京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19958号 / 理博第4225号 / 新制||理||1607(附属図書館) / 33054 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions. Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival. Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

<p> Comprehensive mastery of modern genetics involves a myriad of data processing and analytic techniques; these approaches vary because some genetic conditions are the result of single gene mutations that alter protein function, while other more complex diseases and traits are influenced by many genes. This dissertation will undertake investigation of the heritability and genetic risk of cranial cruciate ligament rupture in Labrador Retrievers, a congenital hypomyelinating polyneuropathy in four Golden Retrievers, and the potential usage of telomere length as a biomarker of welfare in dogs housed in commercial breeding facilities. </p> <p>In the first disease studied, 333 Labrador Retrievers with known torn or healthy cranial cruciate ligament(s) were genotyped via SNP array. Heritability of this polygenic trait was calculated using a variety of programs and including different fixed effects. Overall, heritability was high, ranging from 0.550 to 0.893, with sex and sterilization at a young age (≤12 months) strongly influencing risk of cranial cruciate ligament rupture. Neither genome-wide association analyses using this novel dataset of 333 Labrador Retrievers, nor additional analyses combining this data with publicly-available data, identified any significantly associated SNPs. However, the most associated SNPs were located near biologically relevant genes, such as <em>COL1A2</em> (a collagen gene) and <em>ITGA11</em> (a protein that binds to collagen), as well as genes encoding sex hormone receptors, such as <em>FSHR </em>and <em>LHCCGR</em>. Splitting the data in an attempt to predict phenotypes based on genotype was unsuccessful. Future work focused on parsing out genetic influencers of cranial cruciate ligament rupture risk should continue to collect sex, sterilization status, and age at sterilization data, and larger collaborations and use of publicly-available data will be required to increase the data robustness.</p> <p>For the second study, DNA from four unrelated Golden Retrievers diagnosed with congenital hypomyelinating polyneuropathy via neurological examination, electrodiagnostic evaluation, and peripheral nerve pathology were explored for genetic causes. Whole-genome sequencing was performed on all four dogs to identify potential causative variants. When compared to WGS from >1,000 other dogs who were presumably unaffected by this rare disease, likely causative variants were identified in all four dogs. Two cases shared a homozygous <em>MTMR2 </em>splice donor site variant, with a stop codon introduced within six codons following the inclusion of the intron. One case had a heterozygous <em>MPZ</em> missense mutation leading to an isoleucine to threonine substitution. The last case had a homozygous <em>SH3TC2</em> nonsense variant predicted to truncate approximately one-half of the protein. Haplotype analyses using 524 Golden Retrievers indicated that these variants emerged recently. Each of these variants occurred in genes that are associated with the human Charcot-Marie-Tooth group of heterogeneous peripheral nervous system diseases. Testing a population of unrelated Golden Retrievers (n > 200) did not identify any other dogs with these variants, though breeders should be cautious to avoid propagating these alleles.</p> <p>Finally, the last study within this dissertation investigates the relationship between telomere length and metrics such as age, breed, environment, average breed lifespan, parity, and response to a mild social stressor in a population of dogs housed at commercial breeding facilities. FIDO scores (behavioral phenotypes) were collected for all dogs as a measurement of response to a social stressor. This study is not yet complete; many more dogs remain to be recruited in the near future. Telomere lengths were measured using qPCR and compared to a single-copy gene, <em>36B4</em>, for 309 dogs representing 37 breeds or breed crosses. Age was not significantly associated with telomere length after making appropriate corrections (p-value = 0.077). Breed and facility were significantly associated with telomere length after corrections (p-value = 0.010 and <2.2E-16, respectively). Neither parity nor average breed lifespan were associated with telomere length, however, response to a mild social stressor was, with dogs who responded positively having significantly longer telomeres than dogs who responded negatively across all analyses. This preliminary data indicates that, within this population, breed, environment, and response to stress have strong influences on telomere length, while parity and average breed lifespan did not. As this work continues, increased sample sizes will lead to increased power for detecting associations. Future work should examine these identified relationships in other populations of dogs.</p> <p>Taken together, these studies encompass phenotypes of various complexity, and each study encompassed different methodologies utilized in modern canine genetics. The overall goal for this work was to improve canine health, with the potential for translational implications to human health. The identification of genetic markers associated with or causative of disease, or indicative of health and welfare traits, is necessary for reducing the prevalence of disease and increasing the knowledge of welfare metrics in canines, respectively.</p>

Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.

No description available.

<p>TRF1, a shelterin protein, is a negative mediator of telomere length maintenance. Phosphorylation has been shown to play an important role in modulating TRF1 function. T137 and S249 of TRF1 have been indentified to be candidate phosphorylation sites in vivo, and one of my thesis objectives was to examine their role in regulating TRF1 function. Both T137 and S249 have each been changed to either alanine (nonphosphorylatable) or phosphomimic mutation. The TRF1 mutants were introduced into a TRF1-depleted cell line. Southern analysis revealed that neither T137 nor S249 of TRF1 is involved in telomere length maintenance. Immunoprecipitation studies showed that T137 and S249 are not required for TRF1 interaction with TIN2. In vitro gel-shift assays indicated that T137 and S249 are not important for TRF1 binding to telomeric DNA. Taken together, these results suggest that T137 and S249 may not be required for TRF1 function in telomere length maintenance. Human Est1A has been suggested to play a role in telomere length maintenance. To identify the domain of hEst1A involved in telomere length maintenance, a number of deletion constructs were generated and retrovirally introduced into HT1080 cells. Southern analysis revealed that the RID domain may positively regulate telomere length maintenance whereas the first 220 amino acids at the N-terminus may be a negative mediator of telomere length maintenance. In S. cerevisiae, Est1 recruits telomerase to telomeres in a Tel1- (homolog of ATM) and MRX-dependent manner. To assess whether atm-1 and smg-6 may function in the same genetic pathway of regulation of telomere length in C.elegans, the single mutant strain atm-1(gk186) was crossed with three of smg-6 mutant strains (tm1308, ok1794 and r896) to generate double mutants. Southern analysis revealed that deletion of ATM-1 or SMG-6 (tm1308) results in telomere shortening, suggesting that atm-1 and smg-6 may function in the same genetic pathway to regulate telomere length maintenance.</p> / Master of Science (MSc)

Conventional housing environments for broiler chickens and commercial laying hens are often barren, high-density environments with an emphasis on production efficiency. These housing conditions limit birds' ability to display species-specific behaviors, can negatively impact health, and may contribute to negative cumulative experience. Cumulative experience is the culmination of all positive and negative experienced during an animal's lifetime. However, cumulative experience is difficult to quantify, as no validated measures of cumulative experience exist. Additionally, existing measures of negative animal experience mostly rely on interpretations of animal behavior which can be subjective, time consuming, and difficult to interpret. Therefore, there is scientific need for objective measures that can detect cumulative experience in poultry. Secretory and plasma Immunoglobulin A (IgA), telomere length, feather corticosterone concentrations, and attention bias testing all seem to respond to positive and negative experiences in humans or other non-human animal species, indicating that they may be useful as measures for poultry. Therefore, the objective of this thesis was to determine if these novel measures could be used as indicators of cumulative experience in broiler chickens and laying hens. In chapter 3, secretory and plasma IgA concentrations were measured in broilers raised in either high-complexity or low-complexity environments under either high or low stocking density over three replicated experiments. Birds housed in highly complex environments showed higher concentrations of plasma IgA compared to birds housed in low-complexity environments at day 48 of age, indicating reduced chronic stress in the former. Additionally, day 48 secretory IgA concentrations were decreased in birds housed in high-density environments compared to birds housed in low density environments, indicating birds from high-density environments were more chronically stressed. In chapter 4, gonad and kidney telomere length was measured to determine cumulative experience in broilers raised in the same housing conditions and replicated experiments of chapter 3. Treatment did not impact gonad telomere length, in line with expectations as gonads contain stem cells which produce high concentrations of telomerase. Birds housed in high-complexity pens had longer kidney telomeres compared to birds in low-complexity pens, indicating high-complexity birds had more positive cumulative experience. Stocking density did not impact kidney telomere length. In chapter 5, attention bias, tonic immobility, plasma and secretory IgA concentrations, and feather corticosterone concentrations were determined in laying hens raised in conventional cages or enriched floor pens. Birds in enriched floor pens showed increased attention bias, decreased tonic immobility, increased secretory IgA concentrations at week 22 of age, and decreased feather corticosterone concentrations compared to caged hens. These results indicate that compared to conventional cages, enriched pens in this study improved immune systems, reduced chronic stress, reduced fear, but increased anxiety in hens. In conclusion, secretory and plasma IgA and telomere length show appropriate contrast in response to broiler chicken housing conditions. However, additional work needs to be done before these measures can be widely used as measures of cumulative experience in poultry. Furthermore, attention bias, secretory IgA, and feather corticosterone showed an appropriate contrast between chronic stress responses in laying hens, but confirmation is needed in other contexts. Overall, the results indicate a beneficial relationship between environmental complexity and poultry welfare physiology and affective state, with the exception for anxiety in laying hens. Thus, providing an enriched environment can improve the welfare of commercial poultry and result in positive cumulative experience in most situations. Additionally, these results indicate that stocking density is a negative environment in broilers but potentially less intense than previously thought under experimental conditions. The assessment of behavioral and physiological measures of cumulative and positive animal experience should be included in experiments seeking to determine the impacts of environmental or management conditions to determine the broader impacts on poultry welfare. / Doctor of Philosophy / Conventional housing systems of broiler chickens (raised for meat) and laying hens (raised for egg production) can negatively impact their welfare. Animal welfare, defined as an animal's ability to interact with and cope with their environment, is an individual experience for each animal and fluctuates on a scale from very negative to very positive. Traditionally, measurements of animal welfare have focused only on avoiding the negative aspects of animal welfare such as fear, distress (negative stress), hunger, thirst, pain, and suffering. However, it is important that animals are provided opportunities to experience positive animal welfare to provide a life worth living. So, when measuring animal welfare, all positive and negative experiences (termed cumulative experience) should be included to form an accurate picture of an animal's welfare. However, no validated measures of cumulative experience exist in non-human animals. However, recently, several potential measures of cumulative experience have been proposed in human and non-human animals including secretory and plasma IgA, telomere length, feather corticosterone, and attention bias testing. So, the objective of this thesis was to determine if these proposed measures can be used to determine cumulative experience in commercial broilers and laying hens. In chapters 3 and 4, we investigated if secretory and plasma IgA concentrations (measure of chronic stress; chapter 3) and telomere length (measure of cumulative experience; chapter 4) responded to environmental complexity (positive stimulus) and stocking density (negative stimulus) over three replicated experiments. Broilers were housed in a 2 × 2 factorial study of either high or low complexity or high or low density. This resulted in four treatment groups of high-complexity/high-density, low-complexity/low-density, high-complexity/low-density, and low-complexity/high-density. During chapter 3, environmental complexity increased concentrations of plasma IgA, indicating that birds from high-complexity pens were under less chronic stress compared to birds from low-complexity pens. Alternatively, high density decreased secretory IgA, indicating that birds from high-density pens were under a more chronic stress than birds from low density pens. In chapter 4, environmental complexity increased telomere length in broilers compared to low-complexity pens indicating that environmental complexity positively impacted cumulative experience. However, stocking density did not impact telomere length, indicating that high density did not negatively impact cumulative experience. In chapter 5, we investigated if attention bias (measure of anxiety), tonic immobility duration (measure of fear), plasma and secretory IgA (chronic stress), and feather corticosterone (chronic stress) responded to environmentally enriched floor pens (positive housing system) and conventional caging (negative housing system). We found that birds housed in enriched floor pens were more anxious (increased attention bias), less fearful (decreased tonic immobility duration), and less chronically stressed (increased SIgA concentrations at week 22 and increased feather corticosterone concentrations) compared to birds housed in conventional cages. Overall, IgA concentrations and telomere length (broilers) and attention bias, secretory IgA concentration, and feather corticosterone concentrations (layers) seem useable as measures of animal experience in commercial poultry. Additionally, these results indicate that positive experience has a positive impact on cumulative experience in commercial poultry. Stocking density also seems to contribute to chronic stress in broilers, indicated by decreased SIgA concentrations, but only during the last few weeks of life. These findings should be confirmed by additional studies before common use as measures of cumulative experience in animals. However, the inclusion of measures of cumulative and positive animal experience should be included in experiment which wish to determine the broad impacts of housing system on non-human animals.