Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thesis advisor: Charles S. Hoffman / Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Estudos demonstram que a indução do estresse oxidativo pelo peptídeo beta amiloide (A?) exerce um importante papel no desencadeamento da excitotoxicidade neuronal o que pode resultar no desenvolvimento de doenças neurodegenerativas. A formação do peptídeo A? se deve a alterações na proteína precursora de amiloide (APP) que é clivada para a formação do peptídeo A?. Por sua vez, os mecanismos de ação do A? no S.N.C. ocorrem através da sinalização do receptor NMDA (N-metil D-aspartato) receptor este que quando ativado pelo glutamato exerce importante papel fisiológico no S.N.C., visto que apresenta atividade ionotrópica que permite o influxo de Na+ e Ca2+ para as células neuronais, auxiliando nos processos de formação da memória e aprendizagem. Entretanto, apesar do seu papel fisiológico, a ativação excessiva do receptor NMDA é fortemente correlacionada com lesões no S.N.C. decorrente da excessiva permeabilidade do íon Ca2+ para o citosol das células neuronais. Com isso as concentrações de glutamato na fenda sináptica são estritamente controladas para que não haja ativação excessiva dos receptores com atividade glutamatérgica, como o receptor NMDA. Estudos indicam que o transporte de glutamina/glutamato através da barreira hematoencefálica é menor do que de outros aminoácidos, sendo que cerca de 25% a 30% do transporte de aminoácidos dos vasos sanguíneos para o cérebro através da barreira hematoencefálica é ocupado pelo aminoácido leucina, sendo este um grande responsável pela síntese de glutamato/glutamina no S.N.C. Com isso, estudos tem demonstrado que dietas enriquecidas com aminoácidos de cadeia ramificada, dentre eles a leucina, é responsável por alterar o metabolismo do glutamato e aumentar a susceptibilidade à excitotoxicidade de células neurais. A fim de testar esta hipótese utilizamos um modelo de cultura de células de neuroblastoma humano e realizamos o tratamento com diferentes concentrações de aminoácido leucina associado com o tratamento de peptídeo beta-amilóide. Realizamos as analises de citotoxicidade (LDH), viabilidade celular (MTT) e apoptose celular por citometria de fluxo (marcação com PE Anexina V e 7-AAD). Nossos resultados indicam que houve diferenças apenas entre o controle em relação aos demais grupos de tratamento / Studies demonstrate that induction of oxidative stress by beta amyloid peptide (A?) plays an important role in triggering neuronal excitotoxicity which can result in the development of neurodegenerative diseases. The formation of A? peptide are due to changes in the amyloid precursor protein (APP) which is cleaved to form the peptide A?. On the other hand, the mechanisms of action of A? in the C.N.S. occur through signaling of the NMDA (N-methyl-D-aspartate) receptor that when activated by glutamate plays an important physiological role in the C.N.S., as has inotropic activity that allows the influx of Na+ and Ca2+ into the neuronal cells, assisting in procedures of memory formation and learning. However, despite its physiological role, the excessive activation of the NMDA receptor is strongly correlated with C.N.S. lesions due to excess permeability of Ca2+ ions into the cytosol of neuronal cells. Thus the concentrations of glutamate in the synaptic cleft are strictly controlled so that there is excessive activation of receptors with glutamatergic activity, as the NMDA receptor. Studies indicate that the transport of glutamine/glutamate across the blood brain barrier is lower than that of other amino acids, of which about 25% to 30% of the amino acid transport blood vessels to the brain through the blood brain barrier is occupied by leucine this being one largely responsible for the synthesis of glutamate/glutamine in the C.N.S. Thus, studies have shown that diets enriched in branched chain amino acids, including leucine, are responsible for altering the metabolism of glutamate and excitotoxic increase susceptibility to neural cells. To test this hypothesis we used a cell culture model of human neuroblastoma and carry out the treatment with different concentrations of leucine associated with the processing of amyloid-beta peptide. We performed analysis of cytotoxicity (LDH), cell viability (MTT assay) and apoptosis using flow cytometry (Annexin V staining with PE and 7-AAD). Our results indicate that there were differences only between the control compared to the other treatment groups

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

Neurofibromin (NF1) is a 2,818aa protein encoded by the very large NF1 tumour suppressor gene located on chromosome 17q11.2. Loss of function mutations and deletions in NF1 underlie Neurofibromatosis type-1 (NF-1) - the most common inherited syndrome of the nervous system in humans with a birth incidence of 1:3,000. The most visible feature of NF-1 is the neoplastic manifestations known as neurofibromas, however, the syndrome is also characterized by pigmentary defects, peripheral motor dysfunction, learning disabilities and several developmental abnormalities. The molecular etiology of many of these non-neoplastic phenotypes remains unknown. Here we demonstrate that the Tubulin Binding Domain (TBD) of NF1 is a binding partner of the Leucine Rich Pentatrico Peptide Repeat motif-Containing protein (LRPPRC) and cytoplasmic Dynein Heavy Chain (DHC). The NF1-LRPPRC interaction is of high significance as it links NF-1 with Leigh’s Syndrome, French Canadian variant (LSFC) – an autosomal recessive neurodegenerative disorder that arises due to mutations in the LRPPRC gene. This interaction occurs as part of an RNA granule complex, and use of transgenic mouse models establishes an important role of NF1 and LRPPRC in peripheral nerve development. The NF1-DHC interaction is of importance in melanocytes where our studies suggest a possible role in melanosome localization, disruptions in which may underlie the abnormal pigmentary features known as café-au-lait macules that are commonly associated with NF-1. The validation of LRPPRC and DHC as novel NF1 interactors reveal new roles of NF1, which open the door to better understanding the molecular mechanisms that underlie the myriad of NF-1 manifestations.

Neurofibromin (NF1) is a 2,818aa protein encoded by the very large NF1 tumour suppressor gene located on chromosome 17q11.2. Loss of function mutations and deletions in NF1 underlie Neurofibromatosis type-1 (NF-1) - the most common inherited syndrome of the nervous system in humans with a birth incidence of 1:3,000. The most visible feature of NF-1 is the neoplastic manifestations known as neurofibromas, however, the syndrome is also characterized by pigmentary defects, peripheral motor dysfunction, learning disabilities and several developmental abnormalities. The molecular etiology of many of these non-neoplastic phenotypes remains unknown. Here we demonstrate that the Tubulin Binding Domain (TBD) of NF1 is a binding partner of the Leucine Rich Pentatrico Peptide Repeat motif-Containing protein (LRPPRC) and cytoplasmic Dynein Heavy Chain (DHC). The NF1-LRPPRC interaction is of high significance as it links NF-1 with Leigh’s Syndrome, French Canadian variant (LSFC) – an autosomal recessive neurodegenerative disorder that arises due to mutations in the LRPPRC gene. This interaction occurs as part of an RNA granule complex, and use of transgenic mouse models establishes an important role of NF1 and LRPPRC in peripheral nerve development. The NF1-DHC interaction is of importance in melanocytes where our studies suggest a possible role in melanosome localization, disruptions in which may underlie the abnormal pigmentary features known as café-au-lait macules that are commonly associated with NF-1. The validation of LRPPRC and DHC as novel NF1 interactors reveal new roles of NF1, which open the door to better understanding the molecular mechanisms that underlie the myriad of NF-1 manifestations.

Leucine is a regulator of protein metabolism in vivo, and there is little information regarding its action on cardiac hypertrophy induced by experimental hyperthyroidism and its relationship to serum creatine kinase. The study aimed to verify the effect of leucine in cardiac hypertrophy and serum creatine kinase in rats with hyperthyroidism. 75 animals were used, divided into two large groups according to the length of the experiment, seven days Group (7D) and Group twenty-eight days (D 28), subsequently divided into five subgroups these being the control zero (C0-7 and C028 ) controls (C-7 and C-28), hormone (H-7 and H- 28), leucine (G-7 and G-28) and leucine + hormone (LH-7 and HL-28). Hyperthyroidism was induced by administration of daily 20&#956;g/100 grams of levothyroxine sodium in aqueous suspension by gavage. Leucine was supplemented by adding 5% of the amino conventional diet. Blood was collected by cardiac puncture and analyzes made in kits for TSH, T3, T4 and CK-NAC CK-MB. At the end of the experiment the heart was removed and weighed. Subsequently, the left ventricle was separated together with the interventricular septum and heavy, was also performed to measure the transverse diameter of cardiomyocytes and compared between groups. The exercise tolerance was measured by the swim test and the intensity was determined in 7% of the weight of the animal. Blood pressure and heart rate were measured using a sphygmomanometer to rats tail, 4/25T ADInstruments PowerLab equipment ® and ® software ADInstruments LabChart 7. In statistical comparison was used analysis of variance (ANOVA) and two-way post-Tukey test, considering p values <0.05. In rats treated with thyroid hormone occurs, cardiac hypertrophy with increased weight of the left ventricle, increased heart rate and elevated concentrations of CK-MB after 28 days. The association of leucine seems to modulate hormone-induced cardiac hypertrophy in this experimental model, and reduce blood concentrations of CK-NAC and CK-MB by unknown mechanisms. Thyroxine increases the swimming performance of rats after therapy for 14 and 21 days but with performance drop in 28 days. / A leucina é um regulador do metabolismo proteico in vivo, e existem poucas informações referentes à sua ação na hipertrofia cardíaca induzida pelo hipertireoidismo experimental e sua relação com a creatina quinase sérica. O estudo teve por objetivo verificar a ação da leucina na hipertrofia cardíaca e na concentração sérica de creatina quinase em ratos Wistar em hipertireoidismo. Foram utilizados 75 animais, divididos dois grandes grupos de acordo com o tempo de experimento, Grupo sete dias (7D) e Grupo vinte e oito dias (28 D), posteriormente divididos em cinco subgrupos sendo estes o controle zero (C0-7 e C028) controle (C-7 e C-28), hormônio (H-7 e H-28), leucina (L-7 e L-28) e hormônio + leucina (HL-7 e HL-28). O hipertireoidismo foi induzido administrando-se, diariamente, 20&#956;g/100 gramas de levotiroxina sódica em suspensão aquosa, por gavagem. A leucina foi suplementada adicionando-se 5% do aminoácido à ração convencional. O sangue foi coletado por punção cardíaca e as análises feitas em kits para TSH, T3, T4 CK-NAC e CK-MB. Ao final do período experimental o coração foi removido e pesado. Posteriormente, foi separado o ventrículo esquerdo juntamente com o septo interventricular e pesado, também foi realizada a medida do diâmetro transversal dos cardiomiócitos e os resultados comparados entre os grupos. A tolerância ao esforço foi medida através do teste de natação e a intensidade foi determinada em 7% do peso do animal. A pressão arterial e frequência cardíaca foram aferidas utilizando um esfignomanômetro de cauda para ratos, o equipamento PowerLab 4/25T ADInstruments® e o software LabChart 7 ADInstruments®. Na comparação estatística foi utilizada a análise de variância (ANOVA) de duas vias e pós-teste de Tukey, considerando-se significativos valores de p<0,05. Em ratos tratados com hormônio tireoidiano ocorre, hipertrofia cardíaca com aumento do peso do ventrículo esquerdo, aumento da frequência cardíaca e elevação das concentrações de CK-MB após 28 dias. A associação de leucina ao hormônio parece modular a hipertrofia cardíaca induzida neste modelo experimental, além de reduzir as concentrações sanguíneas de CK-NAC e CK-MB por mecanismos ainda desconhecidos. A tiroxina aumenta o desempenho da natação de ratos após uma terapia de 14 e 21 dias, porém com queda de performance em 28 dias. / Mestre em Ciências da Saúde

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