Echinoderms comprise a group of animals with the capability of replacing complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied, however, the molecular machinery behind this faculty remains to be understood. In this study, a de novo transcriptome was assembled and annotated using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 hours and 3 days regenerating intestine, revealing numerous differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with developmental and cancer-related mechanisms linked with regenerative responses. This study demonstrates the diverse gene expression profile during the early stages of regeneration using the mesentery as a focal point....

The work of Carl Woese based on ribosomal RNA sequence analysis from organisms have led scientist to classify life as we know it into three domains. One such domain is the Archaea. There is a lot of interest in studying ...

Nuclear protein localization-3 (Npl3) is a highly dynamic protein involved in mRNA biogenesis. Previous studies showed that the npl3-95 allele encodes a protein that can aggregate into high molecular weight complexes. Besides, two different bioinformatics analysis scored Npl3 as a potential prion protein in yeast. Npl3 protein was characterized as a novel prion-like protein in Saccharomyces cerevisiae using genetic and biochemical criteria. Results presented in this doctoral dissertation show that; (1) Npl3 harbors a putative prion domain in its RGG domain, (2) the paromomycin sensitivity phenotype associated with the npl3-95 mutant allele is dominant and shows non-mendelian inheritance (cytoplasmic inheritance), (3) GndHCl treatment and HSP104 deletion eliminate the paromomycin sensitivity phenotype, and (4) over-expression of the Npl3 wild type protein induces prion-like aggregates. Interestingly, the presence of the Npl3 prion-like form confers the acquisition of an adenine prototrophy phenotype and a decrease in flocculation in the yeast Saccharomyces cerevisiae. Altogether, the results presented in this dissertation provide new evidence of the biological significance of yeast prions and their regulatory role in gene expression....

Transcriptomic databases have become one of the main sources for protein discovery. In our studies of transcripts from normal echinoderms, we have identified several transcripts that have attracted our attention. One of these is a previously unidentified transcript (Orpin) that appeared to be upregulated during intestinal regeneration. In Chapter 2, using bioinformatics tools we: (1) identified a second Orpin sequence (2) describe their motifs and domains, and perform phylogenetic analyses that suggest that Orpins might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade. Semiquantitative RT-PCR analyses revealed that Orpin mRNAs are expressed in various tissues but no significant differential expression was found in regenerating tissues.<br /> <br /> In Chapter 3 we expressed and purified genetically modified versions of Orpin to further characterize this EF-hand protein. We developed two protocols:<br /> <br /> The first protocol consisted in the production of His-OBSm, which is His tagged with two additional genetic modifications: the deletion of the signal peptide encoding sequence and the addition of a 25 residues peptide from the pET200 plasmid vector. These modifications made possible the purification of a soluble recombinant version of Orpin B.<br /> <br /> The second protocol was developed to produce a soluble recombinant Orpin that best resembled the original protein. This protocol consisted of the expression of a GST-Orpin lacking the signal peptide region. For this protocol, we tested different parameters that affect protein expression, including, additives, host cells, growth media and supplementation, and protein extraction methods. The best parameters were identified and used to obtain a soluble Orpin form. We propose that the developed strategies can be used to increase the soluble expression and purification of other EF-hand proteins that are difficult to express by standard methods.<br /> <br /> In chapter 4 we produced antibodies against His-OBSm and used these to identify the cells expressing the protein in H. glaberrima tissues. Antibody specificity was tested through Western Blots.<br /> <br /> Finally, we investigated the effect of the Wnt signaling pathway on Orpin mRNA expression in regenerating gut explants and found an apparent decrease expression of an Orpin B isoform following Wnt/B-catenin pathway activation....

In this thesis, we explore the cognitive basis for the emergence of individual foraging strategies (IFS) across multiple honey bee subspecies using Reversal Learning assays and Free-Flying foraging problems. In Chapter 1 ...

Butterfly wing patterns have captivated evolutionary and developmental biologists for decades because of their vast diversity. Studies have focused on the molecular basis of pattern variation in the Nymphalidae, with little effort put toward skippers, family Hesperiidae. This study had two aims: identify the presence of Wnt genes through ISH and pharmacological drug injections, and test differentially expressed genes in three developmental stages, between wing types, and between wing compartments of E. clarus using RNA-sequencing. I hypothesized, that a simple yet modular set of genes was involved in wing pattern development of the skipper, similar to that of nymphalid butterflies and that genes such as WntA and Ubx would be differentially expressed as in nymphalids. ISH confirmed the presence of two Wnt genes and RNA-seq provided evidence of differential expression, suggesting that the genes underlining wing pattern diversity are being shared between these two distantly related families....