O estado do Rio Grande do Sul possui as maiores jazidas de ágata de que se tem conhecimento. Este material gemológico é utilizado para uma gama abrangente de aplicações em joalheria e produtos e neste formato pode elevar significativamente o seu valor de mercado. Cerca de 80% da produção de ágata é destinada à exportação, principalmente para a China e a Alemanha. O centro de beneficiamento de ágata no Brasil, que detém a maior concentração de empresas, está localizado na região de Soledade, no Rio Grande do Sul. Este município é conhecido mundialmente como polo industrial de “pedras preciosas”. Porém as tecnologias de beneficiamento de ágata das indústrias locais são bastante limitadas e com pouca inovação, sendo que o trabalho tem maior foco na comercialização direta, ou seja, em bruto, e em alguns produtos manufaturados de baixa complexidade tecnológica. Esta pesquisa trata da produção de camafeus, que são pequenas esculturas feitas normalmente em ágata e que são utilizadas na forma de broches e adornos, que em composição com ouro e prata podem se transformar em produtos de joalheria. Para tal, é proposto o desenvolvimento de um protótipo de equipamento para a confecção de camafeus, por um sistema de cópia, sendo uma adaptação de uma fresadora pantográfica. Esse protótipo foi projetado para ser utilizado pelos pequenos produtores soledadenses, que contabilizam cerca de 150 empresas cadastradas na APPESOL (Associação dos Pequenos Pedristas de Soledade). A proposta é de uma alternativa técnica de baixo custo para a criação de produtos de alto valor agregado e a inserção de esculturas em ágata feitas no Brasil no mercado interno e externo. A copiadora de camafeus faz uso de ferramentas diamantadas e copia um modelo produzido em outro material. Ensaios iniciais apontam que camafeus em ágata podem ser executados com esta técnica, porém há a possibilidade de inúmeros materiais gemológicos serem beneficiados também. / The state of Rio Grande do Sul has the biggest agate deposits, this gemological material is used for a wide range of applications in jewelry and products, increasing significantly its value on the market. About 80% of agate production is exported mainly to China and Germany. The agate processing center in Brazil which holds the largest concentration of companies is located in Soledade, Rio Grande do Sul. This municipality is known worldwide as the gems industrial center. However, the processing technology of the local companies are quite limited, with little innovation, whereas being focused on the direct marketing, in other words, in the raw state, and in some manufactured products with low technological complexity. This research is about the cameo production, which are small sculptures usually made of agate and used in the form of brooches and ornaments, that in composition with gold and silver turn into jewels. In order to achieve this is through the development of an mechanism prototype to produce the cameos, using a copying system by adapting a pantograph milling machine. This prototype was designed to be used by small producers which account for 150 companies registered at APPESOL (Small Gems Producers Association of Soledade). The proposal is a low – cost alternative technique for creating products and insertion of agate sculptures produced in Brazil’s national and international markets. The cameo copy machine makes use of diamond tools, that copies from a model made of different material. Initial tests show that agate cameos can be produced using this technique and other gemological materials can use this technique as well.

O compartilhamento de músicas pela Internet pode ter efeitos negativos ou positivos sobre a indústria musical. Por um lado, pode limitar a poder de incentivos à criação garantidos por direitos autorais. Por outro lado, contribui para a divulgação de artistas e músicas. Este artigo estima os efeitos de downloads de música sobre o consumo de CDs e shows para uma amostra de 7.147 estudantes universitários. Utilizando variáveis instrumentais motivadas por um modelo teórico simples, associadas à habilidade em informática e tolerância à ilegalidade, estimamos que a prática de downloads de música reduz a probabilidade de consumir CDs em até 45%, ao passo que aumenta a probabilidade de ir a shows em até 35%. Nossos resultados também apontam que existe uma relação de substituição entre pirataria física de CDs e downloads de música. Esse último resultado sugere que as estimativas feitas sem levar em conta o consumo de CDs piratas na equação estrutural podem ser viesadas. / Music file sharing may have positive or negative effects over the music industry. On the one hand, it might undermine incentives for music creation guaranteed by copyrights. On the other hand, it contributes to the dissemination of artists and songs. In this paper we estimate the effect of music downloads over the consumption of CDs and concerts for a sample of 7.147 university students. Using theoretically motivated instrumental variables related to computer skills and illegality acceptance, we estimate that the practice of downloads generates a reduction on the probability of buying CDs of up to 45% and an increase in the order of 35% on the probability of going to concerts. We also show the existence of a substitution relationship between physical CD piracy and music downloads. This last result suggests that estimates without controlling for CD piracy may be biased.

Stochastic fluctuations in mRNA and protein copy number between cells are inevitable during the process gene expression, even when cells carry identical chromosomes. Such fluctuations are able to impact the phenotypic fate of the cell, and are known to have greater impact when the copy number of the molecule involved is low. Additionally, up to 50% of proteins in Escherichia coli are present in the cell at a level of 10 molecules per cell or fewer (Taniguchi et al. 2010). As such, quantification of low copy number gene expression products and their distribution in cellular populations is key in understanding the process of gene expression. Currently, there are few techniques that allow investigation with the single cell and single molecule resolution required to study low copy number gene expression products. This work presents a novel method for protein quantification at the single molecule level, Quantitative HaloTag-TMR labelling, and uses the technique to quantify the absolute numbers of the low copy number RecB, RecC and RecD subunits of the bacterial DNA repair enzyme RecBCD, finding each subunit is present at between two and eight molecules per cell with mean numbers per cell of 4.9, 4.7 and 4.5 respectively. Additionally single molecule mRNA FISH was used to quantify the mRNA levels of recB and recD within cells, with means of 0.21 and 0.31 mRNA per cell being observed respectively. Finally this work presents a new method for use detecting both mRNA and protein simultaneously in individual cells by combining the HaloTag and FISH protocols to give HaloFISH. This work introduces two novel techniques that allow for single cell examination of gene expression, and investigates RecBCD expression at the single molecule level.

Clefts of the lip and/or palate (CL/P) are one of the most common birth defects in the world occurring about every 1 in 700 live births. Individuals with non-syndromic clefting (NSCL/P) account for about 70% of all cleft cases and exhibit a cleft only whereas syndromic occurrences (SCL/P) include additional cognitive or structural abnormalities. Linkage, genome-wide association, candidate gene, animal model, sequencing and copy number variant (CNV) analyses have been used to study CL/P and have established that it is a heterogeneous, complex disorder. However, the impact of identified sequence variants on protein structure and the contribution of structural genetic variation to CL/P remains poorly understood. In our first analysis we reassessed the phenotype of a 30-year-old individual of SCL/P and noticed phenotypic overlap with Hartsfield syndrome, a rare syndrome resulting from sequence variants in Fibroblast growth factor 1 (FGFR1). We sequenced the coding region of FGFR1 and identified a novel, de novo variant. Due to the fact sequence variants in FGFR1 contribute to multiple syndromes encompassing a wide phenotypic spectrum, we performed an extensive literature search to record every published sequence variant of FGFR1 and mapped it to the protein structure by disease and phenotype. Although no statistically significant protein domain-phenotype correlations were identified, many regions neared significance. This work stresses the need for systematic, comprehensive phenotyping of patients and provides a method for assessing the impact of the location of sequence variants within the 3D structure of the protein. Although rare and common CNVs have been identified in individuals with CL/P, prior to our work no large-scale studies of rare CNVs for the identification of novel clefting genes had been performed. For our second set of analyses, we conducted two such studies, first focusing on a smaller cohort of 140 individuals with NSCL/P from the Philippines to establish our informatic and functional validation pipeline. We used whole-genome tiling arrays to assess rare deletions overlapping genes not previously implicated in clefting, and identified one deletion overlapping Isthmin1 (ISM1) and a deletion just 3’ of the gene in a second affected individual. Functional validation of Ism1 in Xenopus laevis showed strong expression in structures necessary for craniofacial development, and morpholino and CRISPR/Cas9 knockdown of Ism1 resulted in a median cleft lip in some embryos, establishing ISM1 as a novel craniofacial patterning gene. We then expanded our study and assessed genomic CNVs in 1021 individuals with NSCL/P and 81 individuals with SCL/P, finding no differences in CNV number, load or burden between these groups. We also identified 8 putative clefting genes overlapped by deletions in two or more individuals but at a rare (< 1% frequency) in the cohort. Functional validation of these genes using CRISPR/Cas9 in zebrafish and Xenopus tropicalis is currently underway. This work has identified a novel sequence variant leading to the diagnosis of Hartsfield syndrome in an individual with SCL/P, developed an innovative method for assessing the impact of sequence variation on protein structure, improved our understanding of the contribution of CNVs to SCL/P and NSCL/P and identified several putative novel clefting loci which may help explain a portion of the missing heritability of CL/P.

In recent years, microarray technology has revealed the widespread presence of submicroscopic deletions and duplications throughout the human genome termed copy number variants (CNVs). CNVs have a profound effect on gene expression and are an important source of normal genetic variation. In addition, a small proportion of CNVs contribute to genetically simple and complex disease. This thesis focuses on the identification of pathogenic CNVs contributing to the etiology of diseases with "missing heritability" using a well-planned study design individually tailored to each disease cohort to optimize CNV detection and interpretation. We performed a genome-wide analysis for CNVs in five disease cohorts with genetic etiology: autism, age-related macular degeneration (AMD), glaucoma, clubfoot, and Bardet-Biedl syndrome (BBS). Our results indicate that CNVs likely account for a proportion of cases for each disease cohort reported in this thesis. Approximately 20% of our cohort of individuals with autism from trio pedigrees harbors a CNV known to confer risk to develop autism and we identified other novel and rare variants that may play a role in autism pathogenesis. We also characterized a duplication of 2p25.3 identified in two male half-siblings with autism and determined that their mother was somatic mosaic for the duplication. Our work provides evidence that this novel CNV disrupting the genes PXDN and MYT1L are the autism-causing mutation in this pedigree. A comparative cases experimental design was used in the study of AMD and glaucoma. While no common "risk CNVs" were identified for either eye disorder, we did identify several rare overlapping CNVs disrupting genes known to play a role in the eye that may confer risk to disease in a small proportion of individuals. In a fourth genetically complex disease, clubfoot, we identified a duplication of 17q23.2 disrupting the genes TBX4, NACA2, and BRIP1 that segregates with the autosomal dominant clubfoot phenotype in a large pedigree with 16 affected individuals. In addition, the duplication is within the linkage interval identified for this family. We also applied microarray technology to analyze the genomes of individuals with BBS, an autosomal recessive disorder, for the presence of CNVs in known BBS genes as well as CNVs that elucidate novel candidate genes for BBS. From 34 BBS patients with an unidentified mutation, we observed one CNV, a heterozygous deletion of BBS10, unmasking a BBS10 frameshift mutation. A promising BBS candidate gene also emerged from our studies, implicated by an intragenic deletion of the gene MARK3 predicted to result in a frameshift and premature truncation of the protein. Functional studies utilizing antisense morpholino gene knockdown in the zebrafish provide additional evidence that MARK3 is a BBS gene as knockdown of zebrafish mark3 results in a Kupffer's Vesicle defect and a melanosome transport delay, two cardinal BBS phenotypes in the zebrafish. In addition to identifying CNVs involved in disease, the work outlined in this thesis provides valuable insight into the study design and interpretation of a genome-wide analysis of CNV. This includes the appropriate use of controls and publicly available control databases, methods for enriching for CNVs in a patient cohort to maximize efficiency and discovery, and the importance of analyzing all patient cohorts with heritable disease for the presence of CNVs disrupting known disease genes and CNVs that implicate novel genetic candidates. As the reliability and resolution of CNV detection continue to improve, allowing detection of > 1,000 CNVs in each individual genome, it becomes more important than ever to have a well-defined study design for both the detection and interpretation of CNVs.

Genom tilltal och omtal med olika personliga pronomen kategoriserar och definierar vi oss själva och andra, och förstår på så sätt vårt sociala sammanhang. Inom reklamen, som i högsta grad handlar om identiteter, finns trots detta ett förvånansvärt litet intresse av pronomen, varför denna studie har som ambition att undersöka just detta. Vilket pronomen bör användas i vilket sammanhang? Med avseende på mottagaren undersöker studien ett du-tilltal, ett ni-tilltal och ett tilltal utan pronomen. Med avseende på avsändaren undersöker studien ett vi-omtal och ett omtal utan pronomen. Genom en enkätundersökning och intervjuer fastställs slutsatsen att reklamskribentens val av pronomen bör baseras på en god kännedom om målgrupp, produktgrupp och varumärket ifråga. Som tilltal för mottagaren bör ett du användas, eller inget pronomen alls. Du:et bör användas när annonsen ska spegla en individuell köp- eller användarupplevelse, när produkten är dyr och exklusiv eller när mottagaren ska lyftas fram ur mängden. Att utelämna pronomen kan istället passa när du:et upplevs som alltför påträngande. Studien kommer också fram till att ett ni-tilltal helst bör undvikas. Vad gäller omtalspronomen för avsändaren framkommer att det klassiska vi:et både kan och bör uteslutas i vissa situationer. Vi:et upplevs peka på en avlägsen men mänsklig relation där mottagaren till viss del känner sig exkluderad. Att istället utesluta omtalspronomen leder till att mottagaren känner sig stå närmare avsändaren vars budskap också upplevs som sannare.

The 16p11.2 copy-number variant (CNV) represents a well-characterized, high-risk factor for autism spectrum disorder that additionally predisposes deletion carriers (16pdel) to increased head circumference, known as macrocephaly. The 16p11.2 CNV consists of 29 known genes, many of which are associated with neurobiological processes relevant for macrocephaly such as cell proliferation and apoptosis, differentiation and cell growth. Our lab’s previous work has demonstrated that induced pluripotent stem cell (iPSC)-derived neurons from 16pdel carriers show altered cellular morphology related to growth, which include increased soma size, total dendritic length and dendritic complexity. However, specific CNV genes responsible for these phenotypes have not been established. Here, we investigate the relationship between three 16p11.2 genes and the observed cellular phenotypes. We differentiated neurons from control iPSC-derived neural progenitor cells (NPCs) and used short hairpin RNA (shRNA) to reduce the expression of these CNV genes: KCTD13, MAPK3 and C16ORF53. We then assessed neuronal morphology by evaluating soma size, total dendritic length and dendritic complexity. We demonstrate that knocking down KCTD13 and C16ORF53 increases soma size and total dendrite length, respectively, similar to that observed in 16pdel iPSC-derived neurons. For this reason, we speculate that these genes may have a role in cell growth and might underlie macrocephaly. Thus, our study investigates genes in the 16p11.2 CNV that contribute to neuronal morphology, which may have a role in influencing brain size.

Perception and action are coupled via bidirectional relationships between sensory and motor systems. Motor systems influence sensory areas by imparting a feedforward influence on sensory processing termed “motor efference copy†(MEC). MEC is suggested to occur in humans because speech preparation and production modulate neural measures of auditory cortical activity. However, it is not known if MEC can affect auditory perception. We tested the hypothesis that during speech preparation auditory thresholds will increase relative to a control condition, and that the increase would be most evident for frequencies that match the upcoming vocal response. Participants performed trials in a speech condition that contained a visual cue indicating a vocal response to prepare (one of two frequencies), followed by a go signal to speak. To determine threshold shifts, voice-matched or -mismatched pure tones were presented at one of three time points between the cue and target. The control condition was the same except the visual cues did not specify a response and subjects did not speak. For each participant, we measured f0 thresholds in isolation from the task in order to establish baselines. Results indicated that auditory thresholds were highest during speech preparation, relative to baselines and a non-speech control condition, especially at suprathreshold levels. Thresholds for tones that matched the frequency of planned responses gradually increased over time, but sharply declined for the mismatched tones shortly before targets. Findings support the hypothesis that MEC influences auditory perception by modulating thresholds during speech preparation, with some specificity relative to the planned response. The threshold increase in tasks vs. baseline may reflect attentional demands of the tasks. / acase@tulane.edu

Glaucoma is the second leading cause of irreversible blindness in the United States and is the leading cause of blindness in African Americans. Cupping or excavation of the optic nerve, which sends the visual signal from the photoreceptors in the eye to the brain, is a chief feature of glaucoma. A similar excavated appearance of the optic nerve is also the primary clinical sign of other congenital malformations of the eye including optic nerve head coloboma, optic pit, and morning glory disc anomaly collectively termed cavitary optic disc anomaly (CODA). Clinical similarities between CODA and glaucoma have suggested that these conditions may have overlapping pathophysiology. Although risk factors are known, such as the elevated intraocular pressure (IOP) observed in some glaucoma subjects, the biological pathways and molecular events that lead to excavation of the optic disc in glaucoma and in CODA are incompletely understood, which has hindered efforts to improve diagnosis and treatment of these diseases. Consequently, there is a critical need to clarify the biological mechanisms that lead to excavation of the optic nerve, which will lead to improvements in our understanding of these important disease processes. Because of their similar clinical phenotypes and the limited therapy geared at lowering IOP in glaucoma patients, our central hypothesis is that genes involved in Mendelian forms of CODA would also be involved in a subset of glaucoma cases and may provide insight into glaucomatous optic neuropathy. The purpose of my research project has been to identify and functionally characterize the gene that causes congenital autosomal dominant CODA in a multiplex family with 17 affected members. The gene that causes CODA was previously mapped to chromosome 12q14 and following screening of candidate genes within the region that did not yield any plausible coding sequence mutations, a triplication of a 6KB segment of DNA upstream of the matrix metalloproteinase 19 (MMP19) gene was subsequently identified using comparative genomic hybridization arrays and qPCR. This copy number variation (CNV) was present in all affected family members but absent in unaffected family members, a panel of 78 normal control subjects, and the Database of Genomic Variants. In a case-control study of singleton CODA subjects, CNVs were also detected; we detected the same 6KB triplication in 1 of 24 subjects screened. This subject was part of another 3-generation autosomal dominant CODA pedigree where affected members each have the same CNV identified in the larger CODA pedigree. A separate case-control study with 172 glaucoma cases (primary open angle glaucoma = 84, normal tension glaucoma = 88) was evaluated for MMP19 CNVs, however none were detected. Although our cohort of CODA patients is small limiting our ability to accurately determine the proportion of CODA caused by MMP19 mutations, our data indicates that the MMP19 CNV is not an isolated case and additional CODA subjects may have MMP19 defects. Because of the location of the CNV, we evaluated its effect on downstream gene expression with luciferase reporter gene assays. These assays revealed that the 6KB sequence spanned by the CNV in CODA subjects functioned as a transcriptional enhancer; in particular, a 773bp segment had a strong positive influence (8-fold higher) on downstream gene expression. MMP19, a largely understudied gene, was further characterized by expression studies in the optic nerve and retina. Using frozen sections from normal donor eyes, we demonstrated that MMP19 is predominantly localized to the optic nerve head in the lamina cribrosa region with moderate labeling in the postlaminar region, and weak labeling in the prelaminar region and retina. We also evaluated MMP19 expression in relation to the cell types that populate the optic nerve such as astrocytes and retinal ganglion cells. The pattern of expression is consistent with MMP19 being a secreted protein accumulating in the extracellular spaces and basement membranes of the optic nerve. Our studies have identified the first gene associated with CODA and future research is focused on recapitulating CODA phenotypes in animal models and assessing the mechanism of MMP19 involvement during development.

Malignant glioma have poor prognosis resulting mainly from high level of cell proliferation and invasion and resistance to conventional therapy. Identification of novel targets that are critical elements in gliomagenesis may help improve therapeutic outcome. Using genome-wide explorations of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations in high grade glioma that correlates to patients' outcomes. Our analysis revealed for the first time NOTCH3 as one of the most significant gene amplifications mapped to chromosome 19. This amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH signaling pathway is essential for cell proliferation, stem cell maintenance and differentiation and its deregulation has been reported in several human cancers. NOTCHs are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in gliomagenesis and glioma drug resistance. Our objective is to determine NOTCH3 molecular roles in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a role in glioma cell proliferation, cell migration, invasion and apoptosis. We also found a NOTCH3 glioma addiction phenomenon. Therefore, our study uncovers, for the first time, the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allow the identification of a subset of population that may benefit from GSI-based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized medicine.