MEASURING MITOCHONDRIAL DNA DAMAGE IN CAENORHABDITIS ELEGANS, IMPLICATIONS FOR AGEING AND HEALTHSPAN

Ph.DDOCTOR OF PHILOSOPH

3,5-Dinitro­pyridin-4(1H)-one monohydrate

The three independent organic mol­ecules of 3,5-dinitro­pyridin-4(1H)-one monohydrate, C5H3N3O5·H2O, each feature an N—H⋯Owater hydrogen bond. Each water mol­ecule serves as hydrogen-bond donor to two carbonyl O atoms; these hydrogen bonds give rise to a layer motif. Two of the three formula units lie on special positions of site symmetry 2

Maximizing signal-to-noise ratio in the random mutation capture assay

The ‘Random Mutation Capture' assay allows for the sensitive quantitation of DNA mutations at extremely low mutation frequencies. This method is based on PCR detection of mutations that render the mutated target sequence resistant to restriction enzyme digestion. The original protocol prescribes an end-point dilution to about 0.1 mutant DNA molecules per PCR well, such that the mutation burden can be simply calculated by counting the number of amplified PCR wells. However, the statistical aspects associated with the single molecular nature of this protocol and several other molecular approaches relying on binary (on/off) output can significantly affect the quantification accuracy, and this issue has so far been ignored. The present work proposes a design of experiment (DoE) using statistical modeling and Monte Carlo simulations to obtain a statistically optimal sampling protocol, one that minimizes the coefficient of variance in the measurement estimates. Here, the DoE prescribed a dilution factor at about 1.6 mutant molecules per well. Theoretical results and experimental validation revealed an up to 10-fold improvement in the information obtained per PCR well, i.e. the optimal protocol achieves the same coefficient of variation using one-tenth the number of wells used in the original assay. Additionally, this optimization equally applies to any method that relies on binary detection of a small number of template

Effects of Lithium on Age-related Decline in Mitochondrial Turnover and Function in Caenorhabditis elegans

Aging has been associated with the accumulation of damages in molecules and organelles in cells, particularly mitochondria. The rate of damage accumulation is closely tied to the turnover of the affected cellular components. Perturbing mitochondrial turnover has been shown to significantly affect the rate of deterioration of mitochondrial function with age and to alter lifespan of model organisms. In this study, we investigated the effects of upregulating autophagy using lithium in Caenorhabditis elegans. We found that lithium treatment increased both the lifespan and healthspan of C. elegans without any significant change in the mortality rate and oxidative damages to proteins. The increase in healthspan was accompanied by improved mitochondrial energetic function. In contrast, mitochondrial DNA copy number decreased faster with age under lithium. To better understand the interactions among mitochondrial turnover, damage, and function, we created a mathematical model that described the dynamics of functional and dysfunctional mitochondria population. The combined analysis of model and experimental observations showed how preferential (selective) autophagy of dysfunctional mitochondria could lead to better mitochondrial functionality with age, despite a lower population size. However, the results of model analysis suggest that the benefit of increasing autophagy for mitochondrial function is expected to diminish at higher levels of upregulation due to a shrinking mitochondrial populatio

Masked Gradient-Based Causal Structure Learning

This paper studies the problem of learning causal structures from observational data. We reformulate the Structural Equation Model (SEM) in an augmented form with a binary graph adjacency matrix and show that, if the original SEM is identifiable, then this augmented form can be identified up to super-graphs of the true causal graph under mild conditions. Three methods are further provided to remove spurious edges to recover the true graph. We next utilize the augmented form to develop a masked structure learning method that can be efficiently trained using gradient-based optimization methods, by leveraging a smooth characterization on acyclicity and the Gumbel-Softmax approach to approximate the binary adjacency matrix. It is found that the obtained entries are typically near zero or one, and can be easily thresholded to identify the edges. We conduct experiments on synthetic and real datasets to validate the effectiveness of the proposed method and show that the method can readily include different smooth functions to model causal relationships

Mitochondrial DNA Damage Does Not Determine C. elegans Lifespan

The mitochondrial free radical theory of aging (mFRTA) proposes that accumulation of oxidative damage to macromolecules in mitochondria is a causative mechanism for aging. Accumulation of mitochondrial DNA (mtDNA) damage may be of particular interest in this context. While there is evidence for age-dependent accumulation of mtDNA damage, there have been only a limited number of investigations into mtDNA damage as a determinant of longevity. This lack of quantitative data regarding mtDNA damage is predominantly due to a lack of reliable assays to measure mtDNA damage. Here, we report adaptation of a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the detection of sequence-specific mtDNA damage in C. elegans and apply this method to investigate the role of mtDNA damage in the aging of nematodes. We compare damage levels in old and young animals and also between wild-type animals and long-lived mutant strains or strains with modifications in ROS detoxification or production rates. We confirm an age-dependent increase in mtDNA damage levels in C. elegans but found that there is no simple relationship between mtDNA damage and lifespan. MtDNA damage levels were high in some mutants with long lifespan (and vice versa). We next investigated mtDNA damage, lifespan and healthspan effects in nematode subjected to exogenously elevated damage (UV- or γ-radiation induced). We, again, observed a complex relationship between damage and lifespan in such animals. Despite causing a significant elevation in mtDNA damage, γ-radiation did not shorten the lifespan of nematodes at any of the doses tested. When mtDNA damage levels were elevated significantly using UV-radiation, nematodes did suffer from shorter lifespan at the higher end of exposure tested. However, surprisingly, we also found hormetic lifespan and healthspan benefits in nematodes treated with intermediate doses of UV-radiation, despite the fact that mtDNA damage in these animals was also significantly elevated. Our results suggest that within a wide physiological range, the level of mtDNA damage does not control lifespan in C. elegans

Expression and non-chromatographic purification of 1,3-propanediol oxidoreductase in Escherichia coli

The gene dhaT from Klebsiella pneumoniae encodes 1,3-propanediol oxidoreductase (PDOR). Thermally responsive elastin-like polypeptides (ELPs) was used as a fusion tag to purify the proteins (PDOR). The ELP gene was attached to dhaT and ligated into the pET-22b vector. Different NaCl concentrations were employed to decrease the transition temperature (Tt) which was diminished as salt concentration increased. The optimal final concentration of NaCl was 1 M and the corresponding Tt was 39.5\ubaC. Enzymatic assays were determined via every step for purification of fusion PDOR. PDOR showed good stability during the purification process, the specific activity in the first and second round of inverse transition cycling (ITC) was 276.1 \ub1 13.3 and 213.3 \ub1 10.8 U/mg, respectively. The ELPs fusion PDOR was superior to histidine tagged PDOR in both yield and activity after the purification

Duration of untreated bipolar disorder: A multicenter study

Little is known about the demographic and clinical differences between short and long duration of untreated bipolar disorder (DUB) in Chinese patients. This study examined the demographic and clinical features of short (≤2 years) and long DUB (\u3e2 years) in China. A consecutively recruited sample of 555 patients with bipolar disorder (BD) was examined in 7 psychiatric hospitals and general hospital psychiatric units across China. Patients’ demographic and clinical characteristics were collected using a standardized protocol and data collection procedure. The mean DUB was 3.2 ± 6.0 years; long DUB accounted for 31.0% of the sample. Multivariate analyses revealed that longer duration of illness, diagnosis of BD type II, and earlier misdiagnosis of BD for major depressive disorder or schizophrenia were independently associated with long DUB. The mean DUB in Chinese BD patients was shorter than the reported figures from Western countries. The long-term impact of DUB on the outcome of BD is warranted

Expression of Avian Influenza Virus Receptors and H5N1 Virus Infection In Human Respiratory Tract

Oral Presentations - Virus Structure/Function and Receptor BindingReceptor specificity restricts influenza virus cross species transmission, with SAα2,6 Gal and SAα2,3 Gal sialic acids recognized by human and avian influenza viruses, respectively. This study investigated the distribution of these two species of sialic acids in the human respiratory tract. The SAα2,3 Gal species was infrequently detected in the upper respiratory tract, but prevalent in the lower part, while the SAα2,6 Gal species is more common in the upper respiratory tract. Though alveolus cells are more susceptible than trachea and bronchus epithelial cells to avian influenza H5N1 virus infection in the ex vivo experiment, H5N1 virus was found to infect the upper respiratory tract epithelial cells of a human case. It was also found that H5N1 virus infection occurs in the epithelial cells of the respiratory tract which do not express detectable SA α2,3Gal. These observations may be important to investigate further if the currently-observed limited human to human transmission by H5N1 virus is associated with the differential expression of SAα2,3 Gal in human upper respiratory tract among individuals.postprin

Assessing the Effectiveness of Environmental Training for Diving Tourists Using the DEA Model

This study proposes an approach based on data envelopment analysis to assess the effectiveness of environmental training for tourists. Most studies have considered only outcomes (i.e., the continuance or halting of improper behavior towards the environment) to represent the effectiveness of environmental training but this approach does not consider the amount of resources that have been applied in the process. The model utilizes input and output factors to estimate the index of effectiveness. We used a survey of underwater tourist activity to test the proposed model in the empirical evaluation and explored both the internal and external influences on the effectiveness