1995 — 2021 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Setting the Stage For Replacement of Mitochondrial Genes @ University of Miami School of Medicine
DESCRIPTION (provided by applicant): Our long-term goal is to develop genetic approaches that could be used for the treatment of mitochondrial disorders associated with mitochondrial DNA (mtDNA) mutations. We propose to develop methods that improve oxidative phosphorylation function in cells harboring heteroplasmic mtDNA mutations (i.e. a mixture of wild-type and mutated mtDNA). Our approach is based on the mtDNA heteroplasmy shift concept, where, by genetic manipulation, the levels of the mutated genome are decreased in relation to the wild-type genome, thereby improving cellular health. In the previous funding period we showed that mitochondria-targeted restriction endonucleases are powerful tools to achieve mtDNA heteroplasmy shift. The present proposal will further expand this approach to any mtDNA mutation. To do so, we will take advantage of new developments in the field of designed nucleases. TAL-effector nucleases have emerged as highly flexible molecules that can recognize DNA regions based on a modular arrangement of their DNA binding domains. We have obtained preliminary data showing that this approach is feasible and propose to better characterize the system in vitro, ex vivo and in vivo.
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1997 — 2000 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Exploring Nuclear/Mitochondrial Dna Interactions @ University of Miami School of Medicine
DESCRIPTION (Adapted from the Investigator's Abstract): This proposal is aimed at exploring the detailed molecular interactions between factors encoded in the nuclear genome with factors encoded by the mitochondrial genome. Dr. Moraes, who has extensive experience in characterizing mtDNA mutations in patients with various human mitochondropathies, here aims to study the interaction between factors encoded by the nuclear and mitochondrial genomes by creating and characterizing a heterologous system of primate cell lines harboring nuclear DNA from one species and mitochondrial DNA from another, so-called "xenomitochondrial cybrids." Thus, biological functions that involve coordinated participation of nuclear-coded and mtDNA-coded factors may be affected in these cell lines. The P.I. suggests that biochemical and molecular studies of these cell lines will shed light on many nDNA-mtDNA interactions that are needed for mtDNA transcription and replication, assembly of respiratory complexes and ATP production, mitochondrial protein synthesis, mitochondrial posttranscriptional modifications of RNA, mtDNA heteroplasmy dynamics, and mtDNA recombinations, all of which are essential for the establishment and maintenance of an efficient ATP generating system.
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2001 — 2004 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Exploring Nuclear-Mitochondrial Dna Interactions @ University of Miami School of Medicine
DESCRIPTION (Investigator's abstract): We propose to study nuclear-mitochondrial interactions by using a collection of cell lines and embryos harboring the nuclear background from one species and the mitochondrial genome from a different species. Because oxidative phosphorylation (OXPHOS) depends on the interaction between these two genomes, which have co-evolved to optimize energy production, xenomitochondrial cells are commonly deficient in respiration. In the last three years we produced human xenomitochondrial cybrids harboring non-human primate's mitochondrial genomes and used these cell lines as cellular models of mitochondrial diseases and as tools to study nuclear-mitochondrial interactions, such as mitochondrial DNA maintenance and copy number control. Models of respiratory complexes I and IV deficiencies were also obtained depending on the primate mitochondrial DNA coexisting with the human nuclear DNA. We now propose to expand these preliminary results to other species and to better define the mechanisms associated with defective nuclear-mitochondrial interactions. We also propose to study mechanisms of mtDNA maintenance in early development. We will use cross-species cloned embryos and animals produced by nuclear transfer to assess mtDNA maintenance in a xenomitochondrial environment. We will also study some physical and biochemical properties of oocyte mitochondria and test if these specific features are optimized for mitochondrial maintenance in the egg.
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2001 — 2003 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mouse Models With Defective Respiratory Complexes in Cns @ University of Miami School of Medicine
DESCRIPTION (Adapted from the abstract provided by the applicant): Although defects in mitochondrial respiration have been associated with neurodegenerative disorders, the correlation between specific defects and clinical phenotypes remains unclear. The availability of genetic models with defects in different respiratory chain complexes would provide a powerful model system to assess these associations. We propose to create mouse models bearing defects in specific respiratory complex. These mice will be generated by replacing endogenous genes coding for factors that are essential for the assembly of complex I or IV with genes tagged with loxP sequences. Subsequently, loxP tagged animals will be crossed with mice expressing the Cre recombinase in specific tissues, particularly with ones expressing this recombinase in the central nervous system. We will characterize the defects in such animals at the molecular and biochemical level and study their phenotype at different ages. Since the level of gene disruption by the Cre/loxP system can be titrated, we expect to generate lines of transgenic mice that will be partially deficient thereby mimicking neurodegenerative processes.
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2001 — 2010 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oxidative Phosphorylation in Cell Growth and Death @ University of Miami School of Medicine
The key role of cytochrome c in apoptosis is widely accepted. However, the requirement of cytochrome c for apoptosome formation has been defined mostly by in vitro assays. In vivo, cytochrome c translocation from the mitochondria to the cytosol is an indicator of apoptosis, but this association does not address the actual mechanism, requirements and the sequence of events leading to apoptosis. Proof of cytochrome c requirement for in vivo apoptosis has been hampered by the lack of mutants and its dual role. In addition, we and others have also shown that defects in oxidative phosphorylation can protect cells from an apoptotic death Therefore, it is important to differentiate the oxidative phosphorylation effect from the apoptosome assembly catalysis role of cytochrome c. To better define the mechanisms associated with the intrinsic apoptotic mechanism (the mitochondrial pathway), we propose to develop and analyze cytochrome c deficient cells and mice These studies have far reaching implications to different areas of medical and biological research, including cancer, neurodegeneration and development.
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2005 — 2010 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Mouse Models of Mitochondrial Encephalomyopathies @ University of Miami School of Medicine
[unreadable] DESCRIPTION (provided by applicant): Defects in oxidative phosphorylation (OXPHOS) have been associated with various neuromuscular degenerative processes. We propose to take advantage of current methodology that allows the manipulation of nuclear genes in embryonic stem cells and generate and characterize of mouse models with conditional mutations in nuclear-coded OXPHOS genes. The general hypotheses and questions this proposal will test are: i) What are the specific role of the knocked out factors on OXPHOS assembly and function? ii) Are different cell populations more susceptible to defects in specific OXPHOS complexes? iii) Do defects in specific respiratory chain complexes in muscle or nerves lead to different muscular or neurodegenerative phenotypes? iv) Are these phenotypes associated with different levels of oxidative damage or protein aggregation? Specifically, we will develop knock in mice for three respiratory complex genes in which an evolutionarily conserved exon is flanked by IoxP sites. The following genes will be manipulated: the NDUFS1 gene (complex I), Iron-Sulfur Protein (complex III) and COX10, a protoheme O farnesyl-transferase (required for heme a production and complex IV activity). The knockin animals will be crossed with mice expressing Cre-recombinase driven by CNS and skeletal muscle promoters. We expect these studies to provide evidence that specific defects in the OXPHOS can manifest as distinct phenotypic alterations. [unreadable] [unreadable]
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2010 — 2014 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mitochondrial Dysfunction in Neurodegeneration and Compensatory Approaches @ University of Miami School of Medicine
DESCRIPTION (provided by applicant): A progressive decline in mitochondrial oxidative phosphorylation function during life is a likely contributor to neurodegeneration. However, the understanding of the mechanisms involved in the mitochondrial defects is still rudimentary and practical approaches to mitigate this problem are not available. Our project will study these two aspects of mitochondrial involvement in neurodegeneration and aging. In the first part, we propose to study the role of mitochondrial DNA (mtDNA) deletions in the aging of the brain. We will use a novel mouse developed in our laboratory in which a mitochondria targeted restriction endonuclease (Mito-PstI) is expressed in a tissue-specific and inducible fashion. The double-strand breaks elicited by Mito-PstI lead to recombination and deletion formation. We will generate mtDNA deletions in the CNS or ubiquitously. The role of the mitochondrial polymerase gamma in repairing these double-strand breaks will also be analyzed. The goal of this aim is to study the functional consequences of accumulating different levels of mtDNA deletions during neurodegeneration and aging. In the second part of the proposal, we will develop approaches to mitigate the aging of CNS and other tissues by increasing the expression of PGC-1a, either in skeletal muscle or ubiquitously. This will be achieved by stable and inducible expression. The effect of PGC-1a will be tested both in normal aging mice and in the proof-reading deficient polymerase gamma "mutator mouse". The latter is a model of accelerated aging. Both aims are based on extensive published and unpublished preliminary data. We are confident that the accomplishment of these two aims will lead to not only a better understanding of the role of mitochondrial defects in age-related neurodegeneration but also to novel approaches to counteract these effects. PUBLIC HEALTH RELEVANCE: Mitochondria is believed to play a major role in neurodegeneration and aging. By better understanding the mechanisms involved in this process and by developing approaches to counteract these effects, the debilitating effects of the neurodegenerative process could be mitigated.
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2012 — 2021 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular and Molecular Consequences of Respiratory Chain Defects in Neurons @ University of Miami School of Medicine
Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been associated with various primary mitochondrial diseases as well as with neurodegenerative disorders. Such disorders can be caused by defects in nuclear or mitochondrial DNA (mtDNA). We propose to take advantage of existing and new mouse models created in our lab to manipulate the mtDNA to study fundamental mechanisms of the role of mtDNA mutations in neurodegeneration. We will also use DNA editing enzymes to further modulate mtDNA heteroplasmy in vivo. We will analyze the ability of different neuronal types (glutamatergic and dopaminergic) to accumulate mtDNA deletions (aim#1) and a pathogenic point mutation in a tRNA gene (aim#2). With these models in place, we will also study the susceptibility of glutamatergic and dopaminergic neuronal subtypes to OXPHOS defects.
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2015 — 2019 |
Caicedo, Alejandro (co-PI) [⬀] Moraes, Carlos Torres |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) R33Activity Code Description: The R33 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the R21 mechanism. Although only R21 awardees are generally eligible to apply for R33 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under R33. |
Imaging Mitochondrial Signaling in B-Cells Ectopically Implanted in the Eye @ University of Miami School of Medicine
? DESCRIPTION (provided by applicant): Imaging mitochondrial signaling in beta cells (? cells) ectopically implanted in the eye. In the last 20 years, it became clear that defects in the mitochondrial energy producing system, either genetic or toxin-induced, cause many different phenotypes. Therefore, defects in one of the five mitochondrial oxidative phosphorylation (OXPHOS) complexes likely trigger distinct signaling pathways, which differentially affect specific cell types. This team will use mouse models with specific defects in complexes I, III or IV to explore enzyme-specific signaling events in mitochondrial disorders. A novel model will be used to test this hypothesis in a dynamic, real-time platform. The approach will involve implanting ? cells in the anterior chamber of the mouse eye, which allows for the use of imaging techniques to follow both cellular and mitochondrial function as well as its signaling patterns in vascularized environment akin to the in vivo situation. This is a multiple Principal Investigator application that will use the expertise of the group of Dr. Alejandro Caicedo (?-cell implant model, imaging physiological biomarkers) and the group of Dr. Carlos Moraes (mitochondrial physiology and mouse models of OXPHOS defects) to tease out signaling signatures associated with defects (genetic or toxin-induced) in specific OXPHOS complexes.
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2016 — 2020 |
Moraes, Carlos Torres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Consequences of Mtdna Damage to Age-Related Degenerative Processes @ University of Miami School of Medicine
? DESCRIPTION (provided by applicant): During the course of accomplishing the previous specific aims, we found that a relatively brief expression of a mitochondrial targeted restriction endonuclease, ubiquitously in mice, generates double strand breaks (DSB) in the mtDNA of the different tissues. MtDNA levels recovered from this transient event and no abnormal phenotypes were observed in the weeks following the molecular insult. However, later in life mice developed a phenotype resembling accelerated aging. Initial characterization showed a reduction in progenitor cell pools. These observations, together with other recent reports led us to hypothesize that progenitor cells are targets of mtDNA damage. We also obtained preliminary data showing that p53-associated pathways are involved in the mechanism leading to a reduction of progenitor cell pools after mtDNA transient insults. We propose a series of experiments to rigorously test this hypothesis in mouse and cultured cells. We will also develop a valuable model of mice with heteroplasmic mtDNA deletions, which will be used to unveil the role of mtDNA deletions in aging. These models will be shared with the research community. We expect that these studies will increase our understanding the role of mtDNA damage in aging.
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2017 |
Moraes, Carlos Torres |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb Src On Mitochondrial Biogenesis and Dynamics in Health, Disease and Aging @ Federation of Amer Soc For Exper Biology
Abstract The FASEB conference: ?Mitochondrial Biogenesis and Dynamics in Health, Disease and Aging? has brought together experts in basic biology and aging to discuss the function of mitochondria since 2007. We propose to organize this successful conference for the 6th time, following the tradition of inviting the leaders in these fields. The meeting will take place in Palm Beach in late May 2017 and will include travel awards for junior faculty and trainees, as well as opportunities for interactions during the meeting. Based on previous meetings, we expect approximately 130 participants. The long-term impact of this conference will be to create collaborative multidisciplinary research efforts that will pave the way for development of novel diagnostics and urgently needed therapeutics for patients with age-related conditions associated with mitochondrial dysfunction.
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2020 |
Moraes, Carlos Torres |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Acquisition of a Leica Microdissection System For the University of Miami @ University of Miami School of Medicine
Title: Acquisition of a Leica Microdissection System for the University of Miami Project Abstract/Summary The purpose of this shared S10 instrumentation grant application is the purchase of a laser microdissection microscope (LMD7 from Leica) for the University of Miami (UM) to support the NIH funded research of 7 Major Users, as well as 12 Minor User groups representing 5 Centers and 8 Departments at the University of Miami (UM). Currently, there are no available laser microdissection systems at UM or other research facilities in South Florida that can be used to develop these NIH-funded projects. The user groups have identified a strong need for isolation of cell populations and specific tissue regions in the brain, pancreas and tumor sections for further downstream processing that requires the precise sample collection allowed by the LMD7. This capability is essential for several UM-based NIH-funded investigators to perform, accelerate and expand their research programs.
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