Homologous recombination is critical for generating genetic variation in living organisms by exchange and rearrangement of DNA. Most of our knowledge about homologous recombination is limited to processes in bacteria or in eukaryotic nuclei. In E. coli, homologous recombination is dependent on the RecA protein. Higher plant chloroplasts have RecA-like strand exchange activity. However, little is known about these mechanisms in higher plant mitochondria. I have detected a RecA-like strand exchange activity in soybean mitochondria. This activity forms joint molecules in the presence of ATP, Mg2+, and homologous DNA substrates. In addition, the E. coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for RecA protein-promoted strand exchange reactions. Our lab has identified an Arabidopsis homologue of E. coli SSB that is targeted to mitochondria (mtSSB). The results of my research shows the mtSSB protein has the same properties as the E. coli SSB protein and it can stimulate the E. coli RecA protein-promoted strand exchange reactions. DNA helicases utilize the energy of ATP to separate the two parental DNA strands at the replicating fork or during recombinational strand exchange. Although higher plant chloroplast helicase activity has been reported, no such activity has heretofore been identified in higher plant mitochondria. We report the characterization of a plant mitochondrial DNA helicase isolated from soybean leaves. ATP is required for this enzyme and this enzyme poorly utilizes any other NTPs or dNTPs. The enzyme requires Mg2+ for activity. This enzyme only has 3' to 5'unwinding activity. The optimal conditions for mitochondrial DNA helicase are 2 mM ATP, 8 to 10 mM Mg2+,100 to 200 mM NaCl and 37-42 oC incubation for one hour or longer time.

Dravet syndrome (DS) is a severe, pediatric-onset epilepsy disorder linked to loss-of-function mutations in the sodium channel gene SCN1B. DS patients have a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). Cardiac arrhythmias have been implicated as a potential cause underlying SUDEP. An exact pathway for how mutations in SCN1B leads to arrhythmia in DS is unclear. One cellular component linked to regulation of cardiac homeostasis are mitochondria, known as “the powerhouse of the cell” due to their ability to produce cellular energy (ATP) via the electron transport chain (ETC). The ETC is a major producer of reactive oxygen species (ROS). Typically, ROS are buffered by cellular antioxidants, to prevent oxidative stress, an imbalance of ROS that can lead to cell damage. Our previous work indicates that cardiac arrhythmias may result from mitochondrial instability and imbalances between ROS production and buffering. We analyzed whether Scn1b-/-mice are susceptible to arrhythmias due to altered mitochondrial ATP generation, ROS production, and compromised cellular antioxidant defenses. We isolated cardiac mitochondria from postnatal day (P) 15-20 KO and Scn1b+/+ (WT) mice. To assess mitochondrial ATP and ROS production, high-resolution respirometry (O2k, Oroboros) was used to measure mitochondrial O2 and H2O2 flux. We used a substrate-uncoupler inhibitor (SUIT) protocol to elucidate flux under different ETC pathways, including Complex I- and II-linked respiration. As a next step, we evaluated expression of superoxide dismutase (Sod) proteins associated with mitochondrial antioxidant defenses, including Cu/Zn-Sod (Sod1) and Mn-Sod (Sod2) in hearts from KO and WT mice pre- (P10) and post- (P17) seizure development. After addition of substrates supporting Complex-II linked respiration (succinate, ADP) there were no differences in O2 flux between mitochondria isolated from KO and WT hearts. Upon further addition of pyruvate to mitochondria to stimulate Complex I, O2 flux was significantly reduced (p < 0.0001) in mitochondria from KO mice, when compared to WT. Moreover, upon titration of rotenone (a Complex I inhibitor) its negative effect on O2 flux was not as substantial in KO mitochondria as in WT, suggesting that mitochondria from KO have deficits in Complex-I linked respiration. Furthermore, we detected significant differences in ROS production by mitochondria isolated from KO animals. Under conditions of reverse electron flow (succinate as substrate), a state where ROS production is highest, H2O2 flux was elevated significantly (p = 0.048) in mitochondria isolated from KO mice, compared to those isolated from WT. During our analysis of Sod expression, we found that Sod1 (p = 0.01) and Sod2 (p = 0.01) expression is significantly decreased at P17 in KO hearts compared to WT. Overall, our results suggest imbalances between mitochondrial activity and antioxidant defenses, which may underlie increased arrhythmia susceptibility in KO mice.

No description available.

BACKGROUND: Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. METHODS: Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen-glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. RESULTS: We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen-glucose deprivation, while STAT3 inhibition reduced the protective effect. CONCLUSIONS: We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation.

Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.

The feasibility of using rat liver mitochondria respiratory parameters as a short-term toxicity test was investigated. Mitochondrial fractions were exposed to six concentrations of five chlorophenols. Respiratory parameters were measured and compared to control experiments. The toxicity of the chlorophenos, measured by the 50% uncoupling concentration (UC50), increased with increasing chloro substitution. The UC50 values for the five chlorophenols were compared to six physicochemical parameters for the same chlorophenols and high degrees of correlation were found (r >/- 0.890). The highest correlation coefficient obtained was with the octanol-water partition coefficient. UC50 values were also compared to nine currently existing short-term toxicity tests. High degrees of correlation were obtained with several of these tests, including bacterial and fish bioassays. From the results of these experiments, the measurement and use of mitochondria respiratory parameters as a short-term toxicity test appears to offer an alternative to currently used short-term toxicity tests, particularly with chemicals having physicochemical characteristics similar to mitochondria uncouplers. / Master of Science

<P> Mitochondria contain DNA which is distinct from nuclear DNA. The capacity of isolated rat liver mitochondria to synthesize RNA and the types of RNA synthesized were examined in order to determine the genetic function of mitochondrial DNA. </p> <p> It was demonstrated that isolated rat liver mitochondria synthesize RNA, incorporating [3H]UTP, [3H] ATP,[3H]CTP and [3H]GTP in a DNA-dependent reaction. In addition, the DNA-independent incorporation of [3H]CTP and [3H]ATP suggested metabolic turnover of the CCA end of mitochondrial tRNA. </p> <P> Analysis of the , newly-synthesized RNA by sucrose density gradient centrifugation and agarose-polyacrylamide gel electrophoresis demonstrated that mitochondrial ribosomal RNA was synthesized in a DNA-dependent process. It is concluded that one of the genetic functions of mitochondrial DNA is to code for mitochondrial ribosomal RNA. </p> / Thesis / Doctor of Philosophy (PhD)

<p> The present investigation was concerned with mitochondrial energy transfer reactions and their relationship to mitochondrial structural integrity. Experiments with azide demonstrated a close relationship between oxidative phosphorylation and large amplitude mitochondrial volume changes. Azide inhibited energy transfer and energy-linked mitochondrial swelling by competing with adenine nucleotide for a site on the terminal phosphorylating enzyme. As a permeant anion azide exerted secondary effects on mitochondrial structure and function.</p> <p> Experiments with mitochondria treated with phlorizin and phloretin emphasized the importance of Mg++ as a controlling factor in maintaining the integrity of mitochondrial energy transfer processes. The results indicated that these compounds interfered directly with oxidative phosphorylation, and that mitochondrial swelling was either a consequence of impaired energy transfer, or a separate phenomenon.</p> / Thesis / Doctor of Philosophy (PhD)

Coronary artery disease (CAD) is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified more than 200 loci associated with CAD. Here, we investigated the functional effects of a locus tagged by rs61969072 (T/G), with the common allele (T) associated with protection from CAD. Expression quantitative trait loci (eQTL) analysis demonstrated a strong association between rs61969072 and CARS2 gene expression, which increased with the T allele, in various human tissues. CARS2 encodes the mitochondrial cysteinyl-tRNA synthetase, an enzyme that attaches cysteine to its cognate tRNA. We hypothesized that CARS2 is a candidate causal gene and that CARS2 confers a protective effect against CAD. We characterized CARS2 expression in macrophages and demonstrated decreased expression in pro-inflammatory M1 macrophages. Gene expression profiling following CARS2 siRNA knockdown revealed increased levels of several pro-inflammatory cytokines. Functional enrichment analysis identified the anti-inflammatory IL-10 signaling pathway, and western blotting showed that CARS2 attenuated IL-10 pathway activation through STAT3 phosphorylation. We also demonstrated that macrophage CARS2 knockdown in a macrophage/smooth muscle cell (SMC) co-culture model elicited gene expression changes indicative of a less contractile, pro-inflammatory, SMC phenotype. We then performed an in-depth analysis of differentially expressed genes following CARS2 knockdown. Several inflammatory pathways and functions were affected, particularly Protein Kinase R (PKR), implicated in Interferon Induction and Antiviral Response. Downstream of PKR is the NF-κB signaling pathway; CARS2 knockdown led to increased NF-κB protein expression but not activation, as measured by a luciferase reporter assay. Finally, we investigated potential mitochondrial mechanisms that could lead to inflammation. Reduced CARS2 levels were found to decrease mitochondrial membrane potential. However, there was a decrease in reactive oxygen species (ROS) levels and no changes in mitochondrial DNA release, metabolism, or mitochondrial bioenergetics. While ROS are often considered harmful due to their role in oxidative damage and inflammation, studies have shown that under certain contexts, ROS can have protective effects. Further studies are required to understand the mechanisms underlying the anti-inflammatory effects of CARS2. Overall, my findings highlight a novel anti-inflammatory role of CARS2 in human macrophages, consistent with the CAD protective effect of a common GWAS-identified variant.

Preeclampsia (PE), a hypertensive disease of pregnancy, occurring at or after gestational week 20. PE can have life threating consequences for both the mother and the baby. PE is a highly heterogenous disease which makes it challenging to identify any effective therapeutic interventions. We previously discovered three molecular subclasses of PE disease. One of these subclasses is characterized by heightened placental inflammation (inflammation-driven PE). Since it is a newly identified form of PE, we currently do not know about the molecular mechanisms driving this inflammation-driven form of PE. Interestingly, we have observed that placentas from this inflammatory PE subclass uniquely express higher levels of NAD+ consuming enzymes- PARPs - and thus exhibit a decrease in NAD+ content. NAD+ is a regulator of cellular energy metabolism and mitochondrial function. Several studies in the non-pregnant populations suggested that pro-inflammatory disease conditions can trigger hyperactivation of NAD+ consuming enzymes causing a depletion in total NAD+ content, leading to mitochondrial dysfunction and organ failure. Thus, we tested the hypothesis that NAD+ depletion causes placental mitochondrial dysfunction in the inflammatory subclass of PE and that boosting NAD+ could prevent development of this form of placental disease. We aimed to profile PARP activity, NAD+ availability, and mitochondrial health in human cases of all three PE subclasses. We examined the causal relationship between inflammation and dysregulated NAD+ signalling in both an in vitro human trophoblast culture model and in a rodent model of inflammation-driven PE. We also evaluated the therapeutic potential of NAD+ booster, nicotinamide riboside (NR) to improve placental health and function in the rodent model of inflammation-driven PE. Our results suggest that along with increased activity of PARP enzymes and decreased NAD+ levels, human inflammatory PE placentas also exhibit decreased levels of mitochondrial proteins and increased oxidative DNA damage. Using an in vitro human placental (HTR8 cell line) inflammation model we showed that increasing NAD+ under an inflammatory condition improved trophoblast mitochondrial and cellular function. Using an in vivo LPS induced rat model of inflammation-driven PE, we demonstrated that NAD+ boosting during pregnancy improved placental mitochondrial function, reduced inflammation and oxidative stress. This subsequently resulted in improved pregnancy outcomes demonstrated by reduced maternal blood pressure, increased placental/fetal weights and increased fetal survival in the LPS model. Overall, this study identifies targeting NAD+ signaling as a promising intervention for PE. NAD+ boosting through NR has been tested in non-pregnant human populations and found to be safe and effective in enhancing NAD+ levels. Thus, findings of this thesis lay the ground to test NAD+ boosting strategies in PE patients in near future.