Unlike the central nervous system, axonal regeneration does occur in the peripheral nervous system, however, despite this, functional recovery from nerve transection injury remains dismal. This has been attributed to factors intrinsic to the motor or sensory cell body and to elements in the local site of injury including nerve gaps, scar and a limited time frame in which supportive growth factors and extracellular matrix molecules are expressed. The aim of this thesis is to review the mechanisms behind axonal damage and regeneration in the peripheral nervous system and investigate surgical, pharmacological and biological approaches to overcome limitations in regeneration and functional recovery. By taking a broad approach to the topic, I hoped to gain a greater understanding of the inhibitory and regenerative processes at play and provide a contribution to the understanding in the field of peripheral nerve surgery. / Experimental Surgery

Identifying the mechanisms that regulate neural precursor cell (NPC) proliferation and differentiation is important for understanding CNS development among different vertebrates. My work has focused specifically on understanding how mitogenic factors, like basic fibroblast growth factor (FGF2), regulate the NPC cell cycle. Mitogenic factors and serum are thought to drive cell cycle and therefore proliferation mainly by activating G1-type cyclin-dependent kinases (CDKs). The general hypothesis being addressed here is that FGF2 also promotes cell cycle progression of NPCs through the degradation of the cell cycle inhibitor p27Kip1. I show that, in the presence of FGF2 in vitro, embryonic rat cortical NPCs express high protein levels of the CDC28 protein kinase regulatory subunit 1b (Cks1b), a component of the SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1 for proteasomal degradation. I also show that NPCs maintained in FGF2 express undetectable levels of p27KIP1, while removal of FGF2 results in increased p27Kip1 protein expression and decreased protein expression of Cks1b. RNA expression data shows that Cks1b mRNA is reduced in non-dividing NPCs but is present in dividing NPCs, suggesting that Cks1b is being regulated at the transcriptional level. Analysis of the putative promoter of Cks1b reveals numerous conserved transcription factor consensus sites that could potentially play a role in regulation of Cks1b transcription, including consensus sites for E2F and the cell cycle-dependent element (CDE) cell cycle genes homology region (CHR) tandem repressor element. I use chromatin immunoprecipitation and luciferase assays to identify which E2Fs occupy and regulate the transcription of Cks1b under different conditions of mitogen stimulation. The data show that E2F4 occupies the promoter of Cks1b in non-dividing NPCs while E2F1 binds exclusively in proliferating NPCs. Mutation of either the E2F or CDE/CHR consensus sites independently de-represses the activity of a Cks1b promoter reporter in NPCs in G0/G1, while mutation of both sites delays induction of promoter activity. Finally, I use in ovo electroporation to determine if p27Kip1 has an additional role in neuronal differentiation during early spinal cord development. I show that ectopic expression of p27Kip1 is insufficient to induce neuronal differentiation in spinal cord progenitors.

Skin-derived precursors (SKPs) are multipotent dermal stem cells that reside within a hair follicle niche and that share properties with embryonic neural crest precursors. Here, we have asked whether SKPs and their endogenous dermal precursors originate from the neural crest or whether, like the dermis itself, they originate from multiple developmental origins. To do this, we used two different mouse Cre lines that allow us to perform lineage tracing: Wnt1-cre, which targets cells deriving from the neural crest, and Myf5-cre, which targets cells of a somite origin. By crossing these Cre lines to reporter mice, we show that the endogenous follicle-associated dermal precursors in the face derive from the neural crest, and those in the dorsal trunk derive from the somites, as do the SKPs they generate. Despite these different developmental origins, SKPs from these two locations are functionally similar, even with regard to their ability to differentiate into Schwann cells, a cell type only thought to be generated from the neural crest. Analysis of global gene expression using microarrays confirmed that facial and dorsal SKPs exhibit a very high degree of similarity, and that they are also very similar to SKPs derived from ventral dermis, which has a lateral plate origin. However, these developmentally distinct SKPs also retain differential expression of a small number of genes that reflect their developmental origins. Thus, an adult neural crest-like dermal precursor can be generated from a non-neural crest origin, a finding with broad implications for the many neuroendocrine cells in the body.

Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvii, 292 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Mihael C. Ostrowski, Dept. of Molecular, Cellular and Developmental Biology. Includes bibliographical references (p. 259-292).

Includes bibliographical references (p. 80-91).

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, August 2001. / Includes bibliographical references. Also available on the Internet.

A typical clinical cis-diamminedichloroplatinum (II) (cisplatin) dosing regimen consists of repeated cycles of five to seven daily low dose treatments followed by a one to two week recovery period. While effective, this dosing structure results in a prolonged, and sometimes permanent, infertility in men. Undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. It is proposed that an altered spermatogonial environment during recovery from the initial treatment cycle may drive an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced cell death from subsequent cycles. The undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery period of a clinically-relevant course of one and two cycles of 2.5 mg/kg/d of intraperitoneal cisplatin and were compared to mice receiving an equivalent cumulative dose in a single cycle (5.0 mg/kg/d) and vehicle treated controls. Histological examination of the testicular epithelium revealed an increase in the disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of TUNEL positive cells showed an increase in apoptotic germ cells early in the recovery period in mice exposed to cisplatin compared to control animals. Immunohistochemical (IHC) examination of Foxo1 (undifferentiated spermatogonia marker) showed an increase in the undifferentiated spermatogonia population late in the recovery period in mice exposed to one cycle of 2.5 mg/kg/d, but not following two cycles of 2.5 mg/kg/d. Analysis of BrdU incorporation after dosing indicated a decrease in mitotic activity of early germ cells immediately after cisplatin exposure followed by a return to basal levels by the conclusion of the initial recovery period. No such rebound was observed during the second recovery period. IHC investigation of glial cell line-derived neurotrophic factor (GDNF), a recognized SSC niche factor, revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury. / text

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy