Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

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Made available in DSpace on 2016-04-01T17:55:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-04. Added 1 bitstream(s) on 2016-04-01T18:00:45Z : No. of bitstreams: 1 000860186_20160804.pdf: 237722 bytes, checksum: 99252673c7b9a303744a1706fa82e67b (MD5) Bitstreams deleted on 2016-08-05T12:20:24Z: 000860186_20160804.pdf,. Added 1 bitstream(s) on 2016-08-05T12:20:59Z : No. of bitstreams: 1 000860186.pdf: 1059222 bytes, checksum: e6bebf67ecb2875c260948e57fba3979 (MD5) / A produção de laranja e de suco concentrado é uma atividade agrícola que tem uma grande importância para o Brasil, em especial para o estado de São Paulo. Entretanto, o cancro cítrico é uma doença que causa grandes prejuízos à citricultura brasileira e mundial, sendo que até o momento não há nenhum método curativo para esta doença e o principal controle é a erradicação das plantas contaminadas. O cancro cítrico é originário da Ásia e tem como agente causal a bactéria Xanthomonas citri subsp. citri (Xac), a qual ataca todas as espécies comerciais de citros. Em um estudo de expressão gênica em dois mutantes de Xac que apresentavam diminuição ou ausência total de sintomas de cancro cítrico, a proteína XAC1008 se mostrou altamente expressa, pelo que o objetivo deste trabalho foi a caracterização funcional da ORF XAC1008 de Xac. A estratégia utilizada foi a produção de um mutante desta ORF e avaliação da sua patogenicidade quando comparada com a do isolado 306 de Xac selvagem em plantas de limão 'Cravo' (Citrus limonia Osbek) e laranja Pêra (Citrus sinensis (L.) Osbeck). A técnica utilizada para a obtenção do mutante foi a da mutagênese sítio-dirigida por PCR utilizando o vetor suicida pOK1, seguida de recombinação homóloga. Os ensaios de patogenicidade do mutante ΔXAC1008 mostraram que a mutação alterou a coloração e o formato das colônias e impediu a multiplicação da bactéria in planta, embora ela ainda seja capaz de crescer em meio de cultura. O mutante diminuiu de maneira significativa a formação de biofilme e não foi capaz de causar doença em laranja 'Pêra Rio' (Citrus sinensis (L.) Osbeck) e em limão 'Cravo' (Citrus limonia Osbek), dois de seus hospedeiros citros, enquanto que a Xac 306 selvagem apresentou os sintomas característicos do cancro cítrico nos dois hospedeiros. Os resultados indicam que a proteína XAC1008 não está diretamente relacionada... / The production of frozen concentrated juice from sweet orange is an agricultural activity that is of great importance to Brazil, especially for the state of São Paulo. However, citrus canker is a disease that causes bigger losses to the Brazilian and global citrus industry, and to date there is no curative method for this disease and the main control is the eradication of infected plants. The citrus canker originated in Asia and its causal agent is the bacterium Xanthomonas citri subsp. citri (Xac), which attacks all commercial species of citrus. In a study of gene expression in two Xac mutants with impaired or total absence of symptoms of citrus canker, the ORF XAC1008 was highly expressed, so the aim of this study was the functional characterization of the protein encoded by ORF XAC1008 from Xac. The strategy used was the construction of a mutant of the ORF XAC XAC1008 and evaluation of its pathogenicity in plants of Rangpur lime (Citrus limonia Osbek) and sweet orange 'Pêra Rio' (Citrus sinensis (L.) Osbeck) compared with the wild isolate strain Xac 306. The technique used for obtaining the mutant was PCR-based site-directed mutagenesis using the suicide vector POK1, followed by homologous recombination. Pathogenicity tests of the mutant ΔXAC1008 showed that the mutation affected the color and shape of colonies, and prevent multiplication of bacteria in the plant, though it is still able to grow in the culture medium. The mutant decreased significantly biofilm formation and was not able to cause disease in sweet orange 'Pêra Rio' orange and Rangpur lime, two of its citrus hosts, while the wild isolate strain Xac 306 showed the characteristic symptoms of citrus canker in both hosts. The results indicate that the protein XAC1008 is not directly related to the virulence or pathogenicity of Xac in citrus but is essential for the proliferation and survival of the bacteria in the host. Although the presence of the ...

Orientadores: Marcos Antonio Machado, Alexandre Morais do Amaral / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T03:30:01Z (GMT). No. of bitstreams: 1 Homem_RafaelAugusto_M.pdf: 2248507 bytes, checksum: 707bfda4edde12e0c0ac988130077aa3 (MD5) Previous issue date: 2008 / Resumo: O cancro cítrico é uma das mais sérias doenças de citros no mundo, sobretudo nos países onde a citricultura exerce papel influente na geração de empregos e divisas, como o Brasil, o maior produtor mundial de laranjas. Por esse motivo, o agente causal dessa doença, a bactéria Xanthomonas axonopodis pv. citri (Xac), teve seu genoma completamente sequenciado. No genoma de Xac foram identificados dois agrupamentos gênicos (operons), xcsCDEFGHIJKLMN e xpsEFGHIJKLMND, que codificam para o sistema de secreção do tipo II (SSTII), mecanismo altamente envolvido no processo de patogenicidade em algumas bactérias causadoras de doenças em plantas. Até o momento, não há evidência da função desempenhada por cada conjunto gênico do SSTII de Xac e da relação desses com o processo de interação com a planta de citros. Neste estudo foram analisadas as funcionalidades dos dois agrupamentos gênicos do SSTII de Xac e sua atividade durante a interação com a planta hospedeira. As análises revelaram que a bactéria utiliza os dois sistemas de forma distinta e com relevância diferenciada durante a interação com a planta, com repercussão no crescimento da bactéria no tecido foliar, sobretudo o operon xps. Adicionalmente, foram identificadas contribuições distintas na atividade de degradação dos compostos amido, carboximetilcelulose e proteína. Exames de microscopia revelaram que a bactéria tem sua organização estrutural do biofilme influenciada pelos dois SSTII e análises de expressão gênica revelaram que o operon xps apresenta atividade extremamente maior em relação ao operon xcs. Estes resultados mostram pela primeira vez a influência independente de ambos SSTII na capacidade patogênica de Xac. / Abstract: The citrus canker is a major threat to the citrus industry worldwide, especially where the citriculture plays an important role in employments and revenues, like United States and Brazil, the largest orange producing country. Therefore, the causal agent of citrus canker, the Gram negative bacterium Xanthomonas axonopodis pv. citri (Xac), had its genome completely sequenced. Throughout the genome of Xac, two operons (xps EFGHIJKLMND and xcsCDEFGHIJKLMN) that encompass 11 and 12 different proteins, respectively, for the type two secretion systems (TIISS) were identified. These mechanisms are highly involved in pathogenicity and virulence in some bacteria that cause disease in plants. However, so far, there are no studies on the function of these operons in Xac and their relationship for the infection process in the citrus plant. In this study, the functions of the two operons that code for the TIISS in Xac and their activity during the interaction with the plant host were analyzed. The analyses revealed that the bacterium uses both TIISS, however in a different fashion during the contact with the leaf tissue and the xps operon is highly more active. In addition, the operons were found to be differentially effective on the degradation of starch, carboxymethilcellulose and proteins. Confocal microscopy investigations found that the bacterial organization (biofilm) is influenced by both TIISS and gene expression analyses showed a much higher activity of the xps operon as compared to the xcs group. These results provide the first clear evidence of the independent influence of both TIISS in the pathogenic process of Xac. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Orientadores: Telma Teixeira Franco, Ana Carolina Deckmann / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-21T08:47:54Z (GMT). No. of bitstreams: 1 VargasTapia_Eulalia_M.pdf: 2747166 bytes, checksum: 033342e772fb86dbb8d3a20cf75af774 (MD5) Previous issue date: 2012 / Resumo: Neste trabalho foram desenvolvidos estudos visando o melhoramento genético da linhagem de levedura Lipomyces starkeyi DSM 70296, visando sua utilização na biossíntese de precursores de biocombustíveis a partir de fontes renováveis. Inicialmente foram verificados os parâmetros de mutagênese. O melhoramento genético foi conduzido por mutagênese aleatória de DNA por irradiação ultravioleta. O tempo de exposição foi ajustado de forma a assegurar uma taxa de sobrevivência celular não superior a 5%, para obter indivíduos contendo elevado acúmulo de mutações no DNA. Os mutantes foram selecionados com o uso da cerulenina agente interferente ao metabolismo de interesse, de forma que fossem identificados os mutantes cujas alterações genéticas pudessem estar promovendo efeitos sobre este metabolismo. Os mutantes que demonstraram crescimento normal em meio de cultura suplementado com cerulenina foram considerados bons candidatos para estudos aprofundados. Nesta etapa foram selecionados 90 mutantes, dos quais foram selecionados os oito melhores candidatos para estudo através de fermentação em frascos agitados. A avaliação de desempenho fermentativo foi conduzida a partir da avaliação dos índices de crescimento e produtividade de lipídeo utilizando meio de cultura contendo xilose como única fonte de carbono. A fermentação da cepa padrão foi conduzida nas mesmas condições para permitir uma análise comparativa. Os resultados obtidos mostraram que um dos mutantes (identificado como A1) apresentou aumento significativo nos índices de produtividade de biomassa e lipídeo em relação à cepa padrão (teste de Tukey com 95% de significância). Este mutante foi então selecionado para estudo aprofundado através da fermentação em biorreator utilizando a mesma composição de açúcares observada em bagaço de cana-de-açúcar (30% glicose: 70% xilose), conforme determinado em outros estudos conduzidos em nosso laboratório. Novamente, a fermentação da cepa padrão foi conduzida nas mesmas condições para permitir a análise comparativa. Os resultados confirmaram que o mutante A1 apresenta maior produtividade tanto em biomassa (88 g/L) quanto em fração de lipídeo (54,6%), em comparação à cepa padrão (76 g/L e 47,5%, respectivamente). Estes resultados indicam a viabilidade da estratégia de mutagênese aleatória aliadas à seleção de mutantes por cerulenina para o melhoramento genético desta levedura oleaginosa, visando sua aplicação no processo de biossíntese de precursores de biocombustíveis de segunda geração / Abstract: In this work it were performed studies aiming the genetic improvement of the yeast Lipomyces starkeyi strain DSM 70296 for its utilization in the biosynthesis of biofuels' precursors from renewable sources (sugarcane bagasse). Initially, we defined the parameters of mutagenesis. The genetic breeding was carried out by random mutagenesis of DNA by ultraviolet irradiation. The exposure time was adjusted to ensure a cell survival rate not exceeding 5%, in order to obtaining individuals presenting high rates of DNA mutations. The mutants were selected by using cerulenin, an compound displaying effects on the metabolism of interest (biosynthesis of lipids). Thus, the selected mutants are potentially carriers of genetic alterations in this particular metabolism. The mutants demonstrating normal growth in culture medium supplemented with cerulenin were considered good candidates for in-depth studies. In this step we selected 90 mutants, of which eight were considered the best candidates for further studies by fermentation in shake flasks. The evaluation of fermentative performance was carried out based on growth and lipid productivity rates using culture medium containing xylose as sole carbon source. The fermentation of the wild-type strain was conducted under the same conditions to allow a comparative analysis. The results showed that the mutant identified as A1 presented a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain (Tukey test with 95% significance). This mutant was then selected for detailed study by fermentation in bioreactor using the same carbohydrate composition observed in sugarcane bagasse (30% glucose: 70% xylose), as previously determined by other studies in our laboratory. Again, fermentation of the wild-type and the mutant A1 was performed under the same conditions in order to allow a comparative analysis. The results confirmed that the A1 mutant presents an increased productivity of both biomass (88 g/L) and lipids (54.6%) when compared to the wild-type strain (76 g/L and 47.5%, respectively). These results indicate the feasibility of random mutagenesis strategy coupled with mutant selection employing cerulenin for the genetic improvement of the oleaginous yeast L. starkeyi, focusing its use in the biosynthesis of precursors of second generation biofuels / Mestrado / Desenvolvimento de Processos Químicos / Mestra em Engenharia Química

Mutagenesis screening in mice is an effective means of identifying essential genes in cardiovascular development. The l11Jus8 (L8) mutant mouse line was originally isolated from a region-specific N-ethyl-N-nitrosourea (ENU) chemical mutagenesis screen and exhibited an autosomal recessive mid-gestational embryonic lethal phenotype characterised by haemorrhage in the thoracic cavity, blood pooling in the heart, right ventricular dysmorphology and yolk sac vascular degeneration. Prior work mapped the L8 mutation to a ~2.77Mb region on mouse chromosome 11. The aim of this study was to further characterise the L8 mutant phenotype and identify the L8 causative mutation. Phenotypic characterisation conducted here confirmed mid-gestational lethality, haemorrhage and yolk sac vascular degeneration in L8 mutants. Histological analysis of L8 mutants demonstrated presence of fragmented cell nuclei and loss of myocardial integrity in embryonic atrial myocardium. Areas of fragmented cell nuclei did not exhibit positive staining for apoptosis. Furthermore, L8 mutants did not appear to experience typical cardiac defects in aspects including myocardial or smooth muscle differentiation, cell proliferation, ECM production, myocardial hypoplasia/hyperplasia, basement membrane components or observable aberrations in cardiac conduction. L8 mutants exhibited atypical cardiac defects including sudden cessation of heartbeat with morphological indicators of necrosis such as swelling of mitochondria and release of microparticles both from atrial myocardial cells. The L8 mutant appears to represent a novel combination of cardiac defects or novel defects with secondary cardiac phenotypes. Sequencing of the coding exons and splice junctions of 22 candidate genes within the ~2.77Mb L8 locus did not identify the causative mutation. The L8 locus was therefore further refined to a ~1.16Mb region including 20 genes. Sanger sequencing of 10 of these genes plus targeted sequence capture and SOLiD sequencing of the region did not identify a potential L8 mutation. Given the refinement of the candidate locus and advances in sequencing technology and analysis, further sequencing will likely identify the L8 mutation and confirm the cause of the embryonic lethal phenotype.

Mutagen-sensitive (mus) mutations in Drosophila melanogaster render developing flies hypersensitive to the lethal effects of DNA-damaging agents. In general, mus mutations identify DNA repair-related genes. In this study, 5 new second chromosome mus mutations (mus205B¹, mus208B¹, mus209B¹, mus210B¹ and mus211B¹), selected on the basis of sensitivity to methyl methanesulfonate (MMS), were characterized using a variety of genetic tests. One test measured the MMS-sensitivity of double mutant mus strains compared to their component single mutants. Mutant interactions were examined in 8 double mus and in 2 triple mus strains containing combinations of mus201D¹, mus205B¹, mus208B¹, mus210B¹ and mus211B¹ (or mus211B²). These analyses have revealed predominantly synergistic and epistatic responses to MMS. Taken together with the findings of previous genetic and biochemical studies of Drosophila mus strains, these results suggest that 3 major repair pathways may operate in flies to correct damage caused by MMS. Mutagen cross-sensitivity data and the results of the interaction studies suggest that mus mutations might serve as rapid and sensitive bioassays of somatic genotoxicity caused by mutagens and carcinogens. To explore this possibility, a simple mutagen test system was devised employing triple mutant mus strains. One strain (mus208B¹ mus210B¹ mus211B²) was tested for sensitivity to 14 mutagens/carcinogens and 2 non-carcinogens. Eleven of the mutagens/carcinogens were readily detected as genotoxic. Both non-carcinogens were non-genotoxic. These preliminary results demonstrate the feasibility (and some limitations) of the proposed somatic genotoxicity assay and emphasize the need for further test validation using a larger chemical data base. The temperature-sensitive lethal mutation mus209B¹ was subjected to extensive genetic analyses and to temperature shift experiments during development. This locus was found to encode a product(s) that (1) is essential for viability at virtually all pre-imaginal developmental stages (the latter half of pupation appears to be an exception), (2) is necessary for wildtype levels of resistance to the genotoxic effects of MMS and ionizing radiation, and (3) is required for female fertility. Confirmation of the pleiotropic nature of this mutation was obtained by meiotic and cytogenetic mapping studies and by complementation tests with a series of allelic mutations. The mus209B¹ phenotypes are similar to ones conferred by mutations in Drosophila and yeast that disrupt various aspects of chromosome metabolism. In this context, some possible roles for mus209B¹ are discussed. / Science, Faculty of / Zoology, Department of / Graduate

The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Autism spectrum disorders (ASD) are a group of related neurodevelopmental diseases displaying significant genetic and phenotypic heterogeneity. Despite recent progress in ASD genetics, the nature of phenotypic heterogeneity across probands is not well understood. Notably, likely gene-disrupting (LGD) de novo mutations affecting the same gene often result in substantially different ASD phenotypes. We find that truncating mutations in a gene can result in a range of relatively mild decreases (15-30%) in gene expression due to nonsense-mediated decay (NMD), and show that more severe autism phenotypes are associated with greater decreases in expression. We also find that each gene with recurrent ASD mutations can be described by a parameter, phenotype dosage sensitivity (PDS), which characterizes the relationship between changes in a gene’s dosage and changes in a given phenotype. Using simple linear models, we show that changes in gene dosage account for a substantial fraction of phenotypic variability in ASD. We further observe that LGD mutations affecting the same exon frequently lead to strikingly similar phenotypes in unrelated ASD probands. These patterns are observed for two independent proband cohorts and multiple important ASD-associated phenotypes. The observed phenotypic similarities are likely mediated by similar changes in gene dosage and similar perturbations to the relative expression of splicing isoforms. We also identify patterns of developmental and cell type-specific expression that additionally contribute to the variability of several autism phenotypes.

Bestrophins are a family of calcium (Ca²⁺) -activated chloride (Cl⁻) channels (CaCCs) with functional importance in eye physiology. Mutations to the VMD2 gene, which encodes the Best1 protein, cause an array of degenerative eye disorders called bestrophinopathies, which result from aberrant CaCC activity of the Best1 channel in the pigmented epithelium of the retina. While there are four bestrophin paralogs in mammals (Best1-4), the only current structures are of Best1 homologs. The structure of the prokaryotic homolog of Best1 from Klebsiella pneumonia (KpBest) was previously solved in this lab, representing the first structure of a Best1 homolog at the time. This initial study laid the foundational groundwork in the field and contributed significant knowledge to understanding the bestrophin structure-function relationship. Nevertheless, significant questions remain regarding bestrophin function, such as the molecular determinants underlying its Ca²⁺-dependent gating and anion selectivity. This dissertation uses single-particle cryogenic electron microscopy paired with electrophysiology to probe the structure-function relationship of mammalian bestrophins under different buffer conditions and reveals conformational dynamics involved in gating of wild-type channels. Key regions of the channel contributing to its function are described at the atomic level leading to development of a gating model to explain Ca²⁺-dependent activation and inactivation in mammalian bestrophins.