The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands

The impact of negative selection on thymocyte migration in the medulla

Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 mum in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment

A comparative study of the dissociative patterns of 2-substituted thiazolidine prodrugs

honors thesisCollege of ScienceChemistryJeanette C. RobertsThomas G. RichmondGlutathione is essential in preventing hepatotoxicity. However, it can be depleted as in the case of acetaminophen overdose. The production of glutathion e increases in the presence of cysteine. The potential for toxicity of cysteine is overcome by the administration of cysteine prodrugs in the form of 2-substituted thiazolidines. The 2-substituted cysteine ethyl ester derivatives are essentially pro-prodrugs. They were synthesized in the hope of increasing lipophilicity and thus decreasing excretion through the kidneys. Intracellular esteras es are expected to cleave the ester bond releasing the cysteine prodrug which would further dissociate to release cysteine. These cysteine prodrugs dissociate to release the free amino acid and the aldehyde which corresponds to the substituent at the 2-position of the thiazolidine. Upon studying the dissociation of the 2-substituted thiazolidine esters, peaks appear which correspond to methyl and ethyl alcohols. The appearance the putative alcohol peaks is seen in all the derivatives regardless of the nature of the substituent at the 2-position. In no case was the cysteine ester observed; however, there were indications that the thiazolidine ring was opening to release the aldehyde of the 2-substituent. Clearly the acid and ester derivatives exhibit very different dissociation characteristics although the exact reasons for those differences remain to be elucidated

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37°C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire

Automatic summarization of changes in image sequences using algorithmic information theory

An algorithmic information theoretic method is presented for object-level summarization of meaningful changes in image sequences. Object extraction and tracking data are represented as an attributed tracking graph (ATG), whose connected subgraphs are compared using an adaptive information distance measure, aided by a closed-form multi-dimensional quantization. The summary is the clustering result and feature subset that maximize the gap statistic. The notion of meaningful summarization is captured by using the gap statistic to estimate the randomness deficiency from algorithmic statistics. When applied to movies of cultured neural progenitor cells, it correctly distinguished neurons from progenitors without requiring the use of a fixative stain. When analyzing intra-cellular molecular transport in cultured neurons undergoing axon specification, it automatically confirmed the role of kinesins in axon specification. Finally, it was able to differentiate wild type from genetically modified thymocyte cells