Thesis (M.S.)--Ohio State University, 2005. / Includes bibliographical references (leaves 86-98). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--Indiana University School of Dentistry, 1996. / Includes bibliographical references.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Biological evolution is an inherently non-equilibrium process, by which a population acquires a new genetic composition, optimally suited to its present environment. Far from being the slow process it is traditionally viewed as, the rapid evolution of microbes is causing serious global concern in the acquisition of microbial resistance to antibiotics. Better understanding of the mechanisms that govern the evolution of microbes is therefore of paramount importance. In many traditional models, evolution occurs over the space of all possible genetic states (genotypes). These are assigned a quantity called fitness, which quantifies that genotype's suitability over others to thrive within its present environment. A population of replicating cells can evolve over this space under the competing influences of random variations of the genotype (i.e. mutations) and the increased likelihood of success for fitter genotypes (i.e. selection). Many of these models fail to account for the observation that biological diversity is rife, even amongst genetically identical cells that exist in the same environment. This diversity manifests itself as a difference in phenotype (the observable traits of an organism). It means that organisms with the same genotype, but a different phenotype, may have different fitnesses. Therefore, when phenotypic heterogeneity is apparent, evolution over genotype space should consider different fitness landscapes for each of the distinct phenotypic states that exist. Phenotypic heterogeneity has long been observed in populations of microbes. Often these can switch between different phenotypic states for a number of reasons. A common example of this is stochastic phenotype switching, in which cells randomly switch between two phenotypic states, without any inducing influence. This has been shown to benefit populations of cells that are subject to fluctuating environmental conditions, or by creating a division of labour in the population. In this work, I examine the possibility of another role for stochastic phenotype switching: as a mechanism that can accelerate evolution even in a static environment. During evolution, populations can spend large amounts of time trapped at local peaks on a fltness landscape. A cell that switches phenotype will change to a different fitness landscape, which may allow for faster genetic evolution. I begin this work in Chapters 3 and 4, where I present a model of an evolving population of haploid cells, trapped at a local peak on a 1D fitness landscape. These cells have access to a second phenotypic state, in which the fitness landscape is uniform. The focus of this study is to see the effect that stochastic phenotype switching to this secondary phenotype has on the populations evolution of a target state. In Chapter 3 I study this numerically and identify an optimal range for the rate of phenotype switching, within which the time taken for the process can be reduced by many orders of magnitude. I also find that if the frequency of switching is allowed to evolve, then the likely evolutionary trajectory taken by a population is one that first evolves a switching frequency to within the identified optimal range, before escaping from the local peak. In Chapter 4 I present an analytic study of the same model. The aim here is to recover the numerical results from Chapter 3. I employ numerous analytic techniques to show the existence of the optimal range, while developing an analytic approach that allows a study of the model at parameter values that are otherwise difficult to simulate. This same model is extended in Chapter 5 to consider evolution over a more complex genotype space: that of a hypercube. Here, genotypes correspond to particular binary sequences, which can be used as representations of many biological states of interest; for example, nucleotide sequences in DNA or the presence and absence of important mutations in specific genes. My focus here is again on the effect that stochastic phenotype switching has on how a population of cells evolves over genotype space. This is studied numerically for various kinds of randomly generated fitness landscapes. I find that in some instances phenotype switching can significantly benefit a population. However, in other instances it can significantly hinder the evolution, increasing the time taken for the process by many orders of magnitude. Finally, in Chapter 6 I present a model that explores how a population of the bacterium Escherichia coli (E. coli) evolves resistance to the antibiotic ciprofloxacin. This work is motivated by the observed rapid acquisition of resistance of E. coli when exposed to sub-lethal concentrations of the antibiotic. Upon damage to their DNA, cells can induce a switch to a secondary phenotypic state (as part of the SOS response), in which DNA repair and an increased rate of mutations occur. Using this model, with empirical data for the fitness and susceptibility of genotypes, I numerically explore the dependence of rapid evolution on the existence of this secondary phenotypic state. I find that the model predicts, over the short timescales considered, that the evolution of sufficient resistance requires the existence of the secondary phenotypic state. The findings of this work is that the phenotypic switching of cells can have a significant impact on how populations evolve in static environments. While stochastic phenotype switching can help populations escape from local peaks, it can also trap populations on sub-optimal landscapes if the frequency of switching is too low.

A key to understanding the origin and maintenance of the diversity of life is to understand how phenotypic and genetic variation is partitioned within and among populations. I characterize the spatial partitioning of phenotypic and genetic variation in an old Willamette Basin freshwater distribution of threespine stickleback (Gasterosteus aculeatus) and compare these results to younger populations. Phenotypic variation was measured using 14 phenotypic traits, and genetic variation was assessed using RADseq and Stacks software to identify tens of thousands of single nucleotide polymorphisms. The major partitioning of phenotypic and genetic variation in Oregon is along a stereotypical transition from oceanic to freshwater that has been seen in younger systems. Phenotypic and genetic variation is significantly partitioned between basin populations, and the genetic variation is geographically structured. This work suggests that parallel divergence between oceanic and freshwater forms originated before the end of the last glacial maximum.

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:53:57Z : No. of bitstreams: 1 gamba_bf_me_botib.pdf: 1144074 bytes, checksum: 38b7854a72a3108b08ac952129465214 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A síndrome de Smith-Magenis (SMS) é uma complexa anomalia caracterizada por atraso no desenvolvimento neuro-psico-motor (ADNPM), anomalias craniofaciais, esqueléticas e comportamentais. Dentre as anomalias destacamos braquicefalia, baixa estatura, distúrbio do sono, comportamentos de auto-injúria e movimentos estereotipados. SMS é causada por uma deleção intersticial do cromossomo 17(p11.2) ou mutação no gene RAI1, presente nesta região cromossômica. Sua prevalência é estimada em 1:25.000 nascidos vivos podendo chegar até 1:15.000. O diagnóstico clínico para SMS é confirmado pela técnica de citogenética molecular FISH. Neste trabalho realizamos estudo citogenético por bandamento GTG, GTG em alta resolução e FISH no total de 17 casos com suspeita clínica para SMS. Para realização de FISH foram utilizadas duas sondas comerciais da CYTOCELL ®, uma contendo o gene FU1 e outra o gene RAI1. Os resultados clínicos desta casuística comparados com a literatura internacional mostrou que embora fizemos algumas considerações, demonstramos a similaridade do diagnóstico clínico de SMS nos subgrupos SMS-del e SMS-like tanto brasileiros quanto da literatura. Os resultados citogenéticos do bandamento GTG revelaram que sete casos (7/17) apresentam deleção 17(p11.2) e o FISH confirmou a deleção tanto do gene FU1 como do gene RAI1 nos sete casos identificados. Este trabalho é de grande importância por tratar de uma afecção rara, pouco conhecida, e subdiagnosticada no Brasil. Assim destacamos que o uso de ficha clínica específica para SMS é importante para a definição da hipótese diagnóstica, 7/17 casos avaliados apresentaram deleção 17p11.2, e deleção dos genes... / Smith-Magenis syndrome (SMS) is a complex anomaly characterized by developmental delay (ADNPM), craniofacial anomalies, and behavioral disorders. Among the anomalies highlight brachycephaly, short stature, sleep disturbance, behavior of self-injury and stereotyped movements. SMS is caused by an interstitial deletion of chromosome 17 (p11.2) or mutation in the retinoic acid-induced 1 (RAI1) gene, present in this chromosomal region. Its prevalence is estimated at 1:25.000 live births and may reach up to 1:15.000. The c1inical diagnosis for SMS is confirmed by molecular cytogenetic technique of FISH. In this work we performed a cytogenetic study by GTG, GTG high resolution and FISH total of 17 cases with c1inical suspicion for SMS.To perform FISH probes were used two commercial CYTOCELL ®, one containing the FLl1 gene and another gene RAI1. The c1inical results in this series compared to the literature showed that although we made some considerations, we demonstrate the similarity of the clinical diagnosis of SMS in both sub-del SMS and SMS-like Brazilians and literature. The cytogenetic results of GTG banding revealed that seven cases (7 1 17) have deletion 17(p11.2) and FISH confirmed the deletion of both the FLl1 gene and the gene RAI1 the seven cases identified. This work is of great importance for treating arare, little known and under-diagnosed in Brazil. Thus we emphasize that the use of medical records specific to SMS is important to define the diagnosis, 7 117 cases showed stable 17p11.2 deletion, and deletion of genes FUI and RAI, no significant differences between the c1inicalfeatures of this sample group comparing it with the literature and this is about the largest sample of Brazilian SMS already described

IntroduÃÃo: A beta talassemia à um grupo de distÃrbios, cada um resultando de um defeito genÃtico, na velocidade de sÃntese de uma ou mais cadeias globÃnicas da hemoglobina (Hb). A desproporÃÃo na produÃÃo das cadeias globÃnicas pode resultar em eritropoese ineficaz, produÃÃo insuficiente de Hb, hemÃlise e anemia de grau variado. A beta talassemia à mais frequente nos paÃses banhados pelo mar MediterrÃneo, refletindo a participaÃÃo desses povos na formaÃÃo da populaÃÃo brasileira. As mutaÃÃes predominantes no Brasil sÃo a IVS-I-1, IVS-I-6, IVS-I-110 e o CD 39, as quais estÃo associadas a diversos quadros clÃnicos e sÃo, em sua maioria, regionalmente especÃficas. Objetivo: Caracterizar o perfil clÃnico, hematolÃgico e molecular dos indivÃduos adultos com beta talassemia do Hospital UniversitÃrio Walter CantÃdeo, em acompanhamento no centro de referÃncia de Hematologia e Hemoterapia do estado do Cearà (HEMOCE). Metodologia: Foram analisados 22 indivÃduos portadores de beta talassemia, sendo 7 intermediÃria e 15 menor, de ambos os sexos, no perÃodo de fevereiro de 2008 a setembro de 2009. Os dados clÃnicos e laboratoriais: hemograma, nÃveis de Hb A2 e de Hb F; ferro sÃrico; capacidade total e latente de ligaÃÃo do ferro (CTLFe, CLLFe), ferritina e Ãndice de saturaÃÃo da transferrina (IST), foram obtidos dos prontuÃrios, ao diagnÃstico. Cerca de 5 mL de sangue venoso foi coletado em tubo contendo o anticoagulante EDTA para o estudo molecular. A anÃlise das mutaÃÃes foi realizada por meio da tÃcnica da reaÃÃo em cadeia mediada pela polimerase alelo especÃfico (PCR-AE), onde foram analisadas as seguintes mutaÃÃes: IVS-I-1, IVS-I-6, IVS-I-110 e o CD 39. As anÃlises estatÃsticas foram desenvolvidas no software livre R (versÃo 2.7.0) e o nÃvel de significÃncia estabelecido foi 5%. Resultados: Dos 22 pacientes estudados, 15 eram portadores de β talassemia menor e sete intermediÃria. A idade variou de 18 a 68 anos, com mÃdia de 44,7 anos. 18,2 % do sexo masculino e 81,8% do sexo feminino. As mutaÃÃes foram caracterizadas em 68,2% dos casos, que teve como mais frequente a IVS-I-6, seguida do cÃdon 39. A mutaÃÃo IVS-I-I foi caracterizada em um paciente e a IVS-I-110 nÃo foi encontrada. NÃo houve diferenÃa clÃnica significante entre os parÃmetros hematolÃgicos e bioquÃmicos. Houve discrepÃncia entre o fenÃtipo e o genÃtipo em alguns pacientes, porÃm nÃo houve diferenÃa significativa entre as mutaÃÃes e as manifestaÃÃes clÃnicas. ConclusÃes: Os resultados do presente estudo reforÃam o predomÃnio da mutaÃÃo IVS-I-6 no nordeste do Brasil. Recomendam-se estudos posteriores para investigaÃÃo da co-heranÃa com a α talassemia nesses pacientes para justificar a discrepÃncia entre genÃtipos e fenÃtipos. / Background: Beta thalassemia is a group of disorders, each resulting from a genetic defect in the rate of synthesis of one or more globin chains of hemoglobin (Hb). The imbalance in the production of globin chains can result in ineffective erythropoiesis, insufficient production of hemoglobin, hemolysis and anemia of varying degree. Beta thalassemia is more common in countries bordering the Mediterranean Sea, reflecting the participation of these peoples in the formation of the Brazilian population. The predominant mutations in Brazil are the IVS-I-1, IVS-I-6, IVS-I-110 and CD 39, which are associated with different clinical conditions and are mostly regionally specific. Objective: To characterize the clinical, hematological and molecular adults with beta thalassemia of the University Hospital CantÃdeo Walter and followed at a referral center for Hematology of the state of Cearà (Hemoce). Methods: We analyzed 22 individuals with beta thalassemia, 7 intermediate and 15 minor, in both sexes, from February 2008 to September 2009. Clinical data and laboratory tests: blood count, levels of Hb A2 and Hb F, serum iron, total capacity and latent iron binding (CTLFe, CLLFe), ferritin and transferrin saturation index (IST) were obtained from medical records, diagnosis. About 5 mL of venous blood was collected in tubes containing EDTA anticoagulant for molecular study. The analysis of mutations was performed using the technique of chain reaction mediated by allele specific polymerase (PCR-AE), where we analyzed the following mutations: IVS-I-1, IVS-I-6, IVS-I-110 and CD 39. Statistical analysis was carried out in software R (version 2.7.0) and the level of significance was 5%. Results: Of 22 patients studied, 15 were patients with β thalassemia minor and seven intermediate. The age ranged 18-68 years with a mean of 44.7 years. 18.2% male and 81.8% female. The mutations were characterized in 68.2% of cases, which had the most frequent IVS-I-6, followed by the codon 39. The mutation IVS-I-1 was found in one patient and IVS-I-110 was not found. There was no significant clinical differences between the hematological and biochemical parameters. There was a discrepancy between phenotype and genotype in some patients, but no significant difference between mutations and clinical manifestations. Conclusions: The results of this study reinforce the dominance of the mutation IVS-I-6 in northeastern Brazil. Are recommended further studies to investigate the co-inheritance with α-thalassemia in these patients to justify the discrepancy between genotypes and phenotypes.

Abstract This study describes clinical and biochemical consequences of the 3243A→G mutation in the tRNALeu(UUR) gene of the mitochondrial DNA. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) is usually caused by this mutation. Demyelinating polyneuropathy was observed as a novel feature in a patient with the mutation. Based on electrodiagnostic examination the polyneuropathy was defined as being of the demyelinating, mixed (motor more than sensory) type. In a 1 year follow-up an approximately 7% reduction in both the motor and sensory nerve conduction velocities were observed. The effect and mechanism of action of nicotinamide treatment in a MELAS patient with the 3243A→G was studied. The blood NAD concentration increased linearly, being 24-fold elevated at 6 weeks of treatment. Blood lactate and pyruvate concentration decreased by 50% within three days and 24 h urine lactate content within 2 weeks. A clinical improvement together with a decrease in the lesion volume in magnetic resonance imaging within the first month were observed. Alleviation of the lactate accumulation during the nicotinamide treatment suggested that an increase in the cellular NAD+NADH concentration led to enhancement of the oxidation of reducing equivalents, suggesting that complex I of respiratory chain operates at non-saturating substrate concentration. Myoblasts cultured from patients carrying the 3243A→G mutation and from controls were used to measure ATP, ADP, catalase and superoxide dismutase activity, population growth, apoptotic cell death and the morphology of cytoskeletal components. ATP and ADP concentrations were decreased, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, suggesting an increased production of reactive oxygen species due to respiratory chain dysfunction. No increase in apoptotic cell death was observed in proliferating myoblasts, but randomization of vimentin filament direction and length was observed and decreased population growth was associated with the mutation. The results show that the 3243A→G mutation leads to numerous secondary pathophysiological events. Based on the literature and the results of this study, similarities were found between the pathophysiology of 3243A→G mutation and other neurodegenerative diseases and aging.

Growth phenotype profiling of genome-wide gene-deletion strains over stress conditions can offer a clear picture that the essentiality of genes depends on environmental conditions. Systematically identifying groups of genes from such recently emerging high-throughput data that share similar patterns of conditional essentiality and dispensability under various environmental conditions can elucidate how genetic interactions of the growth phenotype are regulated in response to the environment. In this dissertation, we first demonstrate that detecting such “co-fit” gene groups can be cast as a less well-studied problem in biclustering, i.e., constant-column biclustering. Despite significant advances in biclustering techniques, very few were designed for mining in growth phenotype data. Here, we propose Gracob, a novel, efficient graph-based method that casts and solves the constant-column biclustering problem as a maximal clique finding problem in a multipartite graph. We compared Gracob with a large collection of widely used biclustering methods that cover different types of algorithms designed to detect different types of biclusters. Gracob showed superior performance on finding co-fit genes over all the existing methods on both a variety of synthetic data sets with a wide range of settings, and three real growth phenotype data sets for E. coli, proteobacteria, and yeast.

Please read the abstract in the dissertation Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Physiology / unrestricted