Alumni Dissertations

 

Alumni Dissertations

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  • New Families of Beta-Lactamase Inhibitors

    Author:
    Janet Gonzalez
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Manfred Philipp
    Abstract:

    A traditional approach to improving the efficacy of â-lactam antibiotics has been to modify natural antibiotics by modifying the nucleus and adding various side chains. It is generally believed that â-lactam based antibiotics have a limited future given increased resistance demonstrated by many strains of bacteria. Therefore the lack of performance among commonly used antibiotics against the rise of resistant bacterial strains has propelled researchers to look for novel types of antimicrobial agents and enzyme inhibitors as tools to combat this serious and growing threat. The focus of this research is to investigate novel small-molecules from different families with antibacterial properties such as peptides, aromatic ketones, biphenyl and stilbene compounds and boronic acids that demonstrate an inhibitory effect on class A â-lactamase. Some of these inhibitors were found by screening natural products other were found by screening libraries of compounds and yet others were found by computational approaches involving molecular docking. Of all the compounds tested as long as the molecule was within hydrogen bonding distance of Ser84, Lys87 Ser142, Asn186, Gly252, Glu182 or Asp142, they demonstrated inhibitory effect which demonstrates that docking for this class of compounds is highly correlated with successful experimental inhibition.

  • VEGF signaling mediates neuroprotection against oxidative stress in hippocampal neurons in vivo and in vitro

    Author:
    Tianfeng Hao
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Patricia Rockwell
    Abstract:

    Vascular endothelia growth factor (VEGF) signaling through its cognate receptor VEGFR-2 has been shown to be neuroprotective against stressful stimuli including oxidative stress. Using a rat pilocarpine model of status epilepticus (SE), pilocarpine-induced SE was associated with the induction of the oxidative stress marker Heme oxygenase-1 (HO-1) and increased expression of the pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). Treatments with exogenous VEGF attenuated the induction in HO-1 and the increase in COX-2 levels. Previous studies showed that VEGF protected hippocampal cell death in this model and these results suggest that oxidative stress and inflammation contribute to this neuronal loss. Similarly, in cultured hippocampal neurons, pilocarpine induced neuronal cell death accompanied with increased HO-1 expression, reactive oxygen species (ROS) production, caspase-3 cleavage and a loss of mitochondrial membrane potential (ÄØm) that were all suppressed by VEGF. To delineate the protective mechanisms associated with VEGF-mediated neuroprotection, VEGFR-2 inhibition was investigated with respect to oxidative stress in primary cultures of hippocampal neurons. Treatments with the pharmacological inhibitor SU1498 elicited a cytotoxicity that was prevented by the antioxidant N-acetyl-cysteine (NAC) and accompanied by induction of HO-1, cleavage of caspase-3, production of ROS together with a loss of ÄØm. Knockdown of VEGFR-2 by siRNA generated a similar pattern of ROS and ÄØm loss. Treatments with VEGF or VEGF-B prevented neurons from the cell death and mitochondrial dysfunction induced by SU1498 or siRNA, suggesting that a molecular switch occurs where both growth factors may signal through VEGFR-1 when VEGFR-2 activity is blocked. Consistent with a general role for pro-apoptotic BAD and anti-apoptotic Bcl-xL in neuronal cell death and oxidative stress, the phosphorylation of MEK/ERK1/2 and BAD (inactivation) and the protein levels of Bcl-xL that were attenuated by SU1498 were somewhat increased by VEGF. Our findings support a role for VEGF in signaling BAD inactivation and increased Bcl-xL as a specific mechanism that protects hippocampal neurons against oxidative stress. These findings also suggest that VEGF activates VEGFR-2 to protect neurons against oxidative stress but can signal through alternate receptors to serve the same function.

  • Isolation and Characterization of Immunomodulatory Compounds from Juzen-Taiho-To: Novel Understanding of Phytosteryl Glucosides Nano-Aggregates and Synergism

    Author:
    Tal Hasson
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    This thesis describes the realization of the immunomodulatory properties of four phytosteryl-glucosides betta;-sitosterol-D-Glucoside, campesteryl betta-D-glucoside, 24-epicampestryl betta-D-glucoside, Stigmasteryl betta-D-glucoside) purified from a Japanese Kampo herbal formulation termed Juzen-Taiho-To (JTT). JTT has been used for almost a thousand years and currently is prescribed to people with an impaired immune system. Our screening methodology termed Genomic-Screening allowed us to investigate the immunomodulatory properties of JTT in a new and exciting way. Genomic Screening employs the use of GeneChip® oligonucleotide array, and quantitative reverse-transcriptase polymerase chain reaction qRT-PCR. THP-1 human cancerous monocytic cell line was used in our study with JTT due to their accurate representation of healthy monocytes as well as a large variety of receptors. THP-1 treatment with JTT revealed many genes to be differentially regulated most of which were down stream elements of NFkappa B pathway. We chose (ICAM-1) for our screening since it was the most reproducible assay and it also had a high fold change. By monitoring ICAM-1 response to JTT treatment we were able to uncover known phytosteryl-glucosides that previously were not known for their immunostimulatory properties. In addition our study discovered that a specific ratio of our phytosteryl glucosides that synergistically induced ICAM-1 expression. We have also discovered that the purification approach of the four phytosteryl glucosides greatly affects the immunostimulatory properties. Using Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) we were able to show that nano-aggregated of betta-sitosterol-D-Glucoside formed in a water containing solution diminished ICAM-1 expression in THP-1 cells. These findings underline the importance of natural product chemistry as well as the approach to purifying small organic molecule from natural resources. The underlining mechanism of how our phytosteryl glucosides induce an immune response is still unknown. Nevertheless our finding has opened the door to new research avenues with a family of compound that contain a novel class of pathogen associated molecular patterns.

  • Modeling Membrane Active Peptides with Implicit Membrane models

    Author:
    Yi He
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Themis Lazaridis
    Abstract:

    Membranes are the natural barriers of all cellular organisms. They separate the inner environment from the outer environment. They also divide cell contents into different functional compartments. Many membrane active peptides, however, challenge the function of membranes. They translocate through, form pores inside, or even break down the membranes. Understanding their mechanisms will help us design better drugs. Molecular dynamics (MD) provides a unique way to study these peptides at small time scales. A lot of useful information can therefore be determined: such as, peptide orientation, structure adjustment, insertion into the membrane, and binding energy. Implicit membrane models are particularly useful because their low computational cost allows us to study peptides at longer time scales or larger numbers. The object of the thesis is to study the membrane active peptides using implicit membrane models. The study is focused on three areas: 1) We first examined the transmembrane peptide orientation in both implicit and explicit MD simulations. Using theoretical methods, we tried to explain the gap between the tilt angles predicted by hydrophobic mismatching theory and the ones determined by 2H NMR experiments. 2) To study the interaction between cationic peptides and anionic pores, we extended the current implicit pore model to anionic membranes. This model was applied to two typical antimicrobial peptides &mdash magainin and melittin &mdash and was used to explain their different preferences for anionic lipid fractions. We also evaluated the stability of three protegrin octameric pore models using this model. 3) We then tried to determine the link between binding affinity to membrane surface and biological activities of antimicrobial peptides. We found that both the experimental binding free energy and the theoretical transfer energy correlate with the biological activities, although the correlation is weak. Many other factors may also affect the biological activities of antimicrobial peptides. Moreover, based on a critical evaluation of "carpet" model, we found that most peptides would show higher activity than the prediction of the "carpet" model. The deviation of their biological activities from the "carpet" model correlates with their transfer energies to pores. The knowledge we gained from this study can help us establish quantitative models for predicting antimicrobial peptide activities.

  • PROTEASOME IMPAIRMENT, MITOCHONDRIA DYSFUNCTION, AND TAU PATHOLOGY IN ALZHEIMER DISEASE

    Author:
    QIAN HUANG
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    MARIA FIGUEIREDO-PEREIRA
    Abstract:

    Impairment of the ubiquitin/proteasome pathway is implicated in the pathogenesis of many neurodegenerative disorders, such as Alzheimer disease (AD). This is supported by detection of ubiquitinated protein aggregates in neurofibrillary tangles (NFTs) (Ross and Poirier, 2004) and reduction of proteasome activity in autopsied brains from AD patients (Keller et al., 2000). However, the upstream events leading to impairment of the ubiquitin/proteasome pathway are not fully understood. The ubiquitin/proteasome pathway is a highly regulated and efficient pathway that is critical for degradation of most intracellular proteins (Ciechanover, 2005). Ubiquitination, proteasome assembly and proteasome activity are energy dependent processes that require ATP binding and ATP hydrolysis (Li and Demartino, 2009). In neurons, most ATP is generated by mitochondria. Emerging evidence implicates mitochondrial dysfunction and energy deficit in many neurodegenerative disorders such as AD (Lin and Beal, 2006). There are no current treatments that are neuroprotective in the sense that they slow or prevent further neurodegeneration in AD. To provide new insights for the development of novel and more effective therapeutic strategies that prevent/stop neurodegeneration in AD, the MAJOR AIMS of these studies were: (1) Investigate underlying mechanisms that link mitochondrial dysfunction to impairment of the ubiquitin/proteasome pathway. (2) Evaluate the protective effect of elevating cAMP against an endogenous product of inflammation, i.e. prostaglandin J2 that affects the ubiquitin/proteasome pathway. In our studies we used rat E18 cerebral cortical neuronal cultures to investigate the pathogenesis of AD. For the FIRST AIM, we mimicked mitochondrial dysfunction by treating neurons with the following mitochondrial toxins: (1) oligomycin which inhibits ATP synthase, (2) rotenone which inhibits mitochondrial complex I, and (3) antimycin which inhibits mitochondrial complex III. We demonstrate that mitochondrial impairment affects the ubiquitin/proteasome pathway in the neurons via: (1) Reducing polyubiquitinated protein levels by inhibiting the first step of the ubiquitination cascade that is ATP-dependent: ubiquitin adenylation by the enzyme E1, thus blocking E1-E2 transthiolation. (2) Downregulating 26S proteasomes by promoting their disassembly. Notably, we show for the first time to our knowledge, that calpain activation is one of the factors that contributes to 26S proteasome disassembly by selectively cleaving one of its subunits, Rpn10. Calpain activation is associated with necrosis, an energy-independent cell death pathway activated upon mitochondrial dysfunction. We also demonstrate that calpain cleaves pro-caspase 3 to an inactive fragment, most likely to prevent apoptosis that is an energy-dependent cell death pathway. Furthermore, calpain cleaved the microtubule associated protein TAU that is the major component of neurofibrillary tangles in AD and other tauopathies. (3) Promoting a rise in 20S proteasomes, which degrade most oxidized proteins as well as intrinsically disordered proteins in a ubiquitin- and energy-independent manner. (4) Finally, we show that down-regulation of ubiquitinated proteins and 26S proteasomes with a concomitant increase in 20S proteasomes, occur upon acute (up to 16h) and long-term (up to seven days) mitochondrial dysfunction in the neurons. Overall, these data support the notion that upon mitochondrial dysfunction, neurons initiate a series of mechanisms that converge to optimize unregulated and energy-independent turnover of randomly unfolded oxidized proteins by 20S proteasomes. This adaptive response to energy deficiency may be suitable for short-term periods. However, if maintained chronically it may lead to neurodegeneration, as regulated protein degradation by the ubiquitin/proteasome pathway is essential for neuronal survival. For the SECOND AIM, we treated rat E18 cerebral cortical neurons with prostaglandin J2 (PGJ2), an endogenous product of inflammation that affects mitochondrial function, and inhibits the 26S proteasome by causing its disassembly. We compared the effects of PGJ2 with those of the specific and irreversible proteasome inhibitor epoxomicin. We show that treatment with both drugs induces caspase-activation, TAU cleavage at Asp421 (deltaTAU), TAU and Ub-protein aggregation, and neuronal death. Truncation of TAU at Asp421 (deltaTAU) by caspases is an early event in AD tangle pathology. In addition, deltaTAU is detected in neurofibrillary tangles (NFTs) indicating that the apoptotic cascade is involved in NFT formation. To prevent these deleterious events associated with proteasome inhibition, we tested two drugs that elevate intracellular cAMP: dibutyryl-cAMP (db-cAMP) and the lipophilic peptide PACAP27 that increases intracellular cAMP levels by binding to its seven transmembrane G-coupled receptor PAC1R. Previous studies from our lab showed that a single dose of db-cAMP or PACAP27, blocked caspase-activation and deltaTAU upon short-term (up to 8h) treatment with PGJ2. One single dose of these cAMP-elevating drugs however, failed to protect against longer (24h) treatments with PGJ2. In our current studies, we decided to increase the number of drug treatments to improve protection. We established that three doses of either db-cAMP or PACAP27 successfully diminished caspase-activation, deltaTAU and loss of cell viability promoted by long-term (24h) incubations, as long as these changes were mild. Based on these studies, we propose that targeting cAMP/PKA to boost proteasome activity in a sustainable manner, could offer an effective approach to avoid early accumulation of ubiquitinated proteins and later caspase-activation and TAU cleavage, possibly preventing/ delaying neurodegeneration in AD. In summary, our data address two distinct death pathways in neurons that are associated with mitochondria and proteasome impairment: necrosis via calpain activation and apoptosis via caspase activation. We show that on one hand, mitochondria-dependent energy deficit affects ubiquitination and 26S proteasomes, and promotes cleavage of pro-caspase 3 and TAU via calpain activation. These events are associated with necrotic neuronal death. On the other hand, proteasome impairment induces the accumulation of ubiquitinated proteins and promotes TAU cleavage via caspase 3 activation. These events are associated with neuronal apoptotic death. The dual vulnerability of neurons to calpain- and caspase-mediated cell death needs to be taken into consideration when considering therapeutic approaches to prevent neuronal demise in AD and other chronic neurodegenerative diseases associated with proteasome and mitochondria dysfunction.

  • Role of Fibroblast Growth Factor Homologous Factors in Excitability of Hippocampal Neurons

    Author:
    Xiao Huang
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Mitchell Goldfarb
    Abstract:

    Fibroblast growth factor homologous factors (FHFs) are a family of vertebrate neuronal proteins which function in manners distinct from fibroblast growth factors (FGFs). While bearing substantial sequence and structural homology to FGFs, FHFs reside intracellularly and bind targets unrelated to FGF receptors, which include voltage-gated sodium channels (Navs). FHFs have been shown to control excitability of cerebellar granule cells through modulation of channel inactivation. Since action potentials are initiated at axon initial segment (AIS), we suspected that at least some FHF isoforms would reside at AIS in association with Navs. As expected, a broad repertoire of FHF isoforms colocalizes with Navs at the AIS of cultured hippocampal neurons. Moreover, together with other studies in our laboratory, the present study shows that FHF "b" isoform associates with AIS to a far lesser extent than "a" isoform, demonstrating that there is isoform specificity in FHF targeting. AIS targeting of an FHF requires the protein's channel binding surface, as a mutant derivative of FHF2a deficient for channel binding is also deficient in AIS targeting. The association of FHFs with Navs at AIS suggests that FHFs may modulate channel physiology, thereby controlling the intrinsic excitability of hippocampal neurons in a manner similar to that which has been described for cerebellar granule neurons of Fhf1-/-Fhf4-/- mice. Moreover, real time PCR shows that FHF2 is the most abundant of the FHFs expressed in hippocampal neurons. The requirement of FHF2 for hippocampal neuron excitability was analyzed using RNAi in conjunction with electrophysiological recordings. These studies have demonstrated that knockdown of fhf2 in hippocampal neurons derived from Fhf1-/- mice impairs excitability, with less maximum spike frequency and elevated voltage thresholds for spike induction. Sodium channel inactivation parameters are altered in Fhf1-/-Fhf4-/- granule neurons. In accordance, the colocalization and physical interaction of FHFs with Navs in hippocampal neurons suggest that excitability deficits could reflect altered channel physiology in cells lacking FHF1 and FHF2 function. These are the first data to indicate a role for FHF2 in neuronal excitability. These data suggest a widespread role for FHFs in the control of excitability across the central nervous system (CNS).

  • Uncovering hidden potential of natural products

    Author:
    Maria Iacovidou
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    Herbs and soil bacteria have been arguably the most important sources of therapeutic agents throughout human history. Numerous bioactive compounds have been isolated and characterized from these classical sources of natural products. Because of the long history of research on herbs and soil bacteria, they are sometimes perceived as “exhausted” sources of secondary metabolites. These classical sources of natural products, however, can still include compounds with previously overlooked chemical and biological properties. The unifying theme of this thesis study is to uncover such hidden potential of natural products. To this end, these classical sources of secondary metabolites have been examined from new angles. The first two chapters of this thesis describe the examination of oriental herbal formulations with biomarker–guided screening, which has been established recently in our group. In this method, natural products are screened based on their ability to modulate the expression of mRNA biomarkers, which are the genes associated with therapeutic effects of herbal medicine. Importantly, herbal formulations have never been screened in this manner. Thus, biomarker–guided screening can serve as a powerful approach to uncover hidden therapeutic potential of natural products. Chapter 1 describes the biomarker–guided screening of a blood cleansing herbal formulation called Toki–shakuyaku–san (TSS). This formulation has long been used to alleviate various disorders associated with poor blood circulation. Our screening revealed that a simple polyacetylene compound, (6E,12E)–tetradecadiene–8,10–diyne–1,3–diol diacetate (TDEYA), which has been considered as an “inactive” precursor of other metabolites, regulates the expression of genes associated with blood coagulation, such as COX–2, SerpinB2, and perlecan. Identification of this polyacetylene compound is important because it may open a new paradigm of treating circulation disorders through manipulation of gene expression by small molecules. Chapter 2 describes the biomarker-guided screening of another herbal formulation, Juzen–taiho–to (JTT), which is widely known as “immune booster” formulation. In fact, JTT is clinically used in Japan to reinvigorate the immune system of cancer patients undergoing chemotherapy and radiation therapy. It is believed that many beneficial effects of JTT are mediated by monocytes and macrophages. Key monocyte–stimulants in JTT have been difficult to identify despite many years of intensive studies by us and others. However, JTT biomarker–guided screening identified β–glucosylceramides (cerebrosides) as the first group of monocyte–stimulants in JTT. This discovery is an important first step to gain mechanistic insights into the clinically tested immunostimulatory effects of JTT. Finally, chapter 3 describes the screening of secondary metabolites from culturable bacteria from soil samples collected throughout the State of New York. While traditional screenings of secondary metabolites in soil bacteria focus on the search for new antibiotics, our screening looked for a compound with structural complexity which provides us with an access to the pharmacological spaces that are difficult to reach through organic synthesis. This is an important new goal of the screening of bacterial metabolites, because complex molecules of bacterial origin can now be produced from simple carbon sources through metabolic engineering. This study isolated and structurally characterized cyclooctatin B (COB), a new diterpene with a fused 5–8–5 ring system and unique U–shaped conformation. Our preliminary analyses suggest that COB can undergo remarkably elegant cascade reactions when its environment turns mildly acidic. Thus, COB may constitute a new class of pH–sensing bacterial metabolites with a variety of chemical, pharmaceutical, and engineering applications.

  • Peptide Directed 3D Assembly of Nanoparticles through Biomolecular Interaction

    Author:
    Prerna Kaur
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Hiroshi Matsui
    Abstract:

    The current challenge of the `bottom up' process is the programmed self-assembly of nanoscale building blocks into complex and larger-scale superstructures with unique properties that can be integrated as components in solar cells, microelectronics, meta materials, catalysis, and sensors. Recent trends in the complexity of device design demand the fabrication of three-dimensional (3D) superstructures from multi-nanomaterial components in precise configurations. Bio mimetic assembly is an emerging technique for building hybrid materials because living organisms are efficient, inexpensive, and environmentally benign material generators, allowing low temperature fabrication. Using this approach, a novel peptide-directed nanomaterial assembly technology based on bio molecular interaction of streptavidin and biotin is presented for assembling nanomaterials with peptides for the construction of 3D peptide-inorganic superlattices with defined 3D shape. We took advantage of robust natural collagen triple-helix peptides and used them as nanowire building blocks for 3D peptide- gold nanoparticles superlattice generation. The type of 3D peptide superlattice assembly with hybrid NP building blocks described herein shows potential for the fabrication of complex functional device which demands precise long-range arrangement and periodicity of NPs.

  • An Investigation on the Application of Mass Spectrometry Protein Footprinting Technique to Study Interactions of Collagen and Collagen Receptors

    Author:
    Michele Kirchner
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Yujia Xu
    Abstract:

    Collagen is one of the most abundant proteins in the human body. There are many different types of collagen; they interact with an assortment of other proteins in the extracellular matrix, and with collagen receptors on the surfaces of cells. In this study, oxidative protein footprinting using Fenton chemistry followed by mass spectrometry was explored as a method to investigate collagen interactions. A known collagen binding protein, the A3 domain of the von Willebrand Factor protein (vWF A3), was chosen as a model. This protein association is the first critical step in blood clotting, and has been the target for the development of new drugs to treat von Willebrand disease, a bleeding disorder. Mass spectrometry protein footprinting could potentially aid in these endeavors. In order to accomplish this objective, original studies using Fenton chemistry labeling of triple helical peptides, and mass spectrometry collagen sequencing studies were carried out. The von Willebrand Factor A3 protein was produced from an Escherichia coli expression system. This work discovered many challenges that have to be overcome for the successful approach of mass spectrometry oxidative protein footprinting by Fenton chemistry to investigate full chain collagen interactions. Additionally, the mix of variable post translational modifications of collagen makes the identification of binding sites by footprinting unreliable. Even sequencing collagen proves problematic because of the many post translational modifications. Despite these setbacks, we did; however, find that the von Willebrand Factor A3 protein binds to a triple helix conformation lacking any of the presumed necessary hydroxyproline sites. We were also able to identify regions in full chain collagen where the oxidation content varied from what was expected, assuming all the prolines in the Y position were hydroxylated.

  • Characterization of Mitochondrial DNA Heteroplasmy at Five Hotspots within the HVI Region of Post-Mortem, Formalin Fixed Paraffinized Human Liver Cells

    Author:
    Jason Kolowski
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Margaret Wallace
    Abstract:

    Within the field of mitochondrial DNA (mtDNA) analysis, heteroplasmy is a widely recognized and yet poorly-understood event. Heteroplasmy is defined as the presence of more than one mitochondrial genome within a tissue sample from a single individual, such that the mtDNA sequence shows the presence of a mixed base or regions of homologous bases that vary in length. Due to the highly conserved nature of the mitochondrial genome, these heteroplasmic events occur at a variety of well-documented hotspots, a majority of which occur within the hypervariable control region that flanks the origin of replication. This control region is the same area that is tested in forensic mtDNA analysis, and is the most useful for establishing the link between evidentiary samples and maternally-related individuals due to the polymorphisms that accumulate in this region. However, when heteroplasmic events are uncovered in forensic mtDNA analysis, issues arise due to the lack of clear understanding of the origin of heteroplasmy. The occurrence of mtDNA heteroplasmy between different tissues within a single individual has been well-established, and heteroplasmic events have been shown to increase with age. However, what is presently unclear is whether or not mtDNA heteroplasmy exists within a single cell when heteroplasmy is present within a tissue. In this regard, three possibilities exist; 1) a single cell contains a solitary pool of mtDNA genomes (defined as homoplastic), and heteroplasmy exists as a mixture of different homoplastic cells within tissue, 2) a single cell contains a mixture of multiple mtDNA genomes, and heteroplasmy is present within a single cell, or 3) heteroplasmic tissue contains a mixture of homoplastic and heteroplasmic cells due to random cellular distribution throughout the tissue. To investigate this question, laser dissection microscopy will be used to isolate individual cells from a tissue sample with known mtDNA heteroplasmy. Typing of single nucleotide polymorphisms at specific hotspots within the HVI region will then be done to detect possible mtDNA heteroplasmy within a single cell.