This thesis examines the role of root-sourced abscisic acid in the regulation of stomatal closure and leaf expansion in response to drying approximately half of the roots of Ricinus communis L. plants. Drying part of the root system of Ricinus communis promoted stomatal closure and slowed leaf expansion in the absence of any disturbances in shoot water relations, implying the involvement of chemical rather than hydraulic signalling. Initially root-sourced ABA was believed to be responsible for these responses. Delivery rates (concentration x flow rate) of ABA out of the drying roots were calculated which took into account the effect of dilution on solutes in the well-watered and droughted plants due to different transpiration rates in these plants. The delivery term was further modified to account for the differences in sizes of their roots and shoots. ABA delivery out of the roots of plants with drying upper roots increased within the first 12 h and was maintained over the next 3 days. However, significant decline in stomatal apertures and leaf elongation occurred only 2 - 3 days after root drying began. During the early stages of drying upper roots (2-3 d) xylem sap pH, and delivery rate of nitrate and l-aminocyclopropane-l-carboxylic acid were little changed, while hydraulic conductivity of the root system as a whole was reduced approximately 25%, and ABA accumulation (synthesis?) in roots increased. Increased ABA levels in phloem sap was not found, suggesting no enhanced re-cycling of ABA between shoots and roots was taking place in the plants during this time. Antitranspirant activity in xylem sap of droughted plants that was not ABA was sought as a possible cause of stomatal closure. However, convincing evidence of such activity was not found. Examination of ABA output by roots into shoot compared to that entering lamina of the 5th leaf in the canopy showed the attenuation of the signal as transpiration fluid moved up the plant. These obs~f\ations indicate that ABA from roots is unlikely to be a highly active signal eliciting shoot responses to mild drought in Ricinus communis.

Ras-MAPK signalling regulates key cellular processes such as proliferation, differentiation and survival. Unsurprisingly, mutations in RAS genes are now recognized as potent oncogenic drivers. However, disruption of this pathway during development is associated with a family of disorders termed the Rasopathies. Shared clinical features include cutaneous, neurological and cardiac anomalies. At the outset of this study, the genetic etiology of three neurocutaneous disorders, microcephaly-capillary malformation syndrome (MIC-CAP), encephalocraniocutaneous lipomatosis (ECCL) and PHACE (Posterior fossa malformations, facial Hemangiomas, cerebral Arterial anomalies, Cardiovascular defects and Eye abnormalities) syndrome had not yet been established. This thesis identifies mutations in STAM-binding protein (STAMBP) in a cohort of individuals with MIC-CAP syndrome using whole-exome sequencing (WES). This gene encodes a deubiquitinating isopeptidase that regulates cell surface receptor-mediated endocytosis and sorting. Cell lines of individuals with MIC-CAP show reduced STAMBP expression, associated with accumulation of ubiquitinated protein aggregates, increased apoptosis and constitutive activation of the Ras-MAPK and PI3K-AKT pathways. WES also enabled the identification of post-zygotic mutations within the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) in individuals with ECCL. Fibroblasts from affected individuals showed increased phosphorylation of the FGFRs consistent with receptor activation as well as insensitive signal transduction through the Ras-MAPK pathway. Neurocutaneous syndromes can feature striking vascular lesions such as the cerebral vasculopathy and large segmented facials hemangiomas seen in PHACE syndrome. The asymmetric and patchy vascular malformations coupled with a sporadic incidence and absence of familial recurrence suggested that PHACE might be caused by post-zygotic mutations. Interrogation of a discordant sib-pair using copy number analysis and WES did not identify causative mutations indicating the need for a comprehensive and targeted –omic approach to elucidate the molecular mechanism of this syndrome. Taken together, these findings expand the spectrum of the Rasopathies while providing novel pathomechanistic insights into the regulation of cellular proliferation and survival during development.

We recently reported that intestinal-specific overexpression of the POZ-ZF transcription factor Kaiso produced two prominent phenotypes in 1-year old mice: Kaiso transgenic (KaisoTg) mice presented with chronic intestinal inflammation, and an increase in secretory cell types – a trait typical of Notch signalling inhibition. Despite these findings however, the factor(s) responsible for Kaiso-mediated inflammation and secretory cell increases had not been elucidated. The primary goal of this thesis was to begin filling in this knowledge gap, by shedding mechanistic insight on Kaiso’s role in governing these two prominent phenotypes. First, we elucidated Kaiso’s role in the Notch signalling pathway and found that Kaiso inhibited the expression of the Notch1 receptor, and its ligand Dll-1, but promoted the expression of the Jagged-1 ligand. We postulated that the Kaiso-mediated reduction in Dll-1 might be responsible for the increase in secretory cell types, whereas Kaiso-mediated regulation of Jagged-1, which is dispensable for cell fate decisions, may be implicated in colon cancer progression. Importantly, we also found that Kaiso’s effects on Notch pathway inhibition occurred prior to the onset of chronic intestinal inflammation. Our analyses of the chronic inflammatory phenotype in KaisoTg mice demonstrated that Kaiso overexpression drives pathogenic neutrophil-specific recruitment (as evidenced by increases in neutrophil-specific enzymatic activity, the formation of crypt abscesses, and augmented expression levels of the neutrophils-specific chemokine, MIP2); an increase in the pore-forming Claudin-2; reduction of the cell adhesion protein E-cadherin; and abnormal intestinal epithelial repair mechanisms. Together, these findings imply that the pathogenesis of Kaiso-mediated intestinal inflammation is a multi-factorial process. A secondary goal of this thesis was to initiate studies to elucidate how the Kaiso binding partner, Znf131, might play a role Kaiso-mediated transcriptional regulation. We found that Znf131 indirectly associated with several Kaiso target genes, including Cyclin D1 (CCND1). Importantly, Znf131 activated a minimal CCND1 promoter previously shown to be inhibited by Kaiso. Moreover, Kaiso overexpression attenuated Znf131-mediated transcriptional activation and Znf131 expression in intestinal cells. Together, these findings hint that Znf131 and Kaiso may exert opposing biological functions, which may have implications in Kaiso-mediated intestinal homeostasis and disease. / Thesis / Doctor of Philosophy (PhD)

Yes / A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis. / MRC program grant U1175.01.005.00005.01 from RSC and MRC centre grant G0801130 from JAC. / Erratum: 18 Apr 2016: Penedos A, Johnson AL, Strong E, Goldman AS et al (2016) Correction: Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1. PLOS ONE. 11(4): e0154170. https://doi.org/10.1371/journal.pone.0154170.

Yes / In the section “Generation of phospho-specific Hop1 antibodies” of the Materials and Methods, we made several mistakes when indicating the sequence of the peptides used for the generation of antibodies. / Erratum: Penedos A, Johnson AL, Strong E, Goldman AS, Carballo JA, Cha RS (2015) Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1. PLoS ONE 10(7): e0134297. doi: 10.1371/ journal.pone.0134297

Orientador: Cassio Eduardo Lima de Paiva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo / Made available in DSpace on 2018-08-21T11:29:47Z (GMT). No. of bitstreams: 1 Pestana_FernandoAugustoBaptistini_D.pdf: 7431865 bytes, checksum: 756c064e760b74577c3323d4eb32af83 (MD5) Previous issue date: 2012 / Resumo: A sinalização horizontal realizada com tintas é utilizada na grande maioria dos municípios brasileiros, os quais, devido às exigências do Código de Trânsito Brasileiro devem possuir organismo próprio para o gerenciamento do trânsito em sua circunscrição. Os municípios brasileiros, principalmente aqueles de médio e pequeno porte, não possuem equipe técnica especializada nem tampouco ferramentas adequadas para a realização do correto gerenciamento da sinalização horizontal urbana. Este trabalho apresenta uma análise geral do quesito durabilidade da sinalização horizontal, abordando o cenário internacional e o nacional; apresenta os critérios utilizados por diversos organismos rodoviários e instituições nacionais e internacionais, bem como pesquisas científicas atuais sobre o quesito durabilidade da sinalização. Em função das informações levantadas, foi proposto e avaliado um método visual para análise do desgaste da sinalização urbana. Apresenta o estudo de um caso real de controle de durabilidade de sinalização urbana a partir do método proposto, em alguns locais do município de Sumaré - SP / Abstract: Pavement markings are used in most of the Brazilian cities, which, due to the demands of the Brazilian Traffic Code need to have their own agencies for traffic management. The cities, especially those which are medium and small in size, do not have adequate technical experts and appropriate tools to manage the urban pavement markings correctly. The aim of this thesis is to present an overview of pavement markings durability, addressing the national and international scene. The first part of the research was the analysis of the different criteria used by various road agencies, national and international institutions, as well as updated scientific research on durability of pavement markings. According to the information gathered, it was proposed and evaluated a visual method for the analysis of the worn down pavement markings. This study presents a real case-control durability of urban pavement markings with paints from the proposed visual method in some places of Sumaré - SP city / Doutorado / Edificações / Doutor em Engenharia Civil

P53 is a tetrameric transcription factor which exhibits DNA binding activity through its core domain which encompasses the conserved domains (known as Box II, III, IV and V). The N-terminal domain of p53 provides a scaffold for binding of components of the transcriptional machinery. Phosphorylation at residues within the N-terminal transactivation domain of p53 such as Serine 20 is a crucial event in the activation of p53. It stabilises the binding of the co-activator p300, reduces the binding of the inhibitory partner Mdm2 and enhances activation of p53 target genes. The identification of enzymes that phosphorylate p53 transactivation domain is an important development in the ongoing mapping of signaling pathways that control p53-dependent transcription and resultant tumour suppression. Environmental and physiological stresses activate p53 which has led to the creation of several hypothetical models in which tumour suppressor kinases mediate p53 activation by phosphorylation at Serine 20. Although much researched the identity of the main Serine 20 kinase in cells remains undefined. In this study we have identified Calcium Calmodulin kinase superfamily (CAMK) members as potent Serine 20 kinases in cells and show that the co-transfection of p53 peptides derived from the conserved domains can modify this response. Moreover, we show that the multi-protein docking site, p53 Box V domain, is required for Serine 20 phosphorylation and ubiquitination of p53. To further define the domains required for the interaction of p53 with CAMK superfamily members, mutagenesis of p53 was performed. Using transcriptional and binding based assays we were able to establish that p53 does indeed form an interaction with Chk1 and DAPK1. Development of cell models and gene expression studies demonstrated that depletion of Chk1 and DAPK1 results in activation of the p53 signalling pathway. There may therefore be a role for kinases as negative regulators of p53 and a potential for the development of kinases as drug targets for reactivation of the p53 pathway.

A mis-sense point mutation in the human VAPB gene is associated with a familial form of motor neuron disease that has been classified as Amyotrophic Lateral Sclerosis type VIII. Affected individuals suffer from a spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or an atypical slowly progressing form of ALS. Mammals have two homologous VAP genes, vapA and vapB. VAPA and VAPB share 76% similar or identical amino acid residues; both are COOHterminally anchored membrane proteins enriched on the endoplasmic reticulum. Several functions have been ascribed to VAP proteins including membrane trafficking, cytoskeleton association and membrane docking interactions for cytoplasmic factors. It is shown here that VAPA and VAPB are expressed in tissues throughout the body but at different levels, and that they are present in overlapping but distinct regions of the endoplasmic reticulum. The disease-associated mutation in VAPB, VAPB (P56S) is within a highly conserved N-terminal region of the protein that shares extensive structural homology with the major sperm protein (MSP) from nematodes. The MSP domain of VAPA and VAPB is found to interact with the ERlocalized transcription factor ATF6. Over expression of VAPB or VAPB (P56S) attenuates the activity of ATF6-regulated transcription and the mutant protein VAPB (P56S) appears to be a more potent inhibitor of ATF6 activity. Moreover VAP proteins affect the activity of XBP1 and BiP promoter elements, two major components of the Unfolded Protein Response (UPR) of the Endoplasmic Reticulum and the different domains of VAPB have a differential effect on UPR regulation. Finally, over expression of the MSP domain of VAPB leads to cell death via apoptosis, while overexpression of other VAPB domains renders cells more susceptible to apoptotic death after ER stress. The data presented in this thesis indicate that VAP proteins interact directly with components of ER homeostatic and stress signalling systems and may therefore be parts of a previously unidentified regulatory pathway. The mis-function of such regulatory systems may contribute to the pathological mechanisms of degenerative motor neuron disease.

No description available.

No description available.