoptóticas foron analizadas a distintos tempos de supervivencia despois da lesión por ensaios enzimáticos e análise de doble fluorescencia con microscopía confocal. Os primeiros resultados coa caspasa-3 clivada presentábanse en neuronas dañadas cos núcleos TUNEL+, aunque foi observada maioritariamente no núcleo de astrocitos activados no hemisferio lesionado sen asociación con fragmentación do ADN, dende tempos máis cedos, ás 4 horas post-lesión (hpl) ata a formación da cicatriz glial ós 7-14 días, sen encontrarse asociación co marcaxe con TUNEL. A actividade enzimática da caspasa-3 foi detectada as primeiras horas post-lesión e coincidindo cos fragmentos clivados por caspasas da proteína acídica fibrilar (CCP-GFAP) en astrocitos. Sen embargo, a tempos longos de supervivencia, cando surxe a hipertrofia astroglial, a caspasa-3 astroglial xeralmente non correlacionou co clivaxe de CCP-GFAP, pero si se relacionou coa expresión de novo de vimentina. Por outra banda, a caspasa-3 clivada non se vinculou á proliferación celular. Estes resultados iniciais mostraron a evidencia dun non-tradicional papel das caspasas na función astroglial, suxerindo que a sua activación pode ser importante para a reestructuración do citoesqueleto despois dun dano.Secundariamente, ó evaluar as vías de activación da caspasa-3 en neuronas e nas células gliais, a activación espacio-temporal das vías apoptóticas intrínseca e extrínseca foron analizadas. Expresouse principalmente nas neuronas dañadas corticais e hipocampais, unha importante contribución da vía intrínseca através da gran proporción de caspasa-9 activa coa activación neuronal da caspase-3 e morte celular. Por outra banda, a caspasa-8 presentou unha reducida correlación coa caspasa-3, e característicamente presentou especificidade nas capas corticais. Nas células gliais, a activación da caspasa-9, caspasa-8, e tamén p53, outra molécula que activa a caspasa-3 indirectamente, foi soamente significante na capa cortical VI e no corpus callosum, suxerindo que a caspasa-3 astroglial non se correlaciona xeralmente coas clásicas vías de activación. A terceira parte desta tesis focalizouse no estudio das moléculas antiapoptóticas, coma varios inhibitores de apoptosis (IAPs) e as heat shock proteins (HSPs), coñecidas coma inhibidoras da caspasa-3 clivada noutros tipos celulares. As neuronas presentaron unha importante expresión de HSC70/HSP70 e cIAP-2, anque estas pobremente correlacionábanse coa caspasa-3 neste tipo celular. En astrocitos , a survivina e a chaperona HSP25/27 mostraron unha forte correlación con caspasa-3 en varias rexións cerebrais e a varios tempos de supervivencia, sendo estas proteínas destacadas candidatas para o bloqueo da actividade enzimática da caspasa-3, a cal podría acumularse en ausencia de morte astroglial. En conclusión esta tesis destapou novos roles non apoptóticos da caspasa-3 en células astrogliais, suxerindo mecanismos de bloqueo da caspasa-3 e axudou á mellor comprensión dos mecanismos empregados polos astrocitos para enfrentarse ó dano no cerebro inmaduro. / Although it is well established that apoptotic mechanisms play a central role in neuronal death after different types of CNS damage, little is known about the activation of apoptotic pathways in glial cells which have been postulated to be important for the termination of the glial response. It is remarkable that the classically defined main executioner of programmed cell death, caspase-3, has recently been attributed a non-apoptotic role, participating in cellular events including cell proliferation, cell cycle regulation, or cellular differentiation. Accordingly, the general aim of this thesis was to analyze the putative role of the classic apoptotic and anti-apoptotic pathways after acute excitotoxic damage to the immature rat brain. For this purpose, a well-characterized in vivo model of excitotoxicity was used, where damage was induced by the intracortical injection of N-methyl-D-asparate (NMDA) in postnatal day 9 rats. Cell death, apoptotic and anti-apoptotic proteins were analyzed at different survival times after the lesion by enzymatic assays and double immunofluorescence for confocal microscope analysis. The first set of results demonstrated that although cleaved caspase-3 was found in some damaged neurons showing TUNEL+ nuclei, cleaved caspase-3 was mainly observed in the nucleus of activated astrocytes in the lesioned hemisphere as early as 4 h post-lesion, persisted until the glial scar was formed at 7-14 days, and was marginally associated with TUNEL labeling. Caspase-3 enzymatic activity was significant during the first hours post-lesion and co-localized with the presence of caspase-cleaved fragments of glial fibrillary acidic protein (CCP-GFAP) in astrocytes. However, at longer survival times, when astroglial hypertrophy was observed, astroglial caspase-3 did not generally correlate with GFAP cleavage but instead was associated with de novo expression of vimentin. Moreover, astroglial caspase-3 cleavage was not associated with cell proliferation. These first results provided evidence for a non-traditional role of caspases in astroglial function, suggesting that caspase activation may be important for astroglial cytoskeleton remodeling following injury.Secondly, in order to evaluate upstream pathways activating caspase-3 in neurons and glial cells, the spatio-temporal activation of the intrinsic and extrinsic apoptosis pathways were analyzed. In damaged neuronal cells of the ipsilateral cortex and hippocampus an important contribution of the intrinsic mitochondrial pathway through caspase-9 activation was shown to account for most of neuronal caspase-3 activation and apoptotic nuclei. However, neuronal caspase-8 showed a diminished correlation with caspase-3 and characteristically showed cortical layer specificity. In glial cells, activation of caspase-9, caspase-8, and also p53, another putative caspase-3 activating molecule, was only significant in cortical layer VI and the corpus callosum, suggesting that astroglial caspase-3 does not generally correlate with classical upstream activating pathways.In the third part of this thesis, inhibitor of apoptosis proteins (IAPs) and heat shock proteins (HSPs), known inhibitors of cleaved caspase-3 in other cell types, were analyzed. Whereas neuronal cells showed noticeable expression of HSC70/HSP70 and cIAP-2, these poorly correlated with caspase-3 in this cell type. In astrocytes, survivin and the small HSP25/27 showed a strong correlation with caspase-3 in several regions and throughout different survival times, pointing to these proteins as relevant candidates for the blockade of caspase-3 enzymatic activity, which could account for the absence of astroglial cell death. In conclusion, this thesis has discerned new non-apoptotic roles of caspase-3 in astroglial cells and suggests mechanisms for caspase-3 blockade in these glial cells, improving the understanding of the mechanisms employed by astrocytes to cope with damage in the immature brain.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

7xxx series Al-Zn-Mg-Cu-base wrought Al-alloy products are widely used for aerospace structural applications where monotonic and cyclic mechanical properties are of prime concern. Microstructure of these commercial alloys usually contains brittle coarse constituent particles or inclusions of Fe-rich intermetallic compounds and Mg2Si, typically in the size range of 1 to 50 micron. Plastic deformation and fracture of 7xxx series alloys (as well as of numerous other wrought Al-alloys) is associated with gradual microstructural damage accumulation that involves cracking of the coarse constituent particles, growth of voids around the cracked particles, and the void coalescence. To understand and model the microstructural damage evolution processes such particle cracking, quantitative microstructural data associated with the damage nucleation are required under monotonic as well as cyclic loading conditions. In the past quantitative characterization of particle cracking damage in these alloys has been problematic. However, with recent advances in digital image analysis and stereology based techniques, it is now possible to quantitatively characterize the damage nucleation in hot-rolled 7075(T6) Al-alloy (a typical alloy of 7xxx series) due to cracking of the Fe-rich coarse constituent particles. The objectives of this work are: * Quantitative characterization of the cracking of Fe-rich constituent particles as a function of strain under quasi-static loading. This involves measurements of number density of cracked particles, volume fraction of the cracked particles, their size, shape, and orientation distribution, as well as nearest neighbor distribution and two-point correlation functions to quantify spatial dispersion of the cracked particles in a series of interrupted uniaxial tensile test specimens at different strain levels. * Quantitative characterization of the cracking of Fe-rich constituent particles under cyclic loading to study the differences between the particle cracking damage due to monotonic and cyclic loading.

Matrix acidizing is used in carbonate formations to create wormholes that connect the formation to the wellbore. Hydrochloric acid, organic acids, or mixtures of these acids are typically used in matrix acidizing treatments of carbonate reservoirs. However, the use of these acids in deep wells has some major drawbacks including high and uncontrolled reaction rate and corrosion to well tubulars, especially those made of chrome-based tubulars (Cr-13 and duplex steel), and these problems become severe at high temperatures. Hydrochloric acid (HCl) and its based fluids have a major drawback in stimulating shallow (low fracture gradient) formations as they may cause face dissolution (formation surface washout) if injected at low rates. The objective of stimulation of sandstone reservoirs is to remove the damage caused to the production zone during drilling or completion operations. Many problems may occur during sandstone acidizing with Hydrochloric/Hydrofluoric acids (HCl/HF) mud acid. Among those problems: decomposition of clays in HCl acids, precipitation of fluosilicates, the presence of carbonate can cause the precipitation of calcium fluorides, silica-gel filming, colloidal silica-gel precipitation, and mixing between various stages of the treatment. To overcome problems associated with strong acids, chelating agents were introduced and used in the field. However, major concerns with most of these chemicals are their limited dissolving power and negative environmental impact. Glutamic acid diacetic acid (GLDA) a newly developed environmentally friendly chelate was examined as stand-alone stimulation fluid in deep oil and gas wells. In this study we used GLDA to stimulate carbonate cores (calcite and dolomite). GLDA was also used to stimulate and remove the damage from different sandstone cores containing different compositions of clay minerals. Carbonate cores (calcite and dolomite) of 6 and 20 in. length and 1.5 in. diameter were used in the coreflood experiments. Coreflood experiments were run at temperatures ranging from 180 to 300oF. Ethylene diamine tetra acetic acid (EDTA), hydroxyl ethylethylene diaminetriacetic acid (HEDTA), and GLDA were used to stimulate and remove the damage from different sandstone cores at high temperatures. X-ray Computed Topography (CT) scans were used to determine the effectiveness of these fluids in stimulation calcite and dolomite cores and removing the damage from sandstone cores. The sandstone cores used in this study contain from 1 to 18 wt percent illite (swellable and migratable clay mineral). GLDA was found to be highly effective in creating wormholes over a wide range of pH (1.7-13) in calcite cores. Increasing temperature enhanced the reaction rate, more calcite was dissolved, and larger wormholes were formed for different pH with smaller volumes of GLDA solutions. GLDA has a prolonged activity and leads to a decreased surface spending resulting in face dissolution and therefore acts deeper in the formation. In addition, GLDA was very effective in creating wormholes in the dolomite core as it is a good chelate for magnesium. Coreflood experiments showed that at high pH values (pH =11) GLDA, HEDTA, and EDTA were almost the same in increasing the permeability of both Berea and Bandera sandstone cores. GLDA, HEDTA, and EDTA were compatible with Bandera sandstone cores which contains 10 wt percent Illite. The weight loss from the core was highest in case of HEDTA and lowest in case of GLDA at pH 11. At low pH values (pH =4) 0.6M GLDA performed better than 0.6M HEDTA in the coreflood experiments. The permeability ratio (final/initial) for Bandera sandstone cores was 2 in the case of GLDA and 1.2 in the case of HEDTA at pH of 4 and 300oF. At high pH HEDTA was the best chelating agent to stimulate different sandstone cores, and at low pH GLDA was the best one. For Berea sandstone cores EDTA at high pH of 11 was the best in increasing the permeability of the core at 300oF. The low pH GLDA based fluid has been especially designed for high temperature oil well stimulation in carbonate and sandstone rock. Extensive studies have proved that GLDA effectively created wormholes in carbonate cores, is gentle to most types of casing including Cr-based tubular, has a high thermal stability and gives no unwanted interactions with carbonate or sandstone formations. These unique properties ensure that it can be safely used under extreme conditions for which the current technologies do not give optimal results. Furthermore, this stimulation fluid contributes to a sustainable future as it based on readily biodegradable GLDA that is made from natural and renewable raw material.