Two-stage Turing model for generating pigment patterns on the leopard and the jaguar

Based on the results of phylogenetic analysis, which showed that flecks are the primitive pattern of the felid family and all other patterns including rosettes and blotches develop from it, we construct a Turing reaction-diffusion model which generates spot patterns initially. Starting from this spotted pattern, we successfully generate patterns of adult leopards and jaguars by tuning parameters of the model in the subsequent phase of patterning

The Establishment and Regulation of Melanocyte Stem Cells in Zebrafish

Stem cells are the cells that regulate the growth and repair of tissues in adult organisms. In this thesis, I sought to develop methodologies to dissect the function and regulation of stem cells during zebrafish melanocyte regeneration. In the first part of this thesis, I develop a drug based method of ablating melanocytes in the adult zebrafish body. The drug is a copper chelator, neocuproine: NCP) that I show causes death specifically of melanocytes in adult, which allows for regeneration from melanocyte stem cells: MSCs). In the next part of the thesis, I employ clonal lineage statistical analyses to study the establishment, recruitment, and proliferation, differentiation, and survival of MSC daughter cells during larval melanocyte regeneration. These analyses suggest that MSCs are likely recruited at random for each regeneration event, and that approximately 84% of MSCs are recruited for any regeneration event. I demonstrate that kit signaling has a greater requirement during larval regeneration than during ontogeny and compare the regeneration of kit heterozygotes to wild type. The mutant heterozygotes have normal MSC recruitment and normal proliferation, differentiation, and survival of daughter cells. The mutant has defective MSC establishment, with this defect being quantitatively sufficient to explain the regeneration defect observed. I then used further clonal lineage analysis to suggest that reduction of kit signaling causes inappropriate differentiation of fated MSCs into ontogenetic melanocytes. These analyses are not unique for comparison of kit mutants to wild type, so can easily be applied to dissect any gene or drug which affects regeneration. In the final part of the thesis, I explore how many spermatagonia form the adult zebrafish male germline. An understanding of this number allows for efficient mutant screens, an essential part of the genetic dissection of any process. The zebrafish has approximately 485 spermatagonia, giving each male approximately 970 genomes which can be mutagenized. This number can be considered during mutant screen designs to eliminate redundant screening

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility

African Trypanosomes undermine humoral responses and vaccine development : link with inflammatory responses?

African trypanosomosis is a debilitating disease of great medical and socioeconomical importance. It is caused by strictly extracellular protozoan parasites capable of infecting all vertebrate classes including human, livestock, and game animals. To survive within their mammalian host, trypanosomes have evolved efficient immune escape mechanisms and manipulate the entire host immune response, including the humoral response. This report provides an overview of how trypanosomes initially trigger and subsequently undermine the development of an effective host antibody response. Indeed, results available to date obtained in both natural and experimental infection models show that trypanosomes impair homeostatic B-cell lymphopoiesis, B-cell maturation and survival and B-cell memory development. Data on B-cell dysfunctioning in correlation with parasite virulence and trypanosome-mediated inflammation will be discussed, as well as the impact of trypanosomosis on heterologous vaccine efficacy and diagnosis. Therefore, new strategies aiming at enhancing vaccination efficacy could benefit from a combination of (i) early parasite diagnosis, (ii) anti-trypanosome (drugs) treatment, and (iii) anti-inflammatory treatment that collectively might allow B-cell recovery and improve vaccination

Mitochondrial DNA mutations in disease and aging

The small mammalian mitochondrial DNA (mtDNA) is very gene dense and encodes factors critical for oxidative phosphorylation. Mutations of mtDNA cause a variety of human mitochondrial diseases and are also heavily implicated in age-associated disease and aging. There has been considerable progress in our understanding of the role for mtDNA mutations in human pathology during the last two decades, but important mechanisms in mitochondrial genetics remain to be explained at the molecular level. In addition, mounting evidence suggests that most mtDNA mutations may be generated by replication errors and not by accumulated damage

Tracking the In Vivo Dynamics of Antigenic Variation in the African Trypanosome

Trypanosoma brucei, a causative agent of African sleeping sickness in humans and nagana in animals, constantly changes its dense variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of its mammalian host, using an extensive repertoire of dedicated genes. Although this process, referred to as antigenic variation, is the major mechanism of pathogenesis for T. brucei, the dynamics of VSG expression in T. brucei during an infection are poorly understood. In this thesis, I describe the development of VSG-seq, a method for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Using VSG-seq, I monitored VSG expression dynamics in vivo during both acute and chronic mouse infections. My experiments revealed unexpected diversity within parasite populations, and the expression of as much as one-third of the functional genomic VSG repertoire after only one month of infection. In addition to suggesting that the host-pathogen interaction in T. brucei infection is substantially more dynamic and nuanced than previously expected, this observed diversity highlighted the importance of the mechanisms by which T. brucei diversifies its genome-encoded VSG repertoire. During infection, the parasite can form mosaic VSGs, novel variants that arise through recombination events within the parasite genome during infection. Though these novel variants had been identified previously, little was known about the mechanisms by which they form. VSG-seq facilitated the identification of mosaic VSGs during the infection, which allowed me to track their formation over time. My results provide the first temporal data on the formation of these variants and suggest that mosaic VSGs likely form at sites of VSG transcription. VSG-seq, which is based on the de novo assembly of VSGs, obviates the requirement for a reference genome for the analysis of expressed VSG populations. This allows the method to be used for the high-resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. To this end, I have applied VSG-seq to samples grown in vitro, parasites isolated from natural infections, and extravascular parasites occupying various tissues in vivo. These extensions of the method reveal new aspects of T. brucei biology and demonstrate the potential of high-throughput approaches for studying antigenic variation, both in trypanosomes and in any pathogen that uses antigenic variation as a means of immune evasion

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.