ORCID Profile
0000-0002-1172-4018
Current Organisation
Universidade de Coimbra
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Publisher: Mary Ann Liebert Inc
Date: 09-2005
Abstract: Mitochondrial biogenesis and activation of both oxidative phosphorylation, as well as transcription and replication of the mitochondrial genome, are key regulatory events in cell differentiation. Mitochondrial DNA transcription and replication are highly dependent on the interaction with nuclear-encoded transcription factors translocated from the nucleus. Using a human embryonic stem cell line, HSF 6, we analyzed the proliferation of mitochondria and the expression of mtDNA-specific transcription factors in undifferentiated, migratory embryonic stem cells and spontaneously derived cardiomyocytes. Mitochondrial proliferation and mtDNA transcription are initiated in human embryonic stem cells as they undergo spontaneous differentiation in culture into beating cardiomyocytes. Undifferentiated, pluripotent human embryonic stem cells have few mitochondria, and, as they differentiate, they polarize to one extremity of the cell and then bipolarize the differentiating cell. The differentiated cell then adopts the cytoplasmic configuration of a somatic cell as evidenced in differentiating cardiomyocytes. Transcription and replication of the extranuclear mitochondrial genome is dependent on nuclear encoded factors exported to the mitochondrion. However, the differentiating cardiomyocytes have reduced or absent levels of these transcription and replication factors, namely mitochondrial transcription factors A, B1, B2, and nuclear respiratory factor 1 and polymerase gamma. Therefore, final embryonic stem cell commitment may be influenced by mitochondrial proliferation and mtDNA transcription. However, it is likely that differentiating cardiomyocytes are in mitochondrial arrest, awaiting commitment to a final cell fate.
Publisher: Oxford University Press (OUP)
Date: 05-03-2007
Abstract: Human mitochondrial DNA (mtDNA) encodes 13 polypeptides of the electron transfer chain. Its replication is dependent on the nuclear-encoded polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). For POLG, only the polyglutamine tract, characterized by a series of CAG repeats, has been investigated in human sperm. However, TFAM is associated with the reduction in mtDNA content of testicular sperm. We have determined whether POLG and TFAM have functional roles in post-ejaculatory sperm mtDNA. Sperm s les were categorized as: normals, s les with one or two abnormal sperm parameters and oligoasthenoteratozoospermics (OATs). These were analysed by fluorescent PCR to determine the number of CAG repeats, real-time PCR for mtDNA copy number and immunocytochemistry and western blotting for patterns of expression for POLG, TFAM and the mtDNA-encoded COXI. Only the OAT group presented with a significantly higher incidence of heterozygosity for CAG repeats, higher mtDNA content and a lower percentage of sperm expressing POLG and TFAM. Paradoxically, good-quality sperm had fewer mtDNA copies but significantly more sperm expressed POLG, TFAM and COXI. Our data support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, the biological significance of these variants in male infertility remains unclear, as these do not seem to affect mtDNA maintenance. The reduction in mtDNA content in normal s les likely reflects normal spermiogenesis, whereas increases in POLG and TFAM expression possibly compensate for the low mtDNA content, maintaining mitochondrial homeostasis.
Publisher: Wiley
Date: 23-10-2003
DOI: 10.1002/CM.10151
Abstract: Mutations in the human LIS1 gene cause the devastating brain disorder lissencephaly. LIS1 also regulates microtubule dynamics it interacts with the molecular motor cytoplasmic dynein and its cofactor dynactin, and is necessary for neuronal migration. Recently, LIS1 has been suggested to mediate pronuclear migration during fertilization. Here we use rhesus monkey and bovine oocytes, as well as pronucleate-stage bovine zygotes, to determine: Lis1 RNA expression using reverse transcription-polymerase chain reaction LIS1 protein association with dynactin using immunoprecipitation, Western blot analysis, and immunocytochemistry and LIS1 function in mediating genomic union using antibody transfection. We find that Lis1 RNA expression increases during fertilization, that LIS1 and dynactin subunit p150/(Glued) co-immunoprecipitate and co-localize to pronuclear surfaces, and that anti-LIS1 antibodies transfected into zygotes dramatically inhibit pronuclear migration and apposition. LIS1 is, therefore, essential to mediate genomic union in a process that involves the dynein-dynactin complex. These results shed light on an additional role for LIS1 and raise implications for human reproduction.
Publisher: Mary Ann Liebert Inc
Date: 10-01-2013
Publisher: Oxford University Press (OUP)
Date: 04-2006
DOI: 10.1534/GENETICS.105.055145
Abstract: Mitochondrial DNA is an extranuclear genome normally maternally inherited through the oocyte. However, the use of nuclear transfer can result in both donor cell and recipient oocyte mitochondrial DNA persisting through to blastocyst and being transmitted to the offspring. The degree of donor mitochondrial DNA transmission appears to be random and currently no evidence exists to explain this phenomenon. To determine whether this is a dilution factor or directly related to the transcriptional status of the donor cell in respect of mitochondrial DNA transcription factors, we have generated sheep nuclear transfer embryos using donor cells: (1) possessing their full mitochondrial DNA complement, (2) those partially depleted, and (3) those depleted but containing residual levels. For each donor type, donor mitochondrial DNA persisted in some blastocysts. It is evident from the donor cells used that nuclear-encoded mitochondrial DNA transcription and replication factors persist even after mitochondrial DNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mitochondrial DNA replication and transcription. In nuclear transfer-derived embryos, we have observed the persistence of these nuclear-encoded mitochondrial DNA transcription and replication factors but not in those embryos generated through in vitro fertilization. Consequently, nucleo-mitochondrial interaction following nuclear transfer is out of sequence as the onset of mitochondrial replication is a postimplantation event.
Publisher: Humana Press
Date: 2006
DOI: 10.1385/1-59745-046-4:347
Abstract: As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated in idual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).
Location: United States of America
Location: United States of America
Location: No location found
No related grants have been discovered for João Ramalho-Santos.