mRNA/microRNA Profile at the Metamorphic Stage of Olive Flounder (Paralichthys olivaceus)

Flatfish is famous for the asymmetric transformation during metamorphosis. The molecular mechanism behind the asymmetric development has been speculated over a century and is still not well understood. To date, none of the metamorphosis-related genes has been identified in flatfish. As the first step to screen metamorphosis-related gene, we constructed a whole-body cDNA library and a whole-body miRNA library in this study and identified 1051 unique ESTs, 23 unique miRNAs, and 4 snoRNAs in premetamorphosing and prometamorphosing Paralichthys olivaceus. 1005 of the ESTs were novel, suggesting that there was a special gene expression profile at metamorphic stage. Four miRNAs (pol-miR-20c, pol-miR-23c, pol-miR-130d, and pol-miR-181e) were novel to P. olivaceus; they were characterized as highly preserved homologies of published miRNAs but with at least one nucleotide differed. Representative 24 mRNAs and 23 miRNAs were quantified during metamorphosis of P. olivaceus by using quantitative RT PCR or stem-loop qRT PCR. Our results showed that 20 of mRNAs might be associated with early metamorphic events, 10 of mRNAs might be related with later metamorphic events, and 16 of miRNAs might be involved in the regulation of metamorphosis. The data provided in this study would be helpful for further identifying metamorphosis-related gene in P. olivaceus

Stem Cell Maintenance in Naked Mole Rats and Other Longevity Mechanisms in Rodents

This thesis investigated the stem cell biology of the longest-lived rodent the naked mole rat (Heterocephalus glaber, NMR), compared to the short-lived laboratory mouse. The NMR is one of only two eusocial mammals, shows extreme longevity with a maximum lifespan of 32 years and negligible age-associated degeneration. During aging, stem cells play a pivotal role in maintaining tissue homeostasis by replenishing damaged and dead cells with newly differentiated functional cells. Age-associated stem cell dysfunction is a driving force of aging. What stem cell capacity the NMR has and how the NMR maintains their stem cell pool is unknown. In this thesis, I comparatively studied the hematopoietic stem cell (HSC) properties in the NMR and the mouse. I employed BrdU label retention assays to label HSC and track the HSC turnover. I was able to show that the BrdU label-retaining cells were responsive to fluorouracil induced bone marrow injury. I have discovered that the NMR HSC localizes to a niche with extremely high levels of high molecular weight hyaluronic acid (HA), displays slow turnover and extreme quiescence, compared to the mouse HSC. I have demonstrated that the niche HA levels reversely relate to HSC reactive oxidative species (ROS) levels. In addition, overexpressing NMR HAS2 in mice reduces ROS in the HSC and expands the HSC pool by about 3-fold. I have also shown the inhibition of HA synthesis by 4-MU impaired hematopoiesis in NMRs. With these findings, I propose a model of HSC maintenance that highlights the key role of niche HA in maintaining the quiescent HSC pool. Additionally, I describe the NMR iPSC reprogramming done with extensive collaboration with Dr. Li Tan. We have found the NMR fibroblasts are resistant to both the mouse and the NMR defined factors induced reprogramming, either in primed or naïve culture conditions. We have screened factors enhancing reprogramming and found the large T antigen drastically increased iPSC reprogramming efficiency. We have found that the NMR fibroblasts have a more repressive Rb signal pathway and large T induces massive opening of more closed promoter regions in the NMR, compared to the mouse. These results suggest that NMR displays a more stable epigenome that resists iPSC reprogramming. In another study that I collaborated with Dr. Jorge Azpurua, we have discovered that the NMR shows about 10 times higher translation fidelity than the mouse. However, whether the translation fidelity correlates with species maximum lifespan is unknown. Thus, I examined translation fidelity in 17 rodent species with diverse maximum lifespans. I have found that the fidelity at the first and second codon positions strongly correlates with species maximum lifespan, and that correlation remains significant after phylogenetic correction. This finding suggests that translation fidelity plays a novel role in longevity. Lastly, I had been managing the NMR colonies during my study and developed strategies to improve the husbandry and breeding of captive NMR colonies. As breeding and keeping NMRs in captivity is challenging to researchers, the slow breeding and low survival of NMRs under laboratory condition limits the NMR research. I have optimized the colony setting which allows NMR colonies to settle down more rapidly and established different chambers for different functions. I have found that pairing young NMRs, but not younger than 2 years old, could result in higher successful rate of establishing new colonies. I have also successfully cross-fostered NMR pups in a foreign colony. All of these strategies will help researchers struggling with breeding NMRs in captivity

Stem cell maintenance in naked mole rats and other longevity mechanisms in rodents

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2018.This thesis investigated the stem cell biology of the longest-lived rodent the naked mole rat (Heterocephalus glaber, NMR), compared to the short-lived laboratory mouse. The NMR is one of only two eusocial mammals, shows extreme longevity with a maximum lifespan of 32 years and negligible age-associated degeneration. During aging, stem cells play a pivotal role in maintaining tissue homeostasis by replenishing damaged and dead cells with newly differentiated functional cells. Age-associated stem cell dysfunction is a driving force of aging. What stem cell capacity the NMR has and how the NMR maintains their stem cell pool is unknown. In this thesis, I comparatively studied the hematopoietic stem cell (HSC) properties in the NMR and the mouse. I employed BrdU label retention assays to label HSC and track the HSC turnover. I was able to show that the BrdU label-retaining cells were responsive to fluorouracil induced bone marrow injury. I have discovered that the NMR HSC localizes to a niche with extremely high levels of high molecular weight hyaluronic acid (HA), displays slow turnover and extreme quiescence, compared to the mouse HSC. I have demonstrated that the niche HA levels reversely relate to HSC reactive oxidative species (ROS) levels. In addition, overexpressing NMR HAS2 in mice reduces ROS in the HSC and expands the HSC pool by about 3-fold. I have also shown the inhibition of HA synthesis by 4-MU impaired hematopoiesis in NMRs. With these findings, I propose a model of HSC maintenance that highlights the key role of niche HA in maintaining the quiescent HSC pool. Additionally, I describe the NMR iPSC reprogramming done with extensive collaboration with Dr. Li Tan. We have found the NMR fibroblasts are resistant to both the mouse and the NMR defined factors induced reprogramming, either in primed or naïve culture conditions. We have screened factors enhancing reprogramming and found the large T antigen drastically increased iPSC reprogramming efficiency. We have found that the NMR fibroblasts have a more repressive Rb signal pathway and large T induces massive opening of more closed promoter regions in the NMR, compared to the mouse. These results suggest that NMR displays a more stable epigenome that resists iPSC reprogramming. In another study that I collaborated with Dr. Jorge Azpurua, we have discovered that the NMR shows about 10 times higher translation fidelity than the mouse. However, whether the translation fidelity correlates with species maximum lifespan is unknown. Thus, I examined translation fidelity in 17 rodent species with diverse maximum lifespans. I have found that the fidelity at the first and second codon positions strongly correlates with species maximum lifespan, and that correlation remains significant after phylogenetic correction. This finding suggests that translation fidelity plays a novel role in longevity. Lastly, I had been managing the NMR colonies during my study and developed strategies to improve the husbandry and breeding of captive NMR colonies. As breeding and keeping NMRs in captivity is challenging to researchers, the slow breeding and low survival of NMRs under laboratory condition limits the NMR research. I have optimized the colony setting which allows NMR colonies to settle down more rapidly and established different chambers for different functions. I have found that pairing young NMRs, but not younger than 2 years old, could result in higher successful rate of establishing new colonies. I have also successfully cross-fostered NMR pups in a foreign colony. All of these strategies will help researchers struggling with breeding NMRs in captivity

Short-term calorie restriction enhances DNA repair by non-homologous end joining in mice

Abstract Calorie restriction (CR) improves health, reduces cancer incidence and extends lifespan in multiple organisms including mice. CR was shown to enhance base excision repair and nucleotide excision repair pathways of DNA repair, however, whether CR improves repair of DNA double-strand breaks has not been examined in in vivo system. Here we utilize non-homologous end joining (NHEJ) reporter mice to show that short-term CR strongly enhances DNA repair by NHEJ, which is associated with elevated levels of DNA-PK and SIRT6

Effect of Vibration Procedure on Particle Distribution of Cement Paste

Vibration procedures significantly affect the performances of cement-based materials. However, studies on the distribution of certain particles within cement-based materials are limited due to the complexity and difficulty of identifying each specific particle. This paper presents a new method for simulating and quantifying the movements of particles within cement paste through the use of “tagged materials”. By separating the tagged particles from the cement paste after vibration, the distribution of the particles in the cement paste can be calculated statistically. The effect of the vibration time and frequency, fresh behavior, and powder characteristics of cement paste on particle motions are investigated. The results demonstrate that when the vibration exceeds 1800 s, it induces a significant uneven dispersion of microparticles. This effect is more pronounced at low viscosities (200 Hz). Larger and denser particles exhibit greater dispersion. This method provides a valuable tool for investigating the theory of particle motion in cement paste, which is crucial for understanding the influence of vibration on the properties of cement-based materials