Academic literature on the topic 'Holocentric'

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.Key words: Cuscuta, holocentric chromosomes, heterochromatin.

Holocentric chromosomes assemble kinetochores along their length instead of at a focused spot. The elongated expanse of an individual holocentric kinetochore and its potential flexibility heighten the risk of stable attachment to microtubules from both poles of the mitotic spindle (merotelic attachment), and hence aberrant segregation of chromosomes. Little is known about the mechanisms that holocentric species have evolved to avoid this type of error. Our studies of the influence of KLP-19, an essential microtubule motor, on the behavior of holocentric Caenorhabditis elegans chromosomes suggest that it has a major role in combating merotelic attachments. Depletion of KLP-19, which associates with nonkinetochore chromatin, allows aberrant poleward chromosome motion during prometaphase, misalignment of holocentric kinetochores, and multiple anaphase chromosome bridges in all mitotic divisions. Time-lapse movies of GFP-labeled mono- and bipolar spindles demonstrate that KLP-19 generates a force on relatively stiff holocentric chromosomes that pushes them away from poles. We hypothesize that this polar ejection force minimizes merotelic misattachment by maintaining a constant tension on pole–kinetochore connections throughout prometaphase, tension that compels sister kinetochores to face directly toward opposite poles.

Due to the X-shape formation at somatic metaphase, the arrangement of the sister chromatids is obvious in monocentric chromosomes. In contrast, the sister chromatids of holocentric chromosomes cannot be distinguished even at mitotic metaphase. To clarify their organization, we differentially labelled the sister chromatids of holocentric Luzula and monocentric rye chromosomes by incorporating the base analogue EdU during replication. Using super-resolution structured illumination microscopy (SIM) and 3D rendering, we found that holocentric sister chromatids attach to each other at their contact surfaces similar to those of monocentrics in prometaphase. We found that sister chromatid exchanges (SCEs) are distributed homogeneously along the whole holocentric chromosomes of Luzula, and that their occurrence is increased compared to monocentric rye chromosomes. The SCE frequency of supernumerary B chromosomes, present additionally to the essential A chromosome complement of rye, does not differ from that of A chromosomes. Based on these results, models of the sister chromatid arrangement in mono- and holocentric plant chromosomes are presented.

Abstract Previous studies suggested that holocentric chromosomes may confer a selective advantage under high ionizing or UV-B radiation due to their tolerance of fragmentation, and that the first plant and animal colonizers of land in the Palaeozoic were or may have been holocentric. Holocentric chromosomes could have, therefore, aided terrestrialization of Earth’s biota half a billion years ago, because leaving water meant facing a sharp increase of UV-B. Because we cannot go back in time, the hypothesis needs to be tested with present-day species using an indicator of UV-B stress. We took advantage of the fact that UV-B intensity increases with elevation and tested whether holocentric plants (six species of Cyperaceae and Juncaceae) are less stressed with increasing elevation than monocentric plants (six species of Poaceae). Phylogenetically corrected regression showed that the proxy for UV-B stress (endopolyploidy index from 671 samples measured by flow cytometry) increased with elevation in holocentric and monocentric species, but the increase was more rapid in monocentric species. Although half a billion year elapsed since terrestrialization, holocentric Cyperaceae and Juncaceae still appear less stressed by UV-B than monocentric Poaceae, despite the other counter UV-B adaptations they both have evolved (graminoid morphology, silica bodies).

Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated “Tyba” and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle–dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.

Abstract Background and Aims Ultraviolet-B radiation (UV-B) radiation damages the DNA, cells and photosynthetic apparatus of plants. Plants commonly prevent this damage by synthetizing UV-B-protective compounds. Recent laboratory experiments in Arabidopsis and cucumber have indicated that plants can also respond to UV-B stress with endopolyploidy. Here we test the generality of this response in natural plant populations, considering their monocentric or holocentric chromosomal structure. Methods We measured the endopolyploidy index (flow cytometry) and the concentration of UV-B-protective compounds in leaves of 12 herbaceous species (1007 individuals) from forest interiors and neighbouring clearings where they were exposed to increased UV-B radiation (103 forest + clearing populations). We then analysed the data using phylogenetic mixed models. Key Results The concentration of UV-B protectives increased with UV-B doses estimated from hemispheric photographs of the sky above sample collection sites, but the increase was more rapid in species with monocentric chromosomes. Endopolyploidy index increased with UV-B doses and with concentrations of UV-B-absorbing compounds only in species with monocentric chromosomes, while holocentric species responded negligibly. Conclusions Endopolyploidy seems to be a common response to increased UV-B in monocentric plants. Low sensitivity to UV-B in holocentric species might relate to their success in high-UV-stressed habitats and corroborates the hypothesized role of holocentric chromosomes in plant terrestrialization.

Holocentric chromosomes of six species of Rhynchospora, R. ciliata, R. pubera, R. riparia and R. barbata (2n = 10), R. nervosa (2n = 30) and R. globosa (2n = 36), were stained with CMA3/DAPI fluorochromes or treated with C-banding and sequentially stained with Giemsa or CMA3/DAPI. Variability in banding pattern was found among the species studied. Heterochromatin was observed on terminal and interstitial chromosome regions, indicating that the holocentric chromosomes of Rhynchospora show a heterochromatin distribution pattern similar to those plant monocentric chromosomes.

L'organisation du génome dans le noyau cellulaire a été observée et décrite dans différents organismes depuis plus de 140 ans. La plupart des organismes où cela a été fait sont monocentriques, c’est-à-dire dont les chromosomes n’ont qu’un seul centromère. Il a été montré que les centromères contraignent fortement l'architecture des chromosomes en interphase, et j'ai contribué lors de ma thèse à l’écriture d’une revue décrivant ce phénomène (Muller et al., 2019). Cependant, à travers l'Arbre de la Vie eucaryote, on peut trouver plusieurs exemples d'organismes qui sont holocentriques, c’est-à-dire qui ont plusieurs centromères distribués sur toute la longueur de leurs chromosomes. Pour étudier l'impact des centromères sur ce type de chromosomes, nous avons choisi comme organisme modèle le ver à soie, Bombyx mori. Bien que les chromosomes holocentriques de B. mori aient fait l'objet d'études décrivant l'organisation et la formation de leurs centromères et kinétochores (Cortes-Silva et al., 2020 ; Senaratne et al., 2021), l'organisation de leur génome reste à décrire. Ma thèse vise à caractériser l'organisation du génome de B. mori en utilisant des techniques basées sur le séquençage et des approches bioinformatiques sur deux systèmes expérimentaux.Dans la première partie de ma thèse, j'ai utilisé une combinaison de données Hi-C et ChIP-Seq d'embryons de B. mori pour identifier et caractériser les caractéristiques de l'organisation du génome. En utilisant les techniques Hi-C, j'ai produit des cartes de contact pour les 28 chromosomes de l'assemblage du génome de B. mori et j'ai pu montrer que les chromosomes de B. mori établissent des contacts peu fréquents entre eux, ce qui donne lieu à des territoires chromosomiques forts. J'ai ensuite combiné ces données Hi-C avec des ensembles de données ChIP-Seq correspondant à plusieurs marques épigénétiques de l'embryon de B. mori afin de définir et de caractériser les compartiments chromosomiques. Cette étude a révélé que les chromosomes de B. mori sont organisés en trois compartiments à l'échelle du génome : A, B et X. Les compartiments A et B de B. mori rappellent ceux décrits pour la première fois dans les chromosomes humains. Le compartiment X est composé de régions très compactes et pauvres en gènes qui n'interagissent ni avec les deux autres compartiments, ni avec des compartiments similaires sur le même chromosome. Ces résultats sont inclus dans une étude à laquelle j'ai participé et qui décrit l'organisation du génome des embryons de B. mori (Muller et al., en cours).Dans la deuxième partie de ma thèse, je me suis tourné vers les lignées cellulaires de B. mori afin de déterminer certains des facteurs contribuant à cette organisation du génome. Pour ce faire, j'ai utilisé l'ARN interférence pour perturber les centromères, les cohésines et les condensines, dont l'impact sur l'organisation du génome a été démontré chez d'autres organismes. J'ai acquis des données Hi-C, profilé différentes marques épigénétiques dans chaques conditions, et montré que les centromères et les complexes SMC jouent un rôle dans l'organisation du génome de B. mori, la cohésine et la condensine II ayant des effets opposés sur le repliement des chromosomes à courte et longue distance. Pour analyser correctement ces données, j'ai développé des outils bioinformatiques pour tenir compte de la nature holocentrique des chromosomes de B. mori. J'ai également eu l'occasion de mettre mes compétences en bioinformatiques au service d'une collaboration portant sur l'appariement méiotique des chromosomes de B. mori (Rosin et al., 2021). Ensemble, en utilisant Hi-C et ChIP-Seq et en effectuant l'analyse bioinformatique des deux, j'ai pu décrire pour la première fois l'organisation du génome de B. mori et caractériser les rôles des facteurs qui y contribuent
The genome’s organization within the cell nucleus has been observed and described in various different organisms for over 140 years. Most of the organisms where this has been done are monocentric, or organisms that have chromosomes with a single centromere. Studies have shown that centromeres strongly constrain the architecture of chromosomes in interphase and during my thesis, I contributed to a review describing this phenomenon (Muller et al., 2019). However, across the eukaryotic tree of life you can find several examples of organisms that are holocentric, or organisms that have centromeres distributed along the entire length of their chromosomes. To study the impact of centromeres on these types of chromosomes, we choose the silkworm, Bombyx mori, as our model organism. Although the holocentric chromosomes of B. mori have been subjects of studies describing the organization and formation of their centromeres and kinetochores (Cortes-Silva et al., 2020; Senaratne et al., 2021), their genome organization has yet to be described. My thesis aims at characterizing the genome organization of B. mori with the use of sequencing-based techniques and bioinformatic approaches on two experimental systems.In the first part of my thesis I used a combination of Hi-C and ChIP-Seq data from B. mori embryos to identify and characterize genome organizational features. Using Hi-C, I produced contact maps for all 28 chromosomes of the B. mori genome assembly and I was able to show that B. mori chromosomes make infrequent contacts between themselves resulting in strong chromosome territories. I then combined this Hi-C data with ChIP-Seq data sets corresponding to several B. mori embryo epigenetic marks in order to define and characterize chromosome compartments. This study revealed that B. mori chromosomes are organized into three genome-wide compartments: A, B and X. The A and B compartments in B. mori are reminiscent of those first described in human chromosomes. The X compartment is composed of highly compact, gene-poor regions that do not interact with the other two compartments, nor with like compartments on the same chromosome. These findings are included in a study that I was a part of describing the genome organization of B. mori embryos (Muller et al., in progress).In the second part of my thesis I turned to B. mori cell lines in order to determine some of the factors contributing to this genome organization. In order to do this, I used RNAi to perturb centromeres, cohesins and condensins which have been shown to have an impact on genome organization in other organisms. I acquired Hi-C data and profiled different epigenetic marks in each condition and show that centromeres and SMC complexes play a role in B. mori genome organization with cohesin and condensin II having opposite effects in short- and long-range chromosome folding. To properly analyze this data, I developed bioinformatic tools to account for the holocentric nature of B. mori chromosomes. I even had the chance to contribute my bioinformatic skill set to a collaboration studying meiotic pairing of B. mori chromosomes (Rosin et al., 2021). Taken together, using Hi-C and ChIP-Seq and performing the bioinformatic analysis of both, I was able to describe for the first time the genome organization of B. mori and characterize the roles of factors contributing to it

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os hemipteros da infraordem Cicadomorpha são representados por aproximadamente 30.000 espécies de insetos sugadores distribuídos mundialmente. Apesar de se destacarem por causarem muitos prejuízos na agricultura e pecuária pouco se sabe sobre a variabilidade genética e cromossômica desses animais que apresentam cromossomos holocentricos. Os DNAs repetitivos são uma ferramenta útil em estudos de diversificação cariotípica, organização e evolução dos genomas. Deste modo, este trabalho teve como objetivo contribuir com o conhecimento sobre a dinâmica dos cariótipos de representantes de Cicadomorpha e a organização de DNAs repetitivos, especificamente: I. analisar o cariótipo de espécies pertencentes a quatro famílias de Cicadomorpha, bem como caracterizar a heterocromatina constitutiva quanto a riqueza de pares de base; II. inferir a respeito da dinâmica evolutiva de famílias gênicas por meio do mapeamento cromossômico; III. analisar a organização cromossômica e testar a conservação interespecífica da sequência telomérica TTAGGn do pool de DNAs repetitivos obtidos (fração C0t) de espécies do gênero Mahanarva e das sequências teloméricas; IV. Analisar o satelitoma de Mahanarva quadripunctata e comparar os diferentes DNAs satélites de seu genoma com o de outras espécies do gênero visando entender a organização e evolução dos satDNAs neste grupo. Os dados obtidos revelam ampla variabilidade cromossômica entre as distintas famílias, causada principalmente por fusões cromossômicas, entretanto dentro de uma mesma família os cariótipos tendem a apresentar menos variações ao nível macrocromossômico. Embora os Cicadomorpha apresentem variabilidade em números diplóides a organização dos DNAs repetitivos é bastante conservada, mesmo em famílias distantes filogeneticamente, sugerindo estabilidade. Alguns dados foram analisados baseados na filogenia da infraordem, sendo sugerido os possíveis padrões ancestrais. Além disso, as possíveis causas da variabilidade em relação aos padrões modais são sugeridos. Os dados apresentados são um avanço no conhecimento da organização de DNAs repetitivos em Cicadomorpha, sendo para algumas sequências a primeira vez que se realiza algum estudo.
The hemipterans of the Cicadomorpha infraorder are represented by approximately 30.000 species of sucking insects distributed worldwide. Although they stand out by cause many damages in agriculture and livestock, they are poorly studied regarding the genetic and chromosomal variability of these animals that present holocentric chromosomes. Repetitive DNAs are a useful tools in studies of karyotypic diversification, organization and evolution of genomes. The aim of this work was to contribute with knowledge about the dynamics of the karyotypes of Cicadomorpha and the organization of repetitive DNAs, specifically: I. Analyze the karyotype of species belonging to four families of Cicadomorpha, as well as to characterize the constitutive heterochromatin regarding base pairs richess; II. Infer about the evolutionary dynamics of multigene families through the chromosomal mapping; III. Analyze the chromosomal organization and test the interspecific conservation of the telemetric repeat TTAGGn from the pool of repetitive DNA fraction (C0t fraction) of Mahanarva species and telomeric sequences; IV. Analyze the satellitome of Mahanarva quadripunctata and compare the different satellite DNAs of its genome with that from other species of the genus in order to understand the organization and evolution of satDNAs in this group. The data obtained reveal a wide chromosomal variability among the different families, caused mainly by chromosomal fusions, however within a same family the karyotypes tend to present less variations at the macro-chromosomal level. Although Cicadomorpha exhibit variability in diploid numbers, the organization of repetitive DNAs is highly conserved, even in phylogenetically distant families, suggesting stability. Some data were analyzed based on the phylogeny of the infraorder, suggesting possible ancestral patterns. In addition, the possible causes of variability relative to modal patterns are suggested. The data presented is an advance in the knowledge of the organization of repetitive DNAs in Cicadomorpha, being for some sequences the first time that some study is done.
FAPESP: 2014/06226-3.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Os heterópteros apresentam a meiose cística nos túbulos seminíferos. Esses possuem o cisto espermatogonial envolto pelas células císticas, as quais desenvolvem a função de nutrição das células em divisão celular. Quanto às características citogenéticas, esses insetos apresentam cromossomos holocinéticos, baixa variabilidade cariotípica e meiose invertida dos cromossomos sexuais. No presente trabalho foram caracterizadas as células císticas quanto a sua localização, ultraestrutura e citogenética e, também, foram analisados os aspectos citogenéticos de quatro espécies do gênero Triatoma. Foram utilizadas as técnicas de microscopia eletrônica de transmissão, citogenética convencional (orceína e AgNOR), bandamento C CMA3/DAPI e a técnica de hibridização in situ fluorescente (FISH), com sonda de DNAr 45S de Drosophila melanogaster. Os resultados indicaram que a célula cística envolve um cisto espermatogonial e apresenta um grande núcleo com invaginações citoplasmáticas. Em todas as espécies foram observados vários graus de ploidia da célula cística. Triatoma infestans e T. infestans melanosoma apresentaram vários blocos heterocromáticos com a periferia CMA3 + e o interior DAPI+. Associada às bordas dos blocos heterocromáticos foram observados os segmentos de DNAr 45S, além da presença de vários nucléolos em cada núcleo. Triatoma matogrossensis, T. rubrovaria e T. brasiliensis apresentaram apenas um bloco heterocromático com as mesmas características, com exceção de T. brasiliensis, que apresentou em algumas células vários blocos CMA3 + dispersos. Nessas espécies foi observado apenas um nucléolo com similaridade na localização dos sítios de DNAr. Quanto aos aspectos citogenéticos, todas as espécies apresentaram 2n = 20A + XY, com decréscimo do tamanho relativo dos cromossomos. Em T. infestans melanosoma os cromossomos foram...
Heteroptera, or true bugs, exhibit meiosis in their seminiferous tubules. They posses the spermatogonial cysts that are enclosed by cyst cells, which develop the nutritional function of the cells during cell division. In terms of cytogenetic characteristics, these insects possess holokinetic chromosomes, low karyotype variability, and inverted meiosis in the sex chromosomes. In this study, cyst cells from four species of the genus Triatoma were characterized by their location, superstructure, and cytogenetic makeup. Electronic transmission microscopy techniques were used, as well as conventional cytogenetic techniques (Orcein and AgNOR), C-banding with CMA3 and DAPI banding, and Fluorescence in situ Hybridization (FISH) with a 45S DNA probe of Drosophila melanogaster. The results indicated that the the spermatogonial cyst is enclosed by the cyst cell, and that the cyst cell possesses a large nucleus with cytopasmic invaginations. In all species studied, varying degrees of ploidy were observed in the cyst cells. Triatoma infestans and T. infestans melanosoma presented with various heterochromatic blocks, with CMA3 + at the periphery and DAPI+ at the interior. Segments of rDNA 45S were found along the edges of the heterochromatic blocks, along with the presence of various nucleoli in each nucleus. Triatoma matogrossensis, T. rubrovaria and T. brasiliensis presented with only one heterochromatic block with the same characteristics (with the exception of T. brasiliensis, which presented with various dispersed CMA3 + blocks). In these species, only one nucleolus that was similar to the localization of the rDNA sites was found. All species presented with 2n = 20A + XY, with a decrease in size relative to the chromosomes. In the case of T. infestans melanosoma, the chromosomes were split into groups based on their relative sizes. The heterochromatin of this species presented... (Complete abstract click electronic access below)

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Os triatomíneos são vetores do protozoário Typanosoma cruzi, agente etiológico da moléstia de Chagas. Esses insetos são hematófagos e pertencem à ordem Heteroptera e à família Reduviidae. Disseminada por grandes extensões do Brasil e de outros países latinoamericanos, a doença de Chagas representa um grave e importante problema de saúde pública, caracterizando limitações e dificuldades aos tratamentos. Isso ocorre devido à precariedade apresentada pela vida dos contingentes humanos mais expostos à infecção. Atualmente, a parasitose tem grande participação entre as doenças cardíacas na América do Sul. Segundo a Organização Mundial de Saúde, estima-se que, até a década de 90, por volta de 20 milhões de indivíduos estavam infectados pelo Trypanosoma cruzi nas áreas endêmicas. Dados atuais revelam que o número de pessoas infectadas foi reduzido para 9,8 milhões graças à intensa erradicação desses insetos. No entanto, a vigilância deve continuar, pois é fundamental para se evitar novos casos. Citogeneticamente, o interesse sobre os triatomíneos está em seus cromossomos holocêntricos e no processo incomum da meiose, cuja segregação dos sexuais é pós-reducional. O número básico de cromossomos nos triatomíneos é de 2n=22. No presente trabalho foi analisado a espermatogênese de duas espécies do gênero Triatoma (Triatoma maculata e Triatoma pseudomaculata), com ênfase aos seguintes aspectos: fases da espermatogênese; estrutura cromatínica e dos cromossomos meióticos e acompanhamento do ciclo nucleolar. Essas espécies estão distribuídas principalmente nos estados do Nordeste brasileiro e são consideradas potencialmente vetores do T. cruzi. As espécies analisadas foram cedidas pelo insetário do Serviço Especial de Saúde de Araraquara (SESA), pertencente ao Departamento de Epidemiologia da Faculdade de Saúde Pública da USP. Os...
The triatomines are vectors of the protozoan Typanosoma cruzi, etiological agent of Chagas' disease. These insects are hematophages that belong to the order Heteroptera and to the family Reduviidae. Disseminated through large portions of Brazil and of other Latin American countries, Chagas' disease represents a grave and important public health problem, characterizing limitations and difficulties in treatments. This occurs due to the precariousness presented by life contingent to humans most exposed to the infection. Currently, parasitosis presents high participation among cardiac diseases in South America. According to the World Health Organization, it is estimated that, until the 1990s, approximately 20 million individuals were infected by Trypanosoma cruzi in endemic areas. Current data reveal that the number of persons infected was reduced to 9.8 million by virtue of intense eradiaction of these insects. Nevertheless, vigilance must continue, since it is fundamental to avoiding new cases. Cytogenetically, the interest in triatomines is in its holocentric chromosomes and in its uncommon meiosis process, whose sexual segregation is post-reductional. The basic number of chromosomes in triatomines is 2n=22. The present work analyzed the spermatogenesis of two species of the genus Triatoma (Triatoma maculata and Triatoma pseudomaculata), with emphasis on the following aspects: spermatogenesis phases; structure of chromatin and of meiotic chromosomes and accompaniment of the nucleolar cycle. These species are distributed principally in the states of northeastern Brazil and are considered potential vectors for T. cruzi. The species analyzed were supplied by the insectary of the Special Health Service of Araraquara (SESA), belonging to the Department of Epidemiology in the School of Public Health at USP. The organs studied were...(Complete abstract click electronic access below)

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Banca: Hermione Elly Melara de Campos Bicudo
Banca: Patricia Pasquali Parise Maltempi
Resumo: Os heterópteros apresentam a meiose cística nos túbulos seminíferos. Esses possuem o cisto espermatogonial envolto pelas células císticas, as quais desenvolvem a função de nutrição das células em divisão celular. Quanto às características citogenéticas, esses insetos apresentam cromossomos holocinéticos, baixa variabilidade cariotípica e meiose invertida dos cromossomos sexuais. No presente trabalho foram caracterizadas as células císticas quanto a sua localização, ultraestrutura e citogenética e, também, foram analisados os aspectos citogenéticos de quatro espécies do gênero Triatoma. Foram utilizadas as técnicas de microscopia eletrônica de transmissão, citogenética convencional (orceína e AgNOR), bandamento C CMA3/DAPI e a técnica de hibridização in situ fluorescente (FISH), com sonda de DNAr 45S de Drosophila melanogaster. Os resultados indicaram que a célula cística envolve um cisto espermatogonial e apresenta um grande núcleo com invaginações citoplasmáticas. Em todas as espécies foram observados vários graus de ploidia da célula cística. Triatoma infestans e T. infestans melanosoma apresentaram vários blocos heterocromáticos com a periferia CMA3 + e o interior DAPI+. Associada às bordas dos blocos heterocromáticos foram observados os segmentos de DNAr 45S, além da presença de vários nucléolos em cada núcleo. Triatoma matogrossensis, T. rubrovaria e T. brasiliensis apresentaram apenas um bloco heterocromático com as mesmas características, com exceção de T. brasiliensis, que apresentou em algumas células vários blocos CMA3 + dispersos. Nessas espécies foi observado apenas um nucléolo com similaridade na localização dos sítios de DNAr. Quanto aos aspectos citogenéticos, todas as espécies apresentaram 2n = 20A + XY, com decréscimo do tamanho relativo dos cromossomos. Em T. infestans melanosoma os cromossomos foram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Heteroptera, or "true bugs", exhibit meiosis in their seminiferous tubules. They posses the spermatogonial cysts that are enclosed by cyst cells, which develop the nutritional function of the cells during cell division. In terms of cytogenetic characteristics, these insects possess holokinetic chromosomes, low karyotype variability, and inverted meiosis in the sex chromosomes. In this study, cyst cells from four species of the genus Triatoma were characterized by their location, superstructure, and cytogenetic makeup. Electronic transmission microscopy techniques were used, as well as conventional cytogenetic techniques (Orcein and AgNOR), C-banding with CMA3 and DAPI banding, and Fluorescence in situ Hybridization (FISH) with a 45S DNA probe of Drosophila melanogaster. The results indicated that the the spermatogonial cyst is enclosed by the cyst cell, and that the cyst cell possesses a large nucleus with cytopasmic invaginations. In all species studied, varying degrees of ploidy were observed in the cyst cells. Triatoma infestans and T. infestans melanosoma presented with various heterochromatic blocks, with CMA3 + at the periphery and DAPI+ at the interior. Segments of rDNA 45S were found along the edges of the heterochromatic blocks, along with the presence of various nucleoli in each nucleus. Triatoma matogrossensis, T. rubrovaria and T. brasiliensis presented with only one heterochromatic block with the same characteristics (with the exception of T. brasiliensis, which presented with various dispersed CMA3 + blocks). In these species, only one nucleolus that was similar to the localization of the rDNA sites was found. All species presented with 2n = 20A + XY, with a decrease in size relative to the chromosomes. In the case of T. infestans melanosoma, the chromosomes were split into groups based on their relative sizes. The heterochromatin of this species presented... (Complete abstract click electronic access below)
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Cromossomos holocêntricos são caracterizados pela ausência de constrição primária e apresentam normalmente a proteína centromérica CENH3 distribuída ao longo de um eixo em cada cromátide. Embora muitos organismos com cromossomos monocêntricos apresentem sequências de DNA centroméricas específicas e associadas com a CENH3, nenhuma sequência centromérica havia sido identificada em organismos com cromossomos holocêntricos até o momento. Além disso, vários estudos reportam adaptações meióticas em espécies com cromossomos holocêntricos. Sendo observada em alguns casos uma inversão da ordem dos eventos meióticos (meiose invertida ou pós-reducional). Assim, o presente trabalho objetivou estudar a organização centromérica e a meiose de espécies com cromossomos holocêntricos do gênero Rhynchospora (Cyperaceae). Foi realizada uma análise citogenômica da organização e composição dos holocentrômeros de Rhynchospora pubera (2n = 10), sendo reportada a primeira descoberta de sequências centroméricas em espécies com cromossomos holocêntricos. Foi observado que os holocentrômeros de R. pubera são compostos principalmente por arranjos de DNA satélite (Tyba) e retroelementos centroméricos (CRRh) distribuídos pelo genoma. A análise detalhada da sucessão dos eventos meióticos de R. pubera e R. tenuis (2n = 4) reportou uma prófase inicial semelhante a de monocêntricos. No entanto, foi verificado que as cromátides-irmãs separam para polos opostos durante a anáfase I e os homólogos segregam somente durante a meiose II, comprovando uma meiose invertida para ambas as espécies. Curiosamente, durante a meiose de R. pubera foi observado uma organização diferencial dos centrômeros. Ao contrário do observado em mitose, durante meiose não foi observado a formação de holocentrômeros em forma de linha, sendo, na verdade, observado estruturas centroméricas aglomeradas. O restabelecimento de holocentrômeros em forma de linha se deu durante a primeira mitose do pólen.
Holocentric chromosomes are characterized by the absence primary constriction and normally show the centromeric protein CENH3 distributed along the axis of each chromatid. Although many monocentric organisms show centromere-specific DNA sequences associated to CENH3, no centromeric sequences had been identified in any holocentric organism so far. Furthermore, many studies report meiotic adaptations in holocentric species. In some cases is observed an inversion of the order of meiotic events. This type of meiosis has been named of inverted or post-reductional meiosis and would be exclusive of holocentric organisms. Thus, the present work aimed to study the centromere organization and meiosis of holocentric species of the genus Rhynchospora (Cyperaceae). A cytogenomic analysis of the composition and organization of the holocentromeres of Rhynchospora pubera (2n = 10) has been performed, being reported the first centromeric sequences from a holocentric species. It was observed that the holocentromeres of R. pubera are composed mainly by arrays of satellite DNA (Tyba) and centromeric retrotransposons (CRRh) distributed genomewide. The detailed analysis of the succession of meiotic events of R. pubera and R. tenuis (2n = 4) demonstrated an early meiotic prophase similar to that of monocentric. However, it was verified that sister chromatids separate to opposite poles during anaphase I, while homologs only segregate at meiosis II. These results prove the inverted meiosis for both species. Curiously, it was observed during meiosis of R. pubera a differential organization of centromere units. In contrast to the observed in mitosis, during meiosis we did not observed the formation of line-like holocentromeres, being in fact observed the formation of cluster-like holocentromeres. The reestablishment of a line-like holocentromere occurred during the first pollen mitosis.

Orientador: Maria Tercília Vilela de Azeredo Oliveira
Banca: Alba Regina de Abreu Lima Catelani
Banca: Lilian Castiglioni
Resumo: Os triatomíneos são vetores do protozoário Typanosoma cruzi, agente etiológico da moléstia de Chagas. Esses insetos são hematófagos e pertencem à ordem Heteroptera e à família Reduviidae. Disseminada por grandes extensões do Brasil e de outros países latinoamericanos, a doença de Chagas representa um grave e importante problema de saúde pública, caracterizando limitações e dificuldades aos tratamentos. Isso ocorre devido à precariedade apresentada pela vida dos contingentes humanos mais expostos à infecção. Atualmente, a parasitose tem grande participação entre as doenças cardíacas na América do Sul. Segundo a Organização Mundial de Saúde, estima-se que, até a década de 90, por volta de 20 milhões de indivíduos estavam infectados pelo Trypanosoma cruzi nas áreas endêmicas. Dados atuais revelam que o número de pessoas infectadas foi reduzido para 9,8 milhões graças à intensa erradicação desses insetos. No entanto, a vigilância deve continuar, pois é fundamental para se evitar novos casos. Citogeneticamente, o interesse sobre os triatomíneos está em seus cromossomos holocêntricos e no processo incomum da meiose, cuja segregação dos sexuais é pós-reducional. O número básico de cromossomos nos triatomíneos é de 2n=22. No presente trabalho foi analisado a espermatogênese de duas espécies do gênero Triatoma (Triatoma maculata e Triatoma pseudomaculata), com ênfase aos seguintes aspectos: fases da espermatogênese; estrutura cromatínica e dos cromossomos meióticos e acompanhamento do ciclo nucleolar. Essas espécies estão distribuídas principalmente nos estados do Nordeste brasileiro e são consideradas potencialmente vetores do T. cruzi. As espécies analisadas foram cedidas pelo insetário do Serviço Especial de Saúde de Araraquara (SESA), pertencente ao Departamento de Epidemiologia da Faculdade de Saúde Pública da USP. Os...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The triatomines are vectors of the protozoan Typanosoma cruzi, etiological agent of Chagas' disease. These insects are hematophages that belong to the order Heteroptera and to the family Reduviidae. Disseminated through large portions of Brazil and of other Latin American countries, Chagas' disease represents a grave and important public health problem, characterizing limitations and difficulties in treatments. This occurs due to the precariousness presented by life contingent to humans most exposed to the infection. Currently, parasitosis presents high participation among cardiac diseases in South America. According to the World Health Organization, it is estimated that, until the 1990s, approximately 20 million individuals were infected by Trypanosoma cruzi in endemic areas. Current data reveal that the number of persons infected was reduced to 9.8 million by virtue of intense eradiaction of these insects. Nevertheless, vigilance must continue, since it is fundamental to avoiding new cases. Cytogenetically, the interest in triatomines is in its holocentric chromosomes and in its uncommon meiosis process, whose sexual segregation is post-reductional. The basic number of chromosomes in triatomines is 2n=22. The present work analyzed the spermatogenesis of two species of the genus Triatoma (Triatoma maculata and Triatoma pseudomaculata), with emphasis on the following aspects: spermatogenesis phases; structure of chromatin and of meiotic chromosomes and accompaniment of the nucleolar cycle. These species are distributed principally in the states of northeastern Brazil and are considered potential vectors for T. cruzi. The species analyzed were supplied by the insectary of the Special Health Service of Araraquara (SESA), belonging to the Department of Epidemiology in the School of Public Health at USP. The organs studied were...(Complete abstract click electronic access below)
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