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Newts, such as Notophthalmus viridescens, can regenerate many structures after amputation or injury and have also shown a refractory response to the formation of cancer in tissues that have regenerative capabilities. The mechanisms behind this latter ability have surprisingly not been studied. In the current study, N. viridescens were exposed to a variety of carcinogens in tissue that cannot regenerate with the intention of inducing tumour formation. After testing multiple carcinogens, multiple sites of injection, and two different modes of delivery, no tumours were generated. Consequently, in vitro assays were developed in order to better understand this ability of newt cells to evade transformation. Mouse and newt muscle cells were exposed to DNA damaging agents, such as irradiation and carcinogens, in culture and their response was monitored with respect to the DNA damage response proteins γ-H2AX, p53, and phospho-p53. These proteins are important as they help prevent mutations in the genome from being passed on to daughter cells and potentially generating cells that proliferate uncontrollably, a hallmark of cancer. Preliminary results suggest that after irradiation, γ-H2AX is present in newt cells for a considerably longer period of time in comparison to mouse cells. p53, as well as phospho-p53, appear to be present at a basal level before and after irradiation in newt cells, whereas mouse cells have a distinct increase upon damage and decrease upon repair. The carcinogen treatments also suggest that newt cells have basal levels of expression of these proteins prior to treatment. These studies suggest that newt cells may have a unique profile of these DNA damage response proteins and may be “primed” to repair any future damage. This is a good first step in understanding what is likely a very complicated explanation for newts’ refractory response to cancer formation.

Newts, such as Notophthalmus viridescens, can regenerate many structures after amputation or injury and have also shown a refractory response to the formation of cancer in tissues that have regenerative capabilities. The mechanisms behind this latter ability have surprisingly not been studied. In the current study, N. viridescens were exposed to a variety of carcinogens in tissue that cannot regenerate with the intention of inducing tumour formation. After testing multiple carcinogens, multiple sites of injection, and two different modes of delivery, no tumours were generated. Consequently, in vitro assays were developed in order to better understand this ability of newt cells to evade transformation. Mouse and newt muscle cells were exposed to DNA damaging agents, such as irradiation and carcinogens, in culture and their response was monitored with respect to the DNA damage response proteins γ-H2AX, p53, and phospho-p53. These proteins are important as they help prevent mutations in the genome from being passed on to daughter cells and potentially generating cells that proliferate uncontrollably, a hallmark of cancer. Preliminary results suggest that after irradiation, γ-H2AX is present in newt cells for a considerably longer period of time in comparison to mouse cells. p53, as well as phospho-p53, appear to be present at a basal level before and after irradiation in newt cells, whereas mouse cells have a distinct increase upon damage and decrease upon repair. The carcinogen treatments also suggest that newt cells have basal levels of expression of these proteins prior to treatment. These studies suggest that newt cells may have a unique profile of these DNA damage response proteins and may be “primed” to repair any future damage. This is a good first step in understanding what is likely a very complicated explanation for newts’ refractory response to cancer formation.

Newts, such as Notophthalmus viridescens, can regenerate many structures after amputation or injury and have also shown a refractory response to the formation of cancer in tissues that have regenerative capabilities. The mechanisms behind this latter ability have surprisingly not been studied. In the current study, N. viridescens were exposed to a variety of carcinogens in tissue that cannot regenerate with the intention of inducing tumour formation. After testing multiple carcinogens, multiple sites of injection, and two different modes of delivery, no tumours were generated. Consequently, in vitro assays were developed in order to better understand this ability of newt cells to evade transformation. Mouse and newt muscle cells were exposed to DNA damaging agents, such as irradiation and carcinogens, in culture and their response was monitored with respect to the DNA damage response proteins γ-H2AX, p53, and phospho-p53. These proteins are important as they help prevent mutations in the genome from being passed on to daughter cells and potentially generating cells that proliferate uncontrollably, a hallmark of cancer. Preliminary results suggest that after irradiation, γ-H2AX is present in newt cells for a considerably longer period of time in comparison to mouse cells. p53, as well as phospho-p53, appear to be present at a basal level before and after irradiation in newt cells, whereas mouse cells have a distinct increase upon damage and decrease upon repair. The carcinogen treatments also suggest that newt cells have basal levels of expression of these proteins prior to treatment. These studies suggest that newt cells may have a unique profile of these DNA damage response proteins and may be “primed” to repair any future damage. This is a good first step in understanding what is likely a very complicated explanation for newts’ refractory response to cancer formation.

Newts, such as Notophthalmus viridescens, can regenerate many structures after amputation or injury and have also shown a refractory response to the formation of cancer in tissues that have regenerative capabilities. The mechanisms behind this latter ability have surprisingly not been studied. In the current study, N. viridescens were exposed to a variety of carcinogens in tissue that cannot regenerate with the intention of inducing tumour formation. After testing multiple carcinogens, multiple sites of injection, and two different modes of delivery, no tumours were generated. Consequently, in vitro assays were developed in order to better understand this ability of newt cells to evade transformation. Mouse and newt muscle cells were exposed to DNA damaging agents, such as irradiation and carcinogens, in culture and their response was monitored with respect to the DNA damage response proteins γ-H2AX, p53, and phospho-p53. These proteins are important as they help prevent mutations in the genome from being passed on to daughter cells and potentially generating cells that proliferate uncontrollably, a hallmark of cancer. Preliminary results suggest that after irradiation, γ-H2AX is present in newt cells for a considerably longer period of time in comparison to mouse cells. p53, as well as phospho-p53, appear to be present at a basal level before and after irradiation in newt cells, whereas mouse cells have a distinct increase upon damage and decrease upon repair. The carcinogen treatments also suggest that newt cells have basal levels of expression of these proteins prior to treatment. These studies suggest that newt cells may have a unique profile of these DNA damage response proteins and may be “primed” to repair any future damage. This is a good first step in understanding what is likely a very complicated explanation for newts’ refractory response to cancer formation.

A hallmark of retinal diseases is degeneration of neural cells, leading to subsequent vision loss. For such diseases, replenishment of functional neural cells may be an optimal therapy. Unlike humans, the adult red-spotted newt, Notophthalmus viridescens, possesses the remarkable ability to regenerate a complete retina following its removal or injury. The purpose of this study was to develop a reproducible model of retinal damage and regeneration in the newt to understand the process of retinal regeneration. Intense light, shown in other organisms to be a relevant model of visual cell loss, was tested in the newt and resulted in variable loss of retinal function, correlating with the appearance of apoptotic cells. Due to the variability of damage observed, surgical removal of the retina was used to complement the light-damage model. A novel and non-invasive protocol using full-field electroretinography was developed to assess retinal function in vivo following damage. Measures of retinal function with the electroretinogram protocol successfully showed that photoreceptor function is initially lost and subsequently restored during regeneration. These results enhance our understanding of retinal regeneration in the adult newt and serve as a starting point for further studies aimed at determining the molecular mechanisms involved in the regeneration process.

A hallmark of retinal diseases is degeneration of neural cells, leading to subsequent vision loss. For such diseases, replenishment of functional neural cells may be an optimal therapy. Unlike humans, the adult red-spotted newt, Notophthalmus viridescens, possesses the remarkable ability to regenerate a complete retina following its removal or injury. The purpose of this study was to develop a reproducible model of retinal damage and regeneration in the newt to understand the process of retinal regeneration. Intense light, shown in other organisms to be a relevant model of visual cell loss, was tested in the newt and resulted in variable loss of retinal function, correlating with the appearance of apoptotic cells. Due to the variability of damage observed, surgical removal of the retina was used to complement the light-damage model. A novel and non-invasive protocol using full-field electroretinography was developed to assess retinal function in vivo following damage. Measures of retinal function with the electroretinogram protocol successfully showed that photoreceptor function is initially lost and subsequently restored during regeneration. These results enhance our understanding of retinal regeneration in the adult newt and serve as a starting point for further studies aimed at determining the molecular mechanisms involved in the regeneration process.

Unlike mammals, adult salamanders possess an intrinsic ability to regenerate complex organs and tissue types, making them an exciting and useful model to study tissue regeneration. The aims of this thesis are two fold, (1) to develop and characterize a reproducible cardiac regeneration model system in the newt, and (2) to decipher the cellular and molecular underpinnings involved in regeneration. In Paper I of this thesis we developed a novel and reproducible heart regeneration model system in the red-spotted newt and demonstrated for the first time the newt’s ability to regenerate functional myocardial muscle, following resection injury, without scarring. The observed findings coincide with an increase in several developmental cardiac transcription factors, wide-spread cellular proliferation of cardiomyocytes and non-cardiomyocyte populations in the ventricle and reverse-remodeling at later time points during regeneration. Of further interest was the identification of functionally active Islet1+ve and GATA4+ve cardiac precursor cells in regenerating areas. The observation of such cell types further compels the similarity between mammalian cardiac development and newt cardiac regeneration and justifies these animals as suitable model organisms for studying heart regeneration. In Paper II we wanted to decipher the molecular cues possibly driving cardiac regeneration in newts. Here we used qualitative and quantitative methods to delineate the function microRNAs (miRNAs) have in this process. One interesting candidate, miR-128, a known tumor suppressor miRNA and regulator of myogenesis, was found to have a regulatory role in controlling non-cardiomyocyte hyperplasia during newt cardiac regeneration. Of further interest was the discovery of a novel binding site of miR-128 in the 3’UTR of Islet1. We speculate that the natural increase in miR-128 expression levels during cardiac regeneration functions as a fine-tuning mechanism to control cellular proliferation of precursor cells. In Paper III of my thesis we sought to explore if a link exists between RNA editing, a wide-spread post-transcriptional process and regeneration. We observed that A-to-I editing enzymes (ADARs) are present in regenerating newt tissues and the localization of ADAR1 alternates between nuclear and cytoplasmic compartments during regeneration. This activity of ADAR1 during regeneration may be partly responsible for driving the cellular plasticity that is needed during multiple phases of tissue regeneration in the red-spotted newt. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

A hallmark of retinal diseases is degeneration of neural cells, leading to subsequent vision loss. For such diseases, replenishment of functional neural cells may be an optimal therapy. Unlike humans, the adult red-spotted newt, Notophthalmus viridescens, possesses the remarkable ability to regenerate a complete retina following its removal or injury. The purpose of this study was to develop a reproducible model of retinal damage and regeneration in the newt to understand the process of retinal regeneration. Intense light, shown in other organisms to be a relevant model of visual cell loss, was tested in the newt and resulted in variable loss of retinal function, correlating with the appearance of apoptotic cells. Due to the variability of damage observed, surgical removal of the retina was used to complement the light-damage model. A novel and non-invasive protocol using full-field electroretinography was developed to assess retinal function in vivo following damage. Measures of retinal function with the electroretinogram protocol successfully showed that photoreceptor function is initially lost and subsequently restored during regeneration. These results enhance our understanding of retinal regeneration in the adult newt and serve as a starting point for further studies aimed at determining the molecular mechanisms involved in the regeneration process.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.