Genetic engineering of mesenchymal stem cells and its application in human disease therapy.

The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

Environmental and genetic determinants of colony morphology in yeast.

Nutrient stresses trigger a variety of developmental switches in the budding yeast Saccharomyces cerevisiae. One of the least understood of such responses is the development of complex colony morphology, characterized by intricate, organized, and strain-specific patterns of colony growth and architecture. The genetic bases of this phenotype and the key environmental signals involved in its induction have heretofore remained poorly understood. By surveying multiple strain backgrounds and a large number of growth conditions, we show that limitation for fermentable carbon sources coupled with a rich nitrogen source is the primary trigger for the colony morphology response in budding yeast. Using knockout mutants and transposon-mediated mutagenesis, we demonstrate that two key signaling networks regulating this response are the filamentous growth MAP kinase cascade and the Ras-cAMP-PKA pathway. We further show synergistic epistasis between Rim15, a kinase involved in integration of nutrient signals, and other genes in these pathways. Ploidy, mating-type, and genotype-by-environment interactions also appear to play a role in the controlling colony morphology. Our study highlights the high degree of network reuse in this model eukaryote; yeast use the same core signaling pathways in multiple contexts to integrate information about environmental and physiological states and generate diverse developmental outputs.

Physical and Genetic Analysis of the CUP1 Tandem Array in the Yeast Saccharomyces cerevisiae

The genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations.

In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.

In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis.

The research described in Chapter 2 is published in 2014.

Reward circuitry dysfunction in psychiatric and neurodevelopmental disorders and genetic syndromes: animal models and clinical findings.

This review summarizes evidence of dysregulated reward circuitry function in a range of neurodevelopmental and psychiatric disorders and genetic syndromes. First, the contribution of identifying a core mechanistic process across disparate disorders to disease classification is discussed, followed by a review of the neurobiology of reward circuitry. We next consider preclinical animal models and clinical evidence of reward-pathway dysfunction in a range of disorders, including psychiatric disorders (i.e., substance-use disorders, affective disorders, eating disorders, and obsessive compulsive disorders), neurodevelopmental disorders (i.e., schizophrenia, attention-deficit/hyperactivity disorder, autism spectrum disorders, Tourette's syndrome, conduct disorder/oppositional defiant disorder), and genetic syndromes (i.e., Fragile X syndrome, Prader-Willi syndrome, Williams syndrome, Angelman syndrome, and Rett syndrome). We also provide brief overviews of effective psychopharmacologic agents that have an effect on the dopamine system in these disorders. This review concludes with methodological considerations for future research designed to more clearly probe reward-circuitry dysfunction, with the ultimate goal of improved intervention strategies.