Physicochemical characterization of immortal strand DNA

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references.Adult tissue differentiation involves the generation of distinct cell types from adult stem cells (ASCs). Current understanding of tissue differentiation mechanisms is based on studies of protein and RNAs that asymmetrically segregate between daughter cells during embryogenesis. Whether or not other types of biomolecules segregate asymmetrically has not been widely studied. In 1975, John Cairns proposed that ASCs preferentially segregate the oldest parental template DNA strands to themselves and pass on newly replicated DNA strands to their differentiating progeny in order to protect the stem cell from inheriting DNA replication mutations. This laboratory has shown non-random chromosome segregation in murine fetal fibroblasts that model asymmetric self-renewal like ASCs. In these cells, chromosomes that contain the oldest DNA strands co-segregate to the cycling daughter stem-like cells, while chromosomes with more recently replicated DNA segregate to the non-stem cell daughters. Previously, cytological methods were reported to elucidate non-random segregation in these cells. This dissertation research provides additional confirmation of the mechanism using physicochemical methods. Specifically, buoyant density-shift experiments in equilibrium CsCl density gradients were used to detect co-segregated "immortal DNA strands" based on incorporation of the thymidine base analogue bromodeoxyuridine. In addition, DNA from cells undergoing non-random mitotic chromosome segregation was analyzed for unique DNA base modifications and global structural modifications (by HPLC and melting temperature analyses). To date, these studies show no significant differences compared to control randomly segregated DNA. Components of the mitotic chromosome separation(cont.) apparatus that might play a role in the co-segregation mechanism were also evaluated. Two homologous proteins, essential for proper chromosome segregation and cytokinesis, Aurora A kinase and Aurora B kinase, were highly reduced in expression in cells retaining immortal DNA strands and may indicate a role for them in the immortal strand mechanism. These studies independently confirm the immortal strand mechanism and provide methods for its detection in other cell lines. In addition, observed changes in chromosome segregation proteins that are potential candidates for involvement in the mechanism have revealed a new area of investigation in the laboratory. These findings are relevant to understanding normal tissue development, cancer, and aging.y Janice A. Lansita.Ph.D

Glial contribution to excitatory and inhibitory synapse loss in neurodegeneration

Synapse loss is an early feature shared by many neurodegenerative diseases, and it represents the major correlate of cognitive impairment. Recent studies reveal that microglia and astrocytes play a major role in synapse elimination, contributing to network dysfunction associated with neurodegeneration. Excitatory and inhibitory activity can be affected by glia-mediated synapse loss, resulting in imbalanced synaptic transmission and subsequent synaptic dysfunction. Here, we review the recent literature on the contribution of glia to excitatory/inhibitory imbalance, in the context of the most common neurodegenerative disorders. A better understanding of the mechanisms underlying pathological synapse loss will be instrumental to design targeted therapeutic interventions, taking in account the emerging roles of microglia and astrocytes in synapse remodeling

Current Strategies in the non-clinical safety assessment of biologics: New Targets, New Molecules, New Challenges (A report of the 2016 Annual US BioSafe General Membership meeting)

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Conference on Harmonization (ICH) S1, S3a, S5, S9 and S11and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species