A cachaça é uma bebida típica brasileira produzida a partir da destilação do caldo de canade- açúcar fermentado principalmente por Saccharomyces cerevisiae. Grande parte da produção nacional é artesanal, e não há uma preocupação por parte dos produtores quanto ao controle microbiológico da fermentação. Este trabalho objetivou caracterizar a comunidade bacteriana contaminante do fermento utilizado na produção de cachaça. Foram coletadas quatro amostras em um alambique artesanal. A primeira (NA) foi coletada um ano anterior às outras três e utilizada como controle. As restantes foram coletadas ao final do primeiro dia de fermentação (NP), após quinze (NS) e trinta dias (NT) utilizando o mesmo fermento. Um total de 587 seqüências de 16S rDNA foram analisadas, sendo 81 da amostra NA, 177 da amostra NP, 159 da amostra NS e 170 da amostra NT. As análises das seqüências revelaram a presença de 17 gêneros e 27 espécies, além de 27 bactérias não conhecidas. Cento e setenta unidades taxonômicas operacionais (UTOs) foram identificadas utilizando o software DOTUR com uma distância evolucionária de 0.03. Quarenta e três UTOs foram identificadas na amostra controle (NA), 38 na NP, 57 na amostra NS e 38 na terceira amostra (NT). Das 170 UTOs identificadas, apenas dezessete foram identificadas duas vezes em diferentes amostras e somente uma, identificada como Lactobacillus hilgardii, foi identificada três vezes, evidenciando uma grande dinâmica populacional bacteriana durante o processo fermentativo. Análises estatísticas utilizando o software S-LIBSHUFF indicaram diferenças significativas na composição bacteriana entre amostras. Foram encontradas espécies/gêneros ainda não descritas na literatura em fermentos de cachaça, como Weissella cibaria, Leuconostoc citreum e algumas espécies de Lactobacillus. Além destas, várias bactérias não conhecidas também foram identificadas. Os resultados revelaram que a comunidade de bactérias contaminantes do processo fermentativo é muito mais complexa do que se conhecia, com características heterofermentativas e produção de congêneres que provavelmente refletem na qualidade da bebida. Este é o primeiro relato da utilização do método de seqüenciamento do gene 16S rDNA para determinar contaminantes bacterianos em fermentados de cana-de-açúcar para produção de cachaça. / Cachaça is a typical Brazilian spirit made from sugar cane fermented mainly by Saccharomyces cerevisiae. The production is mostly artisanal, and there is no microbiological control during the fermentation process. The objective of this study was to assess the bacterial community associated with the ferment used in the production of cachaça. Four ferment samples were collected from an artisanal still. The first one (NA), was collected one year previously to the other three and constituted a control reference. The remaining three samples were collected at the end of the first day of fermentation (NP), and fifteen (NS) and thirty days (NT) after using this same ferment. A total of 587 16S rDNA sequences were analyzed, being 81 sequences from the NA sample, 177 from NP, 159 from NS, and 170 from the NT sample. Sequence analyses revealed the presence of 17 genus and 27 species plus 27 unknown species. One hundred and seventy operational taxonomic units (OTUs) were identified using the DOTUR software using a cut-off evolutionary distance of 0.03. Forty three were identified in the control (NA) sample, 38 in the NP, 57 in NS, and 38 in the third (NT) sample. Of the 170 OTUs, only seventeen were detected twice in different samples and only one, identified as Lactobacillus hilgardii, was identified thrice, indicating a dynamic nature of the bacterial community during the fermentation process. Statistical analysis using the software S-LIBSHUFF indicated significant differences in the bacterial composition among samples. Our analyses also allowed to identify several bacteria not yet described as contaminants of the cachaça ferment, such as Weissella cibaria, Leuconostoc citreum and some Lactobacillus species. In addition, several unknown bacteria were also detected. The results revealed a much larger bacterial contaminant community present in the fermentative process of cachaça than previously reported with heterofermentative ability to produce secondary compounds which probably influence the quality of the final beverage. This is the first report on the utilization of the 16S rDNA sequencing method to assess the bacterial diversity of sugar cane fermentation for the production of cachaça.

A pesquisa de linhagens com duplicação cromossômica, como a linhagem A de Aspergillus nidulans, teve oseu início no final da década de 1970. Durante este período foram isolados da linhagem A, diversos variantes deteriorados, que foram caracterizados genética e citologicamente. Neste trabalho de pesquisa, as analises genéticas demonstraram que os determinantes de deterioração ou segmentos de inserção de V5, V101, V102, V103 e V104 estão localizados nos grupos de ligação VIII, III, IV, VII e I respectivamente. As análises citogenéticas revelaram diversas alterações no ciclo celular e migração nucleares nas fases iniciais de desenvolvimento. A duplicação cromossômica da linhagem A e os variantes deteriorados foram investigados a nível molecular, por técnica de PCR. Os resultados mostraram que o segmento de inserção consiste de um provável Elemento de Transposição, denominado de MATE, o qual é característico do fungo Aspergillus nidulans. Os segmentos de inserção analisados apresentam características típicas de MATE, como o motivo “Spe” que é encontrado por toda seqüência dos Elementos MATE. / The research with chromosome duplication strains, as strain A of Aspergillus nidulans, began during the 70's, with isolation of several deteriorates variants of strain A and characterization by genetic and cytological analysis. In this work the genetic analysis has demonstrated that the deterioration determinant or insertion sequence in V5, V101, V102, V103 and V104 deteriorates variants are located in the linkages groups VIII, III, IV, VII and I, respectively. The cytological analyses have demonstrated changes in cellular cycle and nuclear migration in initial phases of development. The chromosome duplication of strain A and the deteriorated variants were investigated by PCR with designed primers to mobile elements, what have resulted in the identification of the transposable element MATE, mainly by great similarity with "Spe" motif sequence that is described as essential in activity of these elements.

Breast cancer (BC) is one of the most common cancers among women worldwide that ~25% of new cancer cases diagnosed every year would be BC; moreover, ~15% of cancer deaths per year caused by BC makes it the leading cause of cancer death among women worldwide. To date, though the cause of a large proportion of BC are still unclear, recent studies have revealed that a supportive breast tissue microenvironment is critical for the development and progression of BC, especially the communication with immune cells within breast tissue. Therefore, breast inflammatory microenvironment is currently received a substantial attention in the prevention and treatment of BC. Research on breast cancer immunology suggests that inflammatory mediators, estrogen and several inflammation-related tumorigenic pathways are potentially contributors for inflammatory breast tumorigenesis. It is evidenced that elevated levels of inflammatory mediators, such as cytokines, chemokines, prostaglandins, and enhanced estrogen production while suffering from chronic inflammation is responsible for not only activating oncogenic pathways, for example NF-κB, STAT3 and Wnt signaling pathways, but also reducing the efficacy of cancer-specific immunity against tumor cells. Accordingly, targeting the chronic inflammatory status in breast tissue has become a promising strategy for breast cancer therapy. Recently, due to the annoying side effects accompanying by traditionally anticancer drugs, there is an increased interest in finding out natural sources to treat BC. Herein, we report that antroquinonol (AQ) and/or 4-acetylantroquinonol B (4-AAQB) isolated from Antrodia Camphorata were able to modulate the expression of several inflammatory mediators, IL-6 and IFN-γ in particular, and downregulate the aromatase expression and Wnt signaling responses induced by inflammatory status. Taken together, the present findings provide new insights into the role of AQ and 4-AAQB in inflammatory breast tumors and also suggest them as promising candidates for breast cancer immunotherapy.

The Ph.D. thesis is a collection of eleven commented articles on the topic of molecular-genetic profiling of urogenital tract tumors published in impact factor journals. In the introduction the origin and development of classification units is described including a brief overview of recent genetic profiles of selected types of renal carcinomas. Reviewed are major signaling pathways exploited in targeted therapy as well as the function of the corresponding drugs. Directions in which the research of new biomarkers is aimed are mentioned and the introduction ends with a short list of molecular genetic methods used in classification of renal carcinomas. In the results section are summarized the most important morphological, immunohistochemical, and molecular genetic findings of the studied sets of renal carcinomas. The data are compared with the present characteristics of known entities and hypotheses of possible new entities or relations between them are inferred. In the conclusions the suitability and limitations of the used molecular genetic methods are discussed in the context of the difficulties of the material analyzed and the designs of the studies.

Androgen receptor (AR) gene CAG polymorphisms may be associated with body shape, and are associated with certain breast and prostate cancers. In addition, body shape is associated with risk for a variety of diseases, including heart disease, diabetes, and certain forms of cancer. The CAG repeat in exon l of the AR gene was quantified using Perkin Elmer Applied Biosystems GeneScan analysis software in 96 and 59 healthy Caucasian men and women, respectively, who were over the age of 50 years. All participants had body measurements taken and donated a blood sample. Waist measurements included circumferences at the 1) umbilicus (wstumb), 2) top of the iliac crest (wstili), and 3) midpoint between the lowest rib and the iliac crest (wstwst). Waist-hip ratio (Wl-IR) was calculated using each corresponding waist measurement, respectively (WHRUMB, WHRILI, WHRWST). Mean repeat length was significantly different (p < 0.01) between men (22 ± 0.3 repeats) and women (23 ± 0.3 repeats). There was a significant relationship (p < 0.05) between mean individual CAG repeat number and tertile of WHRUMB in women based on the mean number of CAG repeats for each woman. Waist measurements in women were significantly different for all pairwise comparisons (p < 0.05). In addition, the three measurements of WHR in women, WHRUMB, WHRILI, and WHRWST, were significantly different from each other (p < 0.05). Thus, lesser numbers of CAG repeats may indicate a more androgenic phenotype in women.

It is well established that l,25(OH)2D3 is a major regulator of calcium homeostasis. The steroid exerts its effects on its target organs by two mechanisms, a slow mechanism mediated by nuclear vitamin D receptors (nVDR), and a rapid mechanism mediated by the l,25(OH)2D3 membrane-associated rapid response steroid binding protein (l,25D3-MARRS bp). In this dissertation, the physiological relevance of membrane initiated steroid signaling was investigated by studying the correlation of age in male and female chickens with the magnitude of responses to l,25(OH)2D3 in duodena from 7-, 14-, 28-, and 58-week-old birds. Measurements of l,25(OH)2D3 (130 pM) responsiveness as a function of age showed an age-related decrease in intestinal Ca2+ transport for both male and female birds. Western analyses on isolated basal lateral membranes (BLM) indicated a decreased expression of 1,25D3-MARRS bp with increasing age in male chickens, while its expression increased with age in female birds. Saturation analyses of [3H]l,25(OH)2D3 binding to BLM revealed an allosteric interaction identified as cooperative binding for both male and female birds. The degree of positive cooperativity increased with increasing age. Both genders showed a significant increase in dissociation constant (Kd) with increasing age, indicating decreasing affinity. No age-related changes in the number of binding sites, Bmax, were observed in male chickens, while female birds showed an increase in Bmax with increasing age. Data obtained in parallel binding analyses of [3H]l,25(OH)2D3 to nuclear fraction nVDR in contrast, indicated an absence of cooperative binding and an absence of significant changes in Kd or Bmax with age for both male and female birds. Membrane-initiated signal transduction by l,25(OH)2D3 was assessed by determination of protein kinase C (PKC) and A (PKA) activities. Both male and female birds showed an age-related decrease in stimulation of PKC activity in response to l,25(OH)2D3, while PKA activity increased. In conclusion, this dissertation demonstrates that there is a decrease in l,25(OH)2D3-induced intestinal calcium uptake as a function of age in duodena of both male and female chickens, which can be correlated to a decreased affinity for l,25(OH)2D3, and a decreased PKC activity.

Five normal healthy young university women while on self-chosen diets and living under normal conditions served as experimental subjects in this study. Urinary pregnanediol was measured for 10 days of the menstrual cycle. Meal frequency ( 3 meals vs. 2 meals with no breakfast) was also included in the study. A modification of the method of Eberlein and Bongiovanni on thin layer chromatography was used to determine pregnanediol in the urine sample. Urine pregnanediol excretion was in the pattern as investigated by other workers. The values rose in the latter half of the menstrual cycle, and fell prior to the onset of the next period. No relationship was found between pregnanediol level and meal frequency. A larger number of subjects are recommended in the further work of this problem.

Low-hierarchy selenoproteins are sensitive to selenium (Se) deficiency and are proposed to confer the protection of body Se against age- and development-related diseases. Selenoprotein H (SELENOH), a low-hierarchy selenoprotein, is greatly downregulated by both dietary Se deficiency and age. To explore physiological roles of SELENOH, Selenoh knockout mice were employed. Segregation analyses demonstrated reduced frequencies of homozygotes and heterozygotes among the neonates of the breeding combinations of Selenoh+/- males vs. Selenoh+/- or Selenoh-/- females, demonstrating essential roles of SELENOH in embryogenesis. Litter sizes from these two breeding groups were comparable with control, suggesting a role of SELENOH in zygotic but not somatic embryogenesis. By contrast, SELENOH was dispensable for longevity in mice. Selenoh-/- males showed azoospermia and a 34%-63% reduction in testis mass in the mice. Histological examination and the analyses of stage-specific markers by qRT-PCR indicated that the Selenoh-/- spermatocytes were arrested in the pachytene stage. Furthermore, GPX4 plausibly accounted for some of the Se loss in the testes because the knockout resulted in a 90% decrease in GPX4 mRNA level. Interestingly, the knockout increased the Selenop and Gpx1 mRNA levels by over 2.3- and 1.9-folds, respectively, but decreased the mRNA amount of Selenov by 99.9% in the testes. As a key component of the meiotic cohesion complex to promote sister chromatid cohesion, Rec8 mRNA amount was reduced by 91.1% in the Selenoh-/- testes aged 42 days. SELENOH knockout did not affect body weight or food intake but resulted in glucose intolerance and insulin resistance in the mice. Additionally, SELENOH knockout reduced the mRNA amounts of Gpx1, Gpx4, and Txnrd1 in the liver and Gpx4 in the skeletal muscle. These findings suggest that SELENOH plays a role in glucose metabolism and regulates body Se metabolism in mice. In summary, results of these three studies unveil essential roles of SELENOH in embryogenesis, spermatogenesis, and glucose tolerance in mice. SELENOH plausibly plays upstream roles to optimize these physiological events in association with the transcriptional regulation of several selenoproteins and meiotic proteins. Future studies are warranted to understand the mechanisms by which SELENOH maintains such developmental and metabolic processes.

The Ph.D. thesis is a collection of eleven commented articles on the topic of molecular-genetic profiling of urogenital tract tumors published in impact factor journals. In the introduction the origin and development of classification units is described including a brief overview of recent genetic profiles of selected types of renal carcinomas. Reviewed are major signaling pathways exploited in targeted therapy as well as the function of the corresponding drugs. Directions in which the research of new biomarkers is aimed are mentioned and the introduction ends with a short list of molecular genetic methods used in classification of renal carcinomas. In the results section are summarized the most important morphological, immunohistochemical, and molecular genetic findings of the studied sets of renal carcinomas. The data are compared with the present characteristics of known entities and hypotheses of possible new entities or relations between them are inferred. In the conclusions the suitability and limitations of the used molecular genetic methods are discussed in the context of the difficulties of the material analyzed and the designs of the studies.

The objective of this study was to examine the influence of age on food intake, tissue and organ mass and NF-ĸB and SIRT6 levels in various tissues. The transcription factor, Nuclear Factor Kappa-B (NF-ĸB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. SIRT6 is a member of the sirtuin family of proteins that function as protein lysine deacetylases and are associated with longevity in a number of organisms. Male Sprague-Dawley rats, aged 6 months (Adult) and 21 months (Old) were fed a commercially available diet for 10-17 days. Old rats consumed less food per body weight (BW) each day than Adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). However, when intake data were expressed as g/diet per day there was no significant difference between groups. For skeletal muscle tissue, the average mass of gastrocnemius and soleus (g muscle/g BW) was significantly lower in Old rats. Levels of NF-ĸB (p65/RelA) and SIRT6 were measured by Western blot analysis in gastrocnemius, tibialis anterior, quadriceps, soleus, lung, heart, kidney and liver. NF-ĸB levels were higher in gastrocnemius of Old rats compared to Adult rats. No significant age-specific differences in SIRT6 protein levels were noted in the tissues examined. Interestingly, when examined independent of age, levels of SIRT6 were significantly different between certain tissues. Data from this study suggest that age affects muscle loss and NF-ĸB in a tissue-specific manner. Furthermore, these findings indicate tissue-specific but not age-specific differences in SIRT6 protein levels.