CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho tem como objetivo avaliar a estabilidade de genes de referÃncia e a expressÃo das proteÃnas morfogenÃticas Ãsseas (BMP-2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e seus mensageiros intracelulares (SMADs-1, 5 e 8) em folÃculos caprinos antes e apÃs cultivo por 18 dias. Para avaliar a estabilidade dos genes de referÃncia e o nÃvel de expressÃo das BMPs, receptores e SMADs, folÃculos com aproximadamente 0,2, 0,5 e 1 mm foram isolados mecanicamente de ovÃrios caprinos. AlÃm disso, folÃculos com aproximadamente 0,2 mm foram isolados e cultivados por 18 dias em meio de cultura suplementado com FSH. ApÃs a extraÃÃo do RNA total e sÃntese de cDNA, foi realizada a quantificaÃÃo do RNAm, por PCR em tempo real, utilizando-se primers especÃficos para genes de referÃncia (β-actina, PGK, GAPDH, β-tubulina, UBQ, RPL-19, rRNA18S), e para as BMPs (2, 4, 6, 7 e 15) receptores de BMPs (BMPR-IA, IB e II) e SMADs (1, 5 e 8). Os resultados mostraram que β-tubulina e PGK sÃo os genes de referÃncia mais estÃveis em folÃculos frescos prÃ-antrais e antrais caprinos. Os RNAs mensageiros para as BMPs (2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e SMADs (1, 5 e 8) sÃo expressos em diferentes nÃveis em folÃculos prÃ-antrais e antrais caprinos, sendo que a expressÃo do RNAm para BMP-4, BMP-6 e BMP-7 em folÃculos de 1 mm sÃo significativamente maiores do que em folÃculos de 0,2 e 0,5 mm. Entretanto, os nÃveis de RNAm para BMP-2 foi reduzido em folÃculos de 1 mm, jà os nÃveis de BMP-15 nÃo diferiram entre as categorias foliculares analisadas. Os nÃveis de RNAm para BMPR-IB foram maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm, enquanto que o RNAm para BMPR-II foi significativamente maior em folÃculos de 0,5 mm do que em folÃculos de 0,2 e 1 mm. Por outro lado, nÃveis de RNAm para BMPR-1A nÃo diferiram entre folÃculos analisados. Os nÃveis de RNAm para SMAD-5 foram significativamente maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm. Contudo, folÃculos de 0,5 mm mostraram nÃveis maiores de RNAm para SMAD-8 do que folÃculos de 0,2 e 1 mm. Os nÃveis de RNAm para SMAD-1 nÃo diferiram entre os folÃculos. ApÃs as comparaÃÃes dentro de cada categoria folÃcular, BMP-15 foi mais expressa do que BMP-7 em folÃculos de 0,2 e 0,5 mm. Em folÃculos de 0,5 mm a expressÃo do BMPR-IB foi maior do que BMPR-II. Em todas as trÃs categorias foliculares estudadas, a expressÃo da SMAD-5 foi superior a SMAD-8. ApÃs o cultivo, os folÃculos apresentaram reduÃÃo dos nÃveis de RNAm para BMP-2, BMP-4, BMP-7, BMPR-IA e SMAD-5. Em conclusÃo, β-tubulina e PGK sÃo os dois genes housekeeping mais estÃveis para folÃculos frescos caprinos com 0,2, 0,5 e 1 mm de diÃmetro. BMPs, seus receptores e SMADs apresentam padrÃes de expressÃo especÃficos em cada categoria folicular estudada. No entanto, em folÃculos cultivados hà uma variaÃÃo na expressÃo dos componentes do sistema BMP, diferindo da expressÃo in vivo de folÃculos com o mesmo tamanho. / The aims this study to evaluate the stability of reference genes and the expression of bone morphogenetic protein (BMP-2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and intracellular messengers (SMADs- 1, 5 and 8) in goat follicles before and after culture for 18 days. To evaluate the stability of reference genes and the expression of BMPs, receptors and SMADs, follicles of approximately 0.2, 0.5 and 1 mm were mechanically isolated from goats ovaries. In addition, approximately 0.2 mm follicles were isolated and cultured for 18 days in culture medium supplemented with FSH. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA, the quantification of mRNA was carried out by real-time PCR using specific primers for genes of reference (GAPDH, β-tubulin, β-actin, PGK, UBQ, RPL - 19, rRNA18S) and BMPs (2, 4, 6, 7 and 15) receptors of BMPs (BMPR-IA, IB and II) and SMADs (1, 5 and 8). Results showed that β-tubulin and PGK are the most stable reference genes in goats preantral and antral follicles. The messengers RNA for BMP (2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and Smads (1, 5 and 8) are expressed at different levels in preantral and antral goats, and mRNA expression for BMP-4, BMP-6 and BMP-7 in 1-mm follicles are significantly higher than in follicles of 0.2 and 0.5 mm. However, the levels of mRNA for BMP-2 were reduced in follicles 1 mm, as BMP-15 did not differ between follicular categories. The levels of mRNA for BMPR-IB were higher in follicles of 0.2 mm than in follicles of 0.5 and 1 mm, whereas the mRNA for BMPR-II was significantly higher in follicles than 0.5 mm in follicles of 0.2 to 1 mm. Moreover, mRNA levels for BMPR-1A did not differ between follicles examined. The levels of mRNA for SMAD-5 were significantly higher in 0.2 mm follicles than in follicles of 0.5 and 1 mm. However, follicles of 0.5 mm showed higher levels of mRNA for SMAD-8 than follicles 0.2 and 1 mm. The levels of mRNA for SMAD-1 did not differ between follicles. After the comparisons within each category follicle, BMP-15 expression was higher than BMP-7 in follicles between 0.2 and 0.5 mm. Follicles 0.5 mm in the expression of BMPR-IB was greater than BMPR-II. In all three follicular categories studied, the expression of SMAD-5 was superior to SMAD-8. After culture, follicles showed reduced levels of mRNA for BMP-2, BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, β-tubulin and PGK genes are the two most stable housekeeping for fresh goat follicles 0.2, 0.5 to 1 mm in diameter. BMPs, their receptors and SMADs have specific expression patterns in each category follicular studied. However, in cultured follicles showed a variation in the variation in the expression of BMP system components, differing from in vivo expression of follicles with the same size.

Much has been improved in the basic and applied aspect of female reproduction after understanding of the wave-like developmental pattern of follicles ¡Ý4 mm. However, it is speculated that the understanding of the developmental pattern of small follicles <4 mm may bring about efficient management of ovarian functions for essential reproductive interventions. Present studies were therefore, carried out to characterize the developmental pattern of 1-3 mm follicles in cattle using ultrasonography and to validate a method to histologically characterize the developmental pattern of follicles <1 mm using non-serial data. Transrectal ultrasonography was used once daily (n=18 Hereford-cross heifers) to examine changes in the diameter of follicles ¡Ý1 mm for one interovulatory interval (IOI), as well as every 6 h (n = 9 Hereford-cross cows) from 5 to 13 days after ovulation to encompass emergence of Wave 2. A periodic shift in the peak number (P < 0.05) of 1-3 mm and ¡Ý4 mm follicles and a significant inverse relationship (P < 0.05) between them suggested a wave-like developmental pattern. The number of 1-3 mm follicles detected in anovulatory waves did not differ (P = 0.53) between 2- versus 3-wave IOIs. A difference (P < 0.05) was noticed between anovulatory and ovulatory waves in 3-wave IOIs but not (P = 0.63) in 2-wave IOIs. The future dominant follicle was identified at 1 mm and was found to emerge 6-12 h earlier than the largest subordinate follicle (P < 0.01). Emergence of the future dominant (r = 0.71) and 1st subordinate (r = 0.78) follicles was temporally associated (P < 0.05) with a rise in circulating concentrations of FSH. <p> The developmental pattern of follicles not detectable by ultrasonography (<1 mm) may be assessed histologically by examining the ovaries from different animals on different days (i.e., non-serial method) using follicle diameter or number profiles. A data set (n = 56 heifers) of follicles ¡Ý4 mm was tabulated in a serial (same set of heifers each day; n = 7/day, N = 7) and non-serial (different set of heifers each day; n = 7/day, N = 56) manner for number and diameter profiles around emergence of the first follicular wave. Profiling of serial and non-serial data (serial and non-serial methods) revealed a change in the number of follicles (4-5 mm, 6-8 mm, ¡Ý9 mm) over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.28). Similarly, the diameter of the dominant and first 2 subordinate follicles changed over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.06), indicating that non-serial data provide wave-like profile of follicles. <p> In conclusion, the hypothesis that follicles 1-3 mm develop in a wave-like manner was supported, and a new non-serial method was validated for the study of follicle dynamics using non-serial (e.g. histologic) data.

Much has been improved in the basic and applied aspect of female reproduction after understanding of the wave-like developmental pattern of follicles ¡Ý4 mm. However, it is speculated that the understanding of the developmental pattern of small follicles <4 mm may bring about efficient management of ovarian functions for essential reproductive interventions. Present studies were therefore, carried out to characterize the developmental pattern of 1-3 mm follicles in cattle using ultrasonography and to validate a method to histologically characterize the developmental pattern of follicles <1 mm using non-serial data. Transrectal ultrasonography was used once daily (n=18 Hereford-cross heifers) to examine changes in the diameter of follicles ¡Ý1 mm for one interovulatory interval (IOI), as well as every 6 h (n = 9 Hereford-cross cows) from 5 to 13 days after ovulation to encompass emergence of Wave 2. A periodic shift in the peak number (P < 0.05) of 1-3 mm and ¡Ý4 mm follicles and a significant inverse relationship (P < 0.05) between them suggested a wave-like developmental pattern. The number of 1-3 mm follicles detected in anovulatory waves did not differ (P = 0.53) between 2- versus 3-wave IOIs. A difference (P < 0.05) was noticed between anovulatory and ovulatory waves in 3-wave IOIs but not (P = 0.63) in 2-wave IOIs. The future dominant follicle was identified at 1 mm and was found to emerge 6-12 h earlier than the largest subordinate follicle (P < 0.01). Emergence of the future dominant (r = 0.71) and 1st subordinate (r = 0.78) follicles was temporally associated (P < 0.05) with a rise in circulating concentrations of FSH. <p> The developmental pattern of follicles not detectable by ultrasonography (<1 mm) may be assessed histologically by examining the ovaries from different animals on different days (i.e., non-serial method) using follicle diameter or number profiles. A data set (n = 56 heifers) of follicles ¡Ý4 mm was tabulated in a serial (same set of heifers each day; n = 7/day, N = 7) and non-serial (different set of heifers each day; n = 7/day, N = 56) manner for number and diameter profiles around emergence of the first follicular wave. Profiling of serial and non-serial data (serial and non-serial methods) revealed a change in the number of follicles (4-5 mm, 6-8 mm, ¡Ý9 mm) over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.28). Similarly, the diameter of the dominant and first 2 subordinate follicles changed over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.06), indicating that non-serial data provide wave-like profile of follicles. <p> In conclusion, the hypothesis that follicles 1-3 mm develop in a wave-like manner was supported, and a new non-serial method was validated for the study of follicle dynamics using non-serial (e.g. histologic) data.

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 2 uppsatser. Även tryckt utgåva.

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA.

Measurements of ovarian, corpus luteum, follicles of Lithuanian Black and White and German Black and White breed cows made by ultrasound scanner were revised. Effect of different factors, such as age, breed and season determining persistence of corpus luteum was statistically evaluated. Validation of treatment efficacy of persistent corpus luteum was carried out based on testing of progesterone concentration in peripheral blood plasma.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders.

Thesis (M.Sc.) Uppsala : Sveriges lantbruksuniversitet.

Comparações da dinâmica ovariana entre mais de duas raças equinas, sob condições padronizadas, não são encontradas na literatura. Objetivando comparar a dinâmica folicular e luteal, foram realizados exames diários de ultrassonografia durante um intervalo interovulatório contemporâneo, em éguas de pequeno porte (grupo Mini Pony – MP; n=10), médio porte (grupo Large Pony – LP; n=9) e grande porte (grupo Brasileiro de Hipismo – BH; n=12). Concluiu-se que os três grupos diferiram quanto ao máximo diâmetro do folículo pré-ovulatório (FPO) (mm) (MP=36,15; LP=40,95; BH=46,66), diâmetro do FPO um dia antes da ovulação (mm) (MP=35,8; LP=40,55; BH=46,48) e crescimento diário médio do FPO (mm/dia) (MP=2,6; LP=3,05; BH=3,51). O grupo MP diferiu dos demais quanto ao número de folículos por onda ovulatória (MP=4,8; LP=10,11; BH=9,75), número de folículos por dia (MP=4,19; LP=10,27; BH=10,63), número de folículos maiores ou iguais a 10mm (MP=2,98; LP=5,88; BH=5,98), diâmetro do FPO à divergência (mm) (MP=22,62; LP=24,81; BH=25,58), diâmetro do segundo maior folículo à divergência (SMF) (mm) (MP=15,56; LP=21,25; BH=21,83), diferença de diâmetro entre FPO e SMF à divergência (mm) (MP=7,25; LP=3,56; BH=3,75) e área do corpo lúteo (CL) (mm2) (MP=436,1; LP=674,4; BH=720,4). Não houve diferença entre os grupos quanto à duração do ciclo (dias) (MP=21,9; LP=20,22; BH=20,58), ocorrência de divergência (MP=80%; LP=88,89%; BH=100%), dias decorridos entre a emergência do FPO e SMF (MP=0,8; LP=0,89; BH=0,92) e diferença de diâmetro entre estes na emergência (mm) (MP=0,4; LP=0,44; BH=0,5) e na divergência (mm) (MP=7,25; LP=3,56; BH=3,75); dias entre emergência e divergência (MP=5,12; LP=5,5; BH=5,5) e divergência e ovulação (MP=7,12; LP=6,62; BH=6,8), número de ondas menores (MP=0,3; LP=0,33; BH=0,42) e duração do CL (dias) (MP=12,4; LP=14,67; BH=13,92). / Comparisons of ovarian dynamics between more than two equine breeds, under standardized conditions, are not found in the literature. The objective of this study was to compare follicular and luteal dynamics during one contemporary intervulatory interval by daily ultrasonography examinations in small size mares (Mini Pony group - MP; n=10), medium size (Large Pony group - LP; n=9) and large size (Brazilian Warmblood group - BH; n=12). It was concluded that all three groups differed regarding maximum diameter of the preovulatory follicle (POF) (mm) (MP=36.15; LP=40.95; BH=46.66), maximum diameter of POF one day before ovulation (mm) (MP=35.8; LP=40.55; BH=46.48) and the mean daily growth of POF (mm / day) (MP=2.6; LP=3.05; BH=3.51). The MP group differed from LP and BH groups regarding number of follicles per ovulatory wave (MP=4.8; LP=10.11; BH=9.75), number of follicles per day (MP=4.19; LP=10, 27; BH=10.63), number of follicles equal or greater than 10mm (MP=2.98; LP=5.88; BH=5.98), diameter of POF at deviation (mm) (MP=22.62; LP=24.81; BH=25.58), diameter of second largest follicle (SLF) at deviation (MP=15.56; LP=21.25; BH=21.83), diameter difference between FPO and SLF at deviation (mm) (MP=7.25; LP=3.56; BH=3.75), corpus luteum (CL) area (mm2) (MP=436.1; LP=674.4; BH=720.4). There was no difference between groups regarding cycle length (MP=21.9; LP=20.22; BH=20.58), occurrence of deviation (MP=80%; LP=88.89%; BH=100%), days between emergence of POF and SLF (MP=0.8, LP = 0.89, BH = 0.92), and diameter difference between POF and SLF at emergence (mm) (MP=0.4; LP=0.44; BH=0.5) and at deviation (mm) (MP=7.25; LP=3.56; BH=3.75); days between emergence and deviation (MP=5.12; LP=5.5; BH=5.5), deviation and ovulation (MP=5.12; LP=6.62; BH=6.8), number of minor waves (MP = 0.3; LP = 0.33; BH = 0.42) and CL lifespan (days) (MP=12.4; LP=14.67; BH=13.92).