Thesis Abstracts

Purification and biochemical characterization of a polypeptide from Phoneutria nigriventer spider venom, which affects rabbit corpus cavernosum (Purificação e caracterização bioquímica de um polipeptídeo do veneno da aranha Phoneutria nigriventer com atividade no corpo cavernoso do coelho)

Evandro José Lima Rego*

*1995. Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil. Master’s thesis. Orienting Professor: Benedito Oliveira Filho.

The venom of the spider Phoneutria nigriventer induces various biological reactions, including priapism, which is characterized by a painful and prolonged penile erection. For effective penile erection the relaxation of the corpus cavernosum smooth muscle is necessary. This is regulated by both the nervous system and nitric oxide. Phoneutria nigriventer venom activates the tissue kallikrein system in the rabbit corpus cavernosum, which produces kinin and consequently permits nitric oxide release, leading to relaxation of this tissue. A polypeptide responsible for the relaxation of the rabbit corpus cavernosum was purified from Phoneutria nigriventer venom by high performance liquid chromatography (HPLC). This polypeptide, called PNV4, was biochemically characterized through polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis and N-terminal amino acid sequencing, using automated Edman degradation. PNV4 has a molecular mass of 16.9 kDa, with 144 amino acid residues and presents the following N-terminal sequence. A E L T S C F P V G H E C D G D A S N C N C C G D D V Y C G C G W G R W N C K C K V A D Q S Y A. Comparison of PNV4 with TX1 (another toxin isolated from this venom but with a molecular mass of 8.1 kDa) showed an identical N-terminal amino acid sequence in the first 48 residues. This similarity could be explained by a post-translational process. Isolation of a polypeptide which induces relaxation of corpus cavernosum muscle provides an important tool for the study of the mehanism of action of penile erection.

Publication supported by FAPESP.

Contribution of molecular cytogenetics to the chromosome evolution understanding of genus Akodon (Rodentia, Sigmodontinae) (Contribuição da citogenética molecular para o entendimento da evolução cromossômica no gênero Akodon (Rodentia, Sigmodontinae))

Valéria Fagundes*

* 1997. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil. Doctoral thesis. Orienting Professor: Dr. Yatiyo Yonenaga-Yassuda.

Cytogenetic analysis of 133 specimens of Brazilian rodents of genus Akodon (Muridae: Sigmodontinae) from several localities of the State of São Paulo, Brazil, was performed in the present study. The analysis was carried out on the species A. cursor (2n = 14, 15 and 16), A. montensis (2n = 23, 24 and 25), A. nigrita (2n = 52) and A. serrensis (2n = 46).

The metaphases obtained from bone marrow and fibroblast culture preparations were analyzed after GTG- and CBG-banding and nucleolus organizer regions after silver-staining (Ag-NORs) techniques. Some specimens were also analyzed by in situ hybridization (FISH) using telomeric and ribosomal probes, and by chromosome painting using probes obtained from microdissected chromosomes amplified by DOP-PCR (degenerated oligonucleotide priming-polymerase chain reaction).

Twenty-four different karyotypes in a total of 291 specimens of A. cursor, including those from this study, have already been described with diploid number ranging from 14 to 16, and fundamental number ranging from 18 to 26. In this study, we found three new cytotypes among 15 different karyotypes analyzed in 46 specimens from the State of São Paulo. The differences among cytotypes were due to pericentric inversions in three autosomal pairs (2n, 3 and 5), associated to a complex rearrangement in par 1. Data from G-banded prometaphasic chromosomes and FISH of telomeric probes in A. cursor provided evidence to suggest that centric fusion and pericentric inversions had occurred in pair 1. The form 2n = 16 is considered to be the likely ancestral diploid number in A. cursor.

The species A. montensis showed diploid number variation (2n = 23, 24 and 25) due to monosomy of X chromosome in some females and presence of supernumerary chromosome in both sexes. A morphological heteromorphism was detected in the X chromosomes, which showed to be acrocentric and subtelocentric. The subtelocentric form presented constitutive heterochromatin in the short arm. Five unexpected XY females were detected and the histological analysis revealed a normal female genital and gonadal constitution. Chromosomal analysis revealed the occurrence of a translocation Y/X in those females of a portion of Y chromosome in X chromosome after chromosome painting using species-specific Y probe.

Akodon cursor (2n = 14, 15 and 16) and A. montensis (2n = 24, 25) had their karyotypes compared by G-banding and chromosome painting. The origin of one A. cursor autosome from three different A. montensis autosomes is suggested based on comparative GTG- banding analysis. The evidence of the chromosome evolution was provided for in situ hybridization of the biotinylated DOP-PCR product obtained from a microdissected chromosome of A. cursor on A. montensis chromosomes, associated with location of telomeric sequences using (TTAGGG)_n oligomer. Furthermore, the complete homology between A. cursor and A. montensis karyotypes was documented, including the occurrence of tandem fusion, pericentric inversions, and loss of telomeres, centromeres and chromosome arms. Evidence of the ancestral origin of A. cursor karyotype was pointed out. The rearrangements of karyological differentiation, the occurrence of three natural hybrids between A. cursor and A. montensis, and the extreme morphological similarities support the assumption that the cladogenesis process could be recent.

Comparative studies between Ag-NOR staining and FISH using ribosomal probes in A. cursor and A. montensis karyotypes showed that sometimes ribosomal genes are not detected by FISH. Moreover, the Ag-NOR variability is due to either the presence or absence of ribosomal genes, or to the differential transcription activity of rDNA genes. Both techniques are very useful to the understanding of gene control process.

Data revision of living species of Akodon group revealed 52 species, and 39 had their karyotypes described. Diploid numbers range from 2n = 14 in A. cursor to 2n = 52 in several species. A scenario of Akodont rodents geographic dispersion, based on chromosome data, including Brazilian species, was suggested in this thesis.

Publication supported by FAPESP.

Cellular and nuclear morphophysiological changes in human breast epithelial cells transformed by benzo(a)pyrene and transfected with the c-Ha-ras oncogene: cytochemical and image analysis aspects (Alterações morfológicas celulares e nucleares em células epiteliais mamárias humanas transformadas por benzo(a)pireno e transfectadas com o oncogene c-Ha-ras: aspectos citoquímicos e de análise de imagem)

Luís Fernando Barbisan*

* 1997. Departamento de Biologia Celular, IB, UNICAMP, Campinas, SP, Brasil. Master’s thesis. Orienting Professor: Maria Luiza S. Mello.

Cellular and nuclear morphophysiological changes were investigated by cytochemistry and image analysis in human breast epithelial cell lines, MCF-10F, transformed by benzo(a)pyrene and expressing different stages of progression to neoplastic transformation (BP1, BP1-E and BP1-E1 cell lines), and additionally transfected with the c-Ha-ras oncogene (BP1-Tras cells). Increased mitotic index was observed in BP1-E, BP1-E1, and BP1-Tras cells. An increase in frequency of apoptotic cells was only observed in BP1-Tras cells.

The patterns of relocation of the RNA metachromasy during mitosis were not found to differ when comparing nontransformed and transformed MCF-10F cells. However, nucleolus-like bodies were found close to the chromosomal plate at metaphase or to the spindle in subsequent mitotic phases. These bodies were not abundant in nontransformed MCF-10F cells, and became much scarcer in the transformed cells, possibly due to a decrease in the surplus RNA involved.

Nuclear pleomorphism was evident in all the transformed cell lines. Nuclear abnormalities such as karyorrhexis, karyolysis, multinucleation and micronucleation were more frequent in BP1-Tras cells. All cell lines exhibited binucleate and giant cells, at similar frequencies. Nuclei with smaller areas and perimeters and a more irregular shape were found in BP-transformed cell lines (BP1, BP1-E and BP1-E1 cells). In particular, the nuclear sizes and perimeter of BP1-Tras cells increased when compared to those of BP1, BP1-E and BP1-E1 cells, but were not found to differ from values of the nontransformed MCF-10F cells. The nuclear area and perimeter increase may involve a certain degree of aneuploidy promoted by the ras transfection. Nucleolus sizes and numbers in BP1-E and BP1-Tras cells were found to differ from those of MCF-10F, BP1 and BP1-E1 cells. A nucleolar size increase with a decrease in nucleolus numbers was observed in BP1-E and BP1-Tras cells. Nucleolus fusion and enlargement possibly reflect metabolic activities enhanced in BP1-E and BP1-Tras cells in comparison with the other cell lines. The morphometric parameters analysis did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of tumorigenesis, with the exception of the nucleolar size, which was associated with expression of the tumorigenic phenotype, and the nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumorigenic phenotypes from one another.

The nuclear morphometric data established for the BP-transformed cells and for those transfected by the c-Ha-ras oncogene indicated the existence of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF-10F cells. The increase in frequency of micronuclei and multinucleate cells and in aneuploidy in the BP1-Tras cell line was assumed to be related to the genomic instability induced by ras transfection.

Publication supported by FAPESP.

Cytological aspects of the genetic variability in Glomerella cingulata (Stonem) Spauld & Schrenck f. sp. phaseoli (Colletotrichum lindemuthianum (Sacc. & Magn.) Scribner) (Aspectos citológicos da variabilidade genética em Glomerella cingulata (Stonem) Spauld & Schrenck f. sp. phaseoli (Colletotrichum lindemuthianum (Sacc. & Magn.) Scribner))

María Gabriela Roca Magallanes*

*1997. Laboratório de Citologia, Departamento de Biologia, Universidade Federal de Lavras (UFLA), Lavras, MG, Brasil. Master’s thesis. Orienting Professor: Lisete Chamma Davide.

Colletotrichum lindemuthianum (Sacc. & Magn.) Scribner (mitosporic form) is found in nature causing anthracnose disease in beans, although the meiosporic form is found in culture as Glomerella cingulata f. sp. phaseoli. The objective of the present study was to determine the variability of Glomerella cingulata f. sp. phaseoli. Cytological studies of the mitosporic and meiosporic forms of the fungus were carried out in culture. The majority of the microscopic observations were based on the fungal structures stained with propiocarmim-orcein.

G. cingulata f. sp. phaseoli presents four chromosomes and the normal meiosis process is more frequent than the abnormal process. In the mitosporic form three phenomena which affect genetic variability were demonstrated: nuclear material changes between conidial anastomoses, chromosome polymorphism and the necessary cytological steps for the parasexual cycle to occur.

Research supported by CNPq.

The biology of Spermologus rufus Boheman (Coleoptera: Curculionidae) on seeds of Araucaria angustifolia (Bert.) Kuntze (Biologia de Spermologus rufus Boheman (Coleoptera: Curculionidae) em sementes de Araucaria angustifolia (Bert.) Kuntze)

Marliton Rocha Barreto*

*1997. Pós-graduação em Entomologia, DBA, CCB, Universidade Federal de Viçosa (UFV), Viçosa, MG, Brasil. Master’s thesis. Orienting Professor: Norivaldo dos Anjos.

Morphology, survival, longevity and behavior in all biological stages of Spermologus rufus Boheman were studied at 24.95 ± 0.91^oC, 78.5 ± 5.9% relative humidity and a 12-h photophase. Adults were reared in Wheaton flasks and fed Araucaria angustifolia seeds. These seeds were dissected every day for evaluation of the biological stages. The eggs were oblong, transparent, membranous, apparently smooth and had a yellowish white chorion. The greatest width and length of the eggs were 0.63 ± 0.03 mm and 1.06 ± 0.06 mm, respectively. Incubation period varied from two to six days and mean viability was 97.91 ± 1.66%. Larvae were subcilindrical, rather curved, with a light beige color, and had three instars. The larval period varied from three to 24 days. Pupae, initially white, darkened gradually during development. They were sexed by a round and elevated protuberance, divided by a central depression, located in the ninth sternite in the female only. Male pupae had, on average, 7.02 ± 0.51 mm, 4.27 ± 0.25 mm and 41.40 ± 4.16 mg of body length, width and weight, respectively. Female pupae had 7.13 ± 0.34 mm, 4.41 ± 0.26 mm and 42.35 ± 5.26 mg, respectively. The pupal period varied from six to 14 days. Adults were reddish brown. The maximum length and width of the males were, on average, 8.18 ± 0.37 mm and 3.33 ± 0.14 mm, respectively, and the females 8.61 ± 0.43 mm and 3.39 ± 0.13 mm, respectively. Females had a longer and narrower rostrum than males. The sexual proportion was 1.0 female to 1.43 males. Mean longevity was 219 ± 66.9 days for males and 166 ± 53 for females. There was only one egg in 80.45% of the seeds. The seed medial region and the insertion point were preferred for oviposition. Eggs were laid individually and the mean number of eggs laid per female was 146.7 ± 43.9. Average periods of preoviposition and oviposition were 11.1 ± 7.5 and 118.1 ± 42.3 days, respectively. Only 2% of the attacked seeds germinated. Adults survived 56.6 days, on average, without feeding. They showed polyvoltinism, thanatosis and negative phototaxis; they did not fly and walked about actively when food became scarce. Predation of S. rufus adults by Nesticodes rufipes was observed (Lucas, 1849) (Araneae: Theridiidae).

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