Mitochondrial DNA rearrangements during human aging: a study on liver, muscle and adipose tissue

Aging is characterized by a chronic, low-grade inflammatory state called “inflammaging”. Mitochondria are the main source of reactive oxygen species (ROS), which trigger the production of pro-inflammatory molecules. We are interested in studying the age-related modifications of the mitochondrial DNA (mtDNA), which can be affected by the lifelong exposure to ROS and are responsible of mitochondrial dysfunction. Moreover, increasing evidences show that telomere shortening, naturally occurring with aging, is involved in mtDNA damage processes and thus in the pathogenesis of age-related disorders. Thus the primary aim of this thesis was the analysis of mtDNA copy number, deletion level and integrity in different-age human biopsies from liver, vastus lateralis skeletal muscle of healthy subjects and patients with limited mobility of lower limbs (LMLL), as well as adipose tissue. The telomere length and the expression of nuclear genes related to mitobiogenesis, fusion and fission, mitophagy, mitochondrial protein quality control system, hypoxia, production and protection from ROS were also evaluated. In liver the decrease in mtDNA integrity with age is accompanied with an increase in mtDNA copy number, suggesting the existence of a “compensatory mechanism” able to maintain the functionality of this organ. Different is the case of vastus lateralis muscle, where any “compensatory pathway” is activated and mtDNA integrity and copy number decrease with age, both in healthy subjects and in patients. Interestingly, mtDNA rearrangements do not incur in adipose tissue with advancing age. Finally, in all tissues a marked gender difference appears, suggesting that aging and also gender diversely affect mtDNA rearrangements and telomere length in the three human tissues considered, likely depending on their different metabolic needs and inflammatory status.

Charakterisierung der Proteine CEP164 und ppdpf sowie deren Einordnung in den mitotischen Kontext

Eine regelgerechte bipolare Mitose und die fehlerfreie Aufteilung duplizierter DNA ist die Voraussetzung für die Entwicklung aller Lebewesen. Treten Fehler bei diesem grundsätzlichen Prozess auf, ist entweder der Zelltod oder die maligne Entartung der Zelle die Folge. Daher ist es von zentraler Bedeutung, die Vorgänge während der Zellteilung zu verstehen und die Funktion der an diesem Prozess beteiligten Proteine aufzudecken. Im Vorfeld dieser Arbeit wurden im Rahmen eines siRNA-Screens genomweit alle Proteine durch RNA-Interferenz depletiert und die Mitosen nach erfolgter RNAi phänotypisch untersucht. Ein besonderes Augenmerk lag dabei auf der Entwicklung multipolarer Spindeln durch Defekte in der Zentrosomenbündelung. Dadurch wurden unter anderem die Proteine CEP164 und ppdpf identifiziert. Da weder für CEP164 noch für ppdpf mitotische Funktionen bekannt sind, war es Ziel dieser Arbeit, die beiden Proteine eingehender zu charakterisieren und in den Kontext der Mitose einzuordnen. rnIm Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass CEP164 einer komplexen mitotischen Regulation unterliegt. Die in Interphase durchweg zentrosomale Lokalisation von CEP164 geht in der Mitose verloren. Es wird demonstriert, dass CEP164 während der Mitose unter anderem von CDK1 phosphoryliert wird und des Weiteren ubiquitinyliert wird. Als Interaktionspartner wurde das zentrosomale Protein Ninein identifiziert und demonstriert, dass sich CEP164 mit diesem in einem Komplex von ~2MDA befindet. Als weiterer Interaktionspartner wurde das Ninein-like-Protein ermittelt. Im Hinblick auf die Induktion multipolarer Mitosen wurde gezeigt, dass die Depletion von CEP164 nicht dafür verantwortlich ist. Die Induktion multipolarer Spindeln ist stattdessen darin begründet, dass durch die Transfektion einer siRNA gegen CEP164 auch ein für die Ausbildung der mitotischen Spindel elementares Protein, Ch-TOG, depletiert wird.rnIm Gegensatz dazu wurde im Rahmen dieser Arbeit bestätigt, dass das Protein ppdpf eine wichtige mitotische Funktion übernimmt. Zwar führt die Depletion von ppdpf nur zu einer sehr geringen Zunahme multipolarer Mitosen, allerdings steigt die Zahl aberranter Mitosen deutlich an, während die Spannung innerhalb mitotischer Spindeln abnimmt. Desweiteren konnte nachgewiesen werde, dass ppdpf-RNAi die Entwicklung von „lagging chromosomes“ und nachfolgend von Mikrokernen begünstigt. Es wurde gezeigt, dass ppdpf während der Mitose an Spindelmikrotubuli lokalisiert und spezifisch acetyliertes Tubulin bindet. Diese Interaktion hatte allerdings keinen Einfluss auf die Stabilität von Mikrotubuli während der Mitose. Das Protein ppdpf interagiert zudem mit dem Kinesin Eg5, wobei ppdpf-RNAi allerdings nicht zu einer Modulation der Aktivität von Eg5 zu führen scheint. Inwiefern diese Eigenschaften die Entwicklung von „lagging chromosomes“ begünstigen ist derzeit noch offen.

Global Search for Minimum Energy (H2O)n Clusters, n = 3−5

The Gaussian-3 (G3) model chemistry method has been used to calculate the relative ΔG° values for all possible conformers of neutral clusters of water, (H2O)n, where n = 3−5. A complete 12-fold conformational search around each hydrogen bond produced 144, 1728, and 20 736 initial starting structures of the water trimer, tetramer, and pentamer. These structures were optimized with PM3, followed by HF/6-31G* optimization, and then with the G3 model chemistry. Only two trimers are present on the G3 potential energy hypersurface. We identified 5 tetramers and 10 pentamers on the potential energy and free-energy hypersurfaces at 298 K. None of these 17 structures were linear; all linear starting models folded into cyclic or three-dimensional structures. The cyclic pentamer is the most stable isomer at 298 K. On the basis of this and previous studies, we expect the cyclic tetramers and pentamers to be the most significant cyclic water clusters in the atmosphere.

Holocentric chromosomes: convergent evolution, meiotic adaptations, and genomic analysis

In most eukaryotes, the kinetochore protein complex assembles at a single locus termed the centromere to attach chromosomes to spindle microtubules. Holocentric chromosomes have the unusual property of attaching to spindle microtubules along their entire length. Our mechanistic understanding of holocentric chromosome function is derived largely from studies in the nematode Caenorhabditis elegans, but holocentric chromosomes are found over a broad range of animal and plant species. In this review, we describe how holocentricity may be identified through cytological and molecular methods. By surveying the diversity of organisms with holocentric chromosomes, we estimate that the trait has arisen at least 13 independent times (four times in plants and at least nine times in animals). Holocentric chromosomes have inherent problems in meiosis because bivalents can attach to spindles in a random fashion. Interestingly, there are several solutions that have evolved to allow accurate meiotic segregation of holocentric chromosomes. Lastly, we describe how extensive genome sequencing and experiments in nonmodel organisms may allow holocentric chromosomes to shed light on general principles of chromosome segregation.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

No longitudinal mitochondrial DNA sequence changes in HIV-infected individuals with and without lipoatrophy

The potential for mitochondrial (mt) DNA mutation accumulation during antiretroviral therapy (ART), and preferential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We sequenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)-infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue-containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, respectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

Mutations within the FGF5 gene are associated with hair length in cats

Hereditary hair length variability in mice and dogs is caused by mutations within the fibroblast growth factor 5 (FGF5) gene. The aim of this study was to evaluate the feline FGF5 orthologue as a functional candidate gene for the long hair phenotype in cats, which is recessive to short hair. We amplified the feline FGF5 cDNA and characterised two alternatively spliced transcripts by RT-PCR. Comparative cDNA and genomic DNA sequencing of long- and short-haired cats revealed four non-synonymous polymorphisms in the FGF5 coding sequence. A missense mutation (AM412646:c.194C>A) was found in the homozygous state in 25 long-haired Somali, Persian, Maine Coon, Ragdoll and crossbred cats. Fifty-five short-haired cats had zero or one copy of this allele. Additionally, we found perfect co-segregation of the c.194C>A mutation within two independent pedigrees segregating for hair length. A second FGF5 exon 1 missense mutation (AM412646:c.182T>A) was found exclusively in long-haired Norwegian Forest cats. The c.182T>A mutation probably represents a second FGF5 mutation responsible for long hair in cats. In addition to the c.194C>A mutation, a frameshift mutation (AM412646:c.474delT) was found with a high frequency in the long-haired Maine Coon breed. Finally, a missense mutation (AM412646:c.475A>C) was also associated with the long-haired phenotype in some breeds. However, as one short-haired cat was homozygous for this polymorphism, it is unlikely that it has a functional role in the determination of hair length.

Koproduktion von Wissen in der Entwicklung des Biolandbaus - Einflüsse von Marginalisierung, Anerkennung und Markt

The empirical analysis of the historical development of transdisciplinary co-production of knowledge (CPK) in organic agriculture (OA) in Switzerland has revealed three distinct phases. The initial phase shows various characteristics of transdisciplinary CPK and a high importance of experiential knowledge. The 2nd and 3rd phases involve an in-creasing segregation of farmers’, extension agents’, and scientists’ knowledge, caused by internal and external impacts, i.e. foundation of independent research institutions, changes in agriculture policy, and new market strategies. These developments strikingly represent an ambiguous trend: the growing societal and political recognition of OA and the increasing market, which are positive achievements, are associated by a gradual loss of very precious forms of CPK. A successful re-establishment of CPK processes and re-integration of experiential knowledge would help to resolve this dilemma and thus improve future research in OA and sustainability research in general.

Static replications with traffic light options

It is well known that sufficiently regular, one-dimensional payoff functions have an explicit static hedge by bonds, forward contracts, and options in a continuum of strikes. An easy and natural extension of the corresponding representation leads to static hedges based on the same instruments along with traffic light options, which have recently been introduced in the market. It is well known that the second strike derivative of non-discounted prices of vanilla options is related to the risk-neutral density of the underlying asset price in the corresponding absolutely continuous settings. Similar statements hold for traffic light options in sufficiently regular, bivariate settings.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has sofar only been found in yeast. Ist function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has so far only been found in yeast. Its function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

Mujeres y trabajo urbano : el caso de las mucamas de hotel (Mar Del Plata, 1960-1980)

A partir del análisis de un caso particular, como lo es el trabajo de las mucamas de hotel en la ciudad de Mar del Plata (entre las décadas de 1960 y 1980), el presente artículo se propone como una contribución al campo de estudios sobre el trabajo urbano femenino. En Mar del Plata, el impulso del turismo de masas a mediados del siglo XX fue acompañado por un importante desarrollo hotelero. Éste supuso una atractiva y amplia oferta de trabajo para hombres y mujeres que encontraron allí una forma de acceso al mercado laboral, principalmente durante la temporada estival. En este espacio, las mujeres se han concentrado en puestos de trabajo determinados. En el artículo observamos cómo las continuidades entre algunas de las labores desarrolladas en el hotel y el trabajo doméstico no remunerado contribuyeron a su feminización e indagamos en las implicancias que conllevó que un empleo presente importantes continuidades con dicho trabajo. El análisis de testimonios orales nos permitió un acercamiento a las experiencias de las trabajadoras y, junto a las sentencias de los Tribunales de Trabajo, contribuyeron a visibilizar los procesos (sociales, culturales y económicos) que intervienen en la generización del trabajo y en la segregación ocupacional que caracteriza al mercado de trabajo urbano.

Un futuro urbano con un corazón antiguo

Tomando el ejemplo de Barcelona, el artículo realiza algunas reflexiones generales acerca de las transformaciones en la ciudad como resultado de grandes proyectos urbanos que a la par de la intervención física, modifican notoriamente su carácter. En el trabajo se sostiene como hipótesis que a expensas de un urbanismo de inspiración globalizante que homogeniza y segrega, la ciudad va mutando en nuevos espacios de desposesión para los ciudadanos. Al correr de los tiempos actuales y bajo el influjo de los nuevos proyectos, su carácter histórico como espacio de intercambio y apropiación, va cediendo lugar a la tendencia a la banalización urbana, la segregación social del territorio y el aumento de las desigualdades. Un segundo tópico se vincula con la respuesta de la ciudadanía frente al problema. Aquí se sostiene que por la vía de la denuncia y la crítica; como de la organización y la acción, los ciudadanos comienzan a construir y consolidar nuevos escenarios para la reapropiación del territorio, de su identidad y de su cohesión socio-cultural. Finalmente, en la conclusión se sintetizan algunas de las propuestas expresadas a lo largo del trabajo, y se reflexiona sobre los desafíos que tienen por delante las administraciones locales y los ciudadanos en la producción de un nuevo urbanismo centrado en el derecho a la ciudad.

Un futuro urbano con un corazón antiguo

Tomando el ejemplo de Barcelona, el artículo realiza algunas reflexiones generales acerca de las transformaciones en la ciudad como resultado de grandes proyectos urbanos que a la par de la intervención física, modifican notoriamente su carácter. En el trabajo se sostiene como hipótesis que a expensas de un urbanismo de inspiración globalizante que homogeniza y segrega, la ciudad va mutando en nuevos espacios de desposesión para los ciudadanos. Al correr de los tiempos actuales y bajo el influjo de los nuevos proyectos, su carácter histórico como espacio de intercambio y apropiación, va cediendo lugar a la tendencia a la banalización urbana, la segregación social del territorio y el aumento de las desigualdades. Un segundo tópico se vincula con la respuesta de la ciudadanía frente al problema. Aquí se sostiene que por la vía de la denuncia y la crítica; como de la organización y la acción, los ciudadanos comienzan a construir y consolidar nuevos escenarios para la reapropiación del territorio, de su identidad y de su cohesión socio-cultural. Finalmente, en la conclusión se sintetizan algunas de las propuestas expresadas a lo largo del trabajo, y se reflexiona sobre los desafíos que tienen por delante las administraciones locales y los ciudadanos en la producción de un nuevo urbanismo centrado en el derecho a la ciudad.