INTRODUÇÃO: A prolactina é um hormônio polipeptídico que possui reconhecida ação sistêmica, principalmente no sistema reprodutor. O papel desse hormônio no desenvolvimento e na extensão do câncer da mama ainda é muito debatido. A progressão do câncer de mama em grande parte depende do movimento celular e da capacidade da célula em remodelar seu citoesqueleto de actina. Nesse processo, proteínas envolvidas na migração celular, como moesina, FAK e c-Src, são influenciadas por vários hormônios, incluindo a prolactina. O presente estudo teve por objetivo avaliar os efeitos da PRL na migração de células T47D, MCF-7 e ZR75-1 de câncer de mama, bem como os mecanismos envolvidos. MÉTODOS: As células foram cultivadas em placas de cultura com meio suplementado e divididas em oito grupos diferentes de tratamento: Grupo I (veículo); Grupo II (PRL na concentração de 25 ng/mL); Grupo III (PRL na concentração de 50 ng/mL), Grupo IV (PRL na concentração de 100 ng / mL), Grupo V (RNAi + veículo); Grupo VI (RNAi + PRL na concentração de 25 ng/mL); Grupo VII (RNAi + PRL na concentração de 50 ng/mL) e Grupo VIII (RNAi + PRL na concentração de 100 ng / mL). Nos Grupos de I a IV, a reorganização da actina do citoesqueleto foi analisada por imunofluorescência após 30 minutos do tratamento. Em todos os grupos estudados foram realizadas análise da migração horizontal com auxílio de microscopia de luz e avaliadas as expressões de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src por Western Blot após 48 horas do tratamento. RESULTADOS: As células de câncer de mama expostas à prolactina apresentaram um aumento da expressão de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src. Essas alterações moleculares estão associadas à reorganização da actina do citoesqueleto e ao aumento da mobilidade das células. CONCLUSÕES: Nossos dados sugerem que a prolactina aumenta a migração das células T47D, MFC-7 e ZR75-1 de câncer de mama e remodela a actina do citoesqueleto pela via de sinalização intracelular das proteínas c-Src, FAK e moesina / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. The role of this hormone on breast cancer development and progression has been debated a lot yet. Breast cancer invasion largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, proteins involved in cell migration, such as moesin, FAK and c-Src, are influenced by a large number of hormones, such as prolactin. The present study was aimed for evaluating the effects of PRL on migration of T47D, MCF-7 and ZR75-1 breast cancer cells as well as the molecular mechanisms in this process. METHODS: The cells were cultured in dishes with supplemented medium and were divided in eight different assays: Group I (control); Group II (25ng/ml of prolactin); Group III (50ng/ml of prolactin); Group IV (100ng/ml of prolactin); Group V (RNAi + control); Group VI (RNAi + 25ng/ml of prolactin); Group VII (RNAi + 50ng/ml of prolactin); Group VIII (RNAi + 100ng/ml of prolactin). In Groups I to IV, the actin cytoskeletal reorganization was analyzed by immunofluorescence 30 minutes after the treatment. In all groups, were performed the horizontal migration analysis with light microscopy and evaluated the expression of moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src by Western blot after 48 hours of treatment. RESULTS: Breast cancer cells exposed to prolactin display an elevated moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src expression. These molecular changes are associated with the reorganization of actin cytoskeleton and increased mobility of cells. CONCLUSION: Our data suggest that prolactin enhances the migration of T47D, MFC-7 and ZR75-1 breast cancer cells through the actin cytoskeleton remodeling by intracellular signaling pathway of c-Src, FAK and moesin proteins

INTRODUÇÃO: A prolactina é um hormônio polipeptídico que possui reconhecida ação sistêmica, principalmente no sistema reprodutor. O papel desse hormônio no desenvolvimento e na extensão do câncer da mama ainda é muito debatido. A progressão do câncer de mama em grande parte depende do movimento celular e da capacidade da célula em remodelar seu citoesqueleto de actina. Nesse processo, proteínas envolvidas na migração celular, como moesina, FAK e c-Src, são influenciadas por vários hormônios, incluindo a prolactina. O presente estudo teve por objetivo avaliar os efeitos da PRL na migração de células T47D, MCF-7 e ZR75-1 de câncer de mama, bem como os mecanismos envolvidos. MÉTODOS: As células foram cultivadas em placas de cultura com meio suplementado e divididas em oito grupos diferentes de tratamento: Grupo I (veículo); Grupo II (PRL na concentração de 25 ng/mL); Grupo III (PRL na concentração de 50 ng/mL), Grupo IV (PRL na concentração de 100 ng / mL), Grupo V (RNAi + veículo); Grupo VI (RNAi + PRL na concentração de 25 ng/mL); Grupo VII (RNAi + PRL na concentração de 50 ng/mL) e Grupo VIII (RNAi + PRL na concentração de 100 ng / mL). Nos Grupos de I a IV, a reorganização da actina do citoesqueleto foi analisada por imunofluorescência após 30 minutos do tratamento. Em todos os grupos estudados foram realizadas análise da migração horizontal com auxílio de microscopia de luz e avaliadas as expressões de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src por Western Blot após 48 horas do tratamento. RESULTADOS: As células de câncer de mama expostas à prolactina apresentaram um aumento da expressão de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src. Essas alterações moleculares estão associadas à reorganização da actina do citoesqueleto e ao aumento da mobilidade das células. CONCLUSÕES: Nossos dados sugerem que a prolactina aumenta a migração das células T47D, MFC-7 e ZR75-1 de câncer de mama e remodela a actina do citoesqueleto pela via de sinalização intracelular das proteínas c-Src, FAK e moesina / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. The role of this hormone on breast cancer development and progression has been debated a lot yet. Breast cancer invasion largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, proteins involved in cell migration, such as moesin, FAK and c-Src, are influenced by a large number of hormones, such as prolactin. The present study was aimed for evaluating the effects of PRL on migration of T47D, MCF-7 and ZR75-1 breast cancer cells as well as the molecular mechanisms in this process. METHODS: The cells were cultured in dishes with supplemented medium and were divided in eight different assays: Group I (control); Group II (25ng/ml of prolactin); Group III (50ng/ml of prolactin); Group IV (100ng/ml of prolactin); Group V (RNAi + control); Group VI (RNAi + 25ng/ml of prolactin); Group VII (RNAi + 50ng/ml of prolactin); Group VIII (RNAi + 100ng/ml of prolactin). In Groups I to IV, the actin cytoskeletal reorganization was analyzed by immunofluorescence 30 minutes after the treatment. In all groups, were performed the horizontal migration analysis with light microscopy and evaluated the expression of moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src by Western blot after 48 hours of treatment. RESULTS: Breast cancer cells exposed to prolactin display an elevated moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src expression. These molecular changes are associated with the reorganization of actin cytoskeleton and increased mobility of cells. CONCLUSION: Our data suggest that prolactin enhances the migration of T47D, MFC-7 and ZR75-1 breast cancer cells through the actin cytoskeleton remodeling by intracellular signaling pathway of c-Src, FAK and moesin proteins

Botulinum toxin type A (BTTA) is a polypeptide with 1296 amino acids and its used in some pathologies like hyperhidrosis and migraine, but the cosmetic area is the most important application. BTTA is produced from broth-culture synthesized by the Clostridium botulinum as a single peptide with 150 kDa. Reversed-phase liquid chromatography (RP-LC) method was developed and validated for the assessment of botulinum toxin type A in biopharmaceutical formulations. A RP-LC method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.8, and the mobile phase B was acetonitrile, run isocratically at flow rate 0.3 mL/min. Cromatographic separation was obtained with retention time of 11.4 min, and was linear over the concentration range of 0.2-100 IU/mL (r2 = 0.9999) with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.04 and 0.16 IU/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.31% with bias lower than 0.80%. The method was correlated with in vivo and in vitro bioassays. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p <0.05) compared to intact molecule. The validated methods were applied for the determination of BTTA in biopharmaceutical-derived products, giving mean values of the estimated content/potencies 1.16% lower compared to the in vivo bioassay. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure efficacy of the biotechnology-derived product. / A toxina botulínica tipo A (BTTA) é um polipeptídio constituído de 1296 aminoácidos e é recomendada para patologias, como hiperhidrose e enxaqueca, mas é especialmente usada como cosmético. A toxina botulínica é produzida através do processo de fermentação bacteriana e após sucessivas etapas de purificação resulta em uma proteína com 150 kDa responsável pelo seu efeito biológico. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de potência de BTTA em formulações de produtos biofarmacêuticos. No método foi utilizada coluna Zorbax 300 SB C18 (150 mm x 4,6 mm d.i.), mantida a 45 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,8, e a fase móvel B por acetonitrila, eluídas em vazão isocrática de 0,3 mL/min. O método utilizou detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida em 11,4 min, sendo linear na faixa de concentração de 0,2-100 UI/mL (r2 = 0,9999). Os limites de detecção e quantificação foram 0,04 e 0,16 UI/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,31 com bias inferior a 0,80. O método validado foi correlacionado com bioensaios in vivo e in vitro. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais apresentaram diferença significativa em relação a forma intacta (p <0,05). O método proposto foi aplicado para avaliação da potência de BTTA em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio in vivo, observando-se diferença das médias de teor/potência de 1,16% inferior para o método cromatográfico. Contribuíu-se assim para estabelecer procedimentos que aprimoram a caracterização ou o controle da qualidade, garantindo a segurança e eficácia do produto biotecnológico.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Para avanzar en el entendimiento de las vías de señalización moleculares involucradas en la progresión de cáncer tumoral, se construyo una plataforma personalizada de cDNAs, la cual contiene genes de vías de señalización representativas para investigar la respuesta dinámica temporal a hormonas (progesterona y estradiol) empleando como modelo la línea celular T47D-MTVL e inhibidores específicos de las vías de señalización. Adicionalmente, se realizó un análisis de los perfiles de expresión de un grupo de tumores de mama encontrando buena correlación con los datos clínico-histopatológicos y mostrando como fenotipos específicos correlacionan con mal pronóstico. Los genes más significativos capaces de discriminar entre los fenotipos de tumor fueron determinados, y probados sobre un nuevo grupo de muestras, asignándolas a los subtipos predichos. El análisis de las vías de señalización de los genes más significativos de cada fenotipo fue realizado para elucidar las vías moleculares más representativas afectadas en cada clase de tumor.

Reports of endocrine disrupting potential of common environmental chemicals and the effects on reproductive health are well documented in literature. It has been suggested that deteriorating male reproductive health could be due to in utero exposures to these chemicals. The effects mediated through endocrine disrupting chemicals (EDCs) are on the fetus and may therefore be trans-generational. Ultimately, these chemicals land up in aquatic systems and affect wildlife and humans. Humans are exposed to these chemicals through multiple routes including atmosphere, water, occupational, domestic and food consumption. South Africa (SA) is an important livestock producer with about 13.8 million cattle within the feedlot industry contributing up to 80% of the total beef production. Veterinary growth stimulants (VGS) are used by beef producers to enhance growth in cattle. In SA, the following five VGS have been approved for use in beef products under the Register Act 36 of 1947, estradiol, progesterone and testosterone (natural), α'-zearalanol and trenbolone (synthetic). These VGS and their metabolites are environmentally stable compounds. The excretions from the animals are not treated and land up in the local aquatic systems, indirectly posing a health risk. In SA no research has been done on VGS associated with feedlot activities. The aim of this study was to investigate the effects of a mixture of VGS, as possible EDCs on the reproductive health and thyroid function in male rats in utero, during lactation and life-time exposure. The (anti)estrogenic and (anti)androgenic activity in water from specific feedlots was determined by using a battery of screening bio-assays. Water samples were collected over a period of a year and assessed for EDC activity in the recombinant yeast screen (YES), the T47D-KBluc (estrogenic) and the MDA-Kb2 (androgenic) bioassays. The OECD (Organization for Economic Co-operation and Development) 415 protocol, (1983) for a one-generation reproduction toxicity study, was modified to accommodate one control and three experimental groups. The experimental groups were orally gavaged with mixtures of: zilpaterol, diethylstilbestrol (DES) and α-zearalanol (Group 2; estogenic); with β'-trenbolone and methyltestosterone (MT) (Group 3; androgenic); a combination of compounds (Group 4; estrogenic and androgenic) and the Control group received cottonseed oil only. The bio-assay results indicated that water samples analysed from selected feedlots contained compounds with estrogenic activity. The shorter anogenital distance (AGD) (Group 3), decreased seminal vesicle mass (Group 4), decreased prostate mass (Group 4), increased lumen diameter (Group 3 and 4), lowered sperm concentration (Group 3), and increased T4 (Group 2 and 3) differed significantly from the control. The body weight of the males in Group 2 in the F2 generation was significantly lower than the control. The F2 females in Group 2, 3 and 4 were also significantly lower than the control. The reduced AGD, decreased seminal vesicle and increased T4 (thyroxine) might be the result of an estrogenic effect. The reduced sperm concentration might be the result of in utero and lactation exposure to these VGS. The bio-assays confirmed estrogenic activity in the feedlot water sources. The reproductive toxicology study findings confirm the hypothesis that VGS can act as EDCs and could therefore be responsible for negative reproductive effects and thyroid function. More research is needed to investigate the effects of VGS mixtures at different concentrations on male reproductive health, thyroid function and their offspring. AFRIKAANS : Goed gedokumenteerde literatuur dui aan dat chemikalieë wat algemeen in die omgewing gevind word, die potensiaal het om die manlike voortplantingstelsel aan te tas. Dit word gespekuleer dat in utero blootstelling verantwoordelik kan wees vir hierdie agteruitgang. Die fetus en daarop- volgende geslagte se gesondheid kan ook beÏnvloed word deur chemikalieë. Hierdie chemikalieë het die potensiaal om die watersisteme te bereik en gevolglik dier en menslike gesondheid te beÏnvloed. Blootstelling kan plaasvind deur verskeie roetes wat die atmosfeer, water, werksomstandighede, huishoudelike produkte en gekontamineerde voedsel insluit. Suid-Afrika (SA) is 'n belangrike produsent van vleisprodukte met omtrent 13.8 miljoen beeste wat bydra tot 80% van die vleisproduksie. Veterinêre-groei-stimulante (VGS) word gebruik om die vleisproduksie te verbeter. Vyf groei stimulante naamlik estradiol, progesteroon, testosteroon (natuurlike), α-zearalanol en trenboloon (sinteties) is goedgekeur onder die Wet 36 van 1947, vir groei produksie van beeste. Hierdie VGS en hul metaboliete is stabiel in die natuur. Die fekale en urinere uitskeidingsprodukte van die diere word nie behandel nie en eindig op in ons waterstelsels. Geen navorsing is nog in SA gedoen om die potensiële bydraes wat voerkrale tot die besoedeling van water lewer, te bestudeer nie. Die doel van die studie was om die gesamentlike effekte van mengsels VGS as moontlike endokrien-ontwrigtende chemicalieë (EOC) op die manlike voortplantingstelsel en tiroïdhormone van mannetjiesrotte na in utero-, gedurende laktasie- en na 'n leeftyd-blootstelling te bepaal. Die (anti)estrogeniese en (anti)androgeniese aktiwiteit in water vanaf spesifieke voerkrale is met behulp van 'n reeks biologiese seltoetse bepaal. Watermonsters is geanaliseer met die gisseltoets (YES)(estrogenies), die T47D-KBluc (estrogenies) en die MDA-Kb2 (androgenies). Die OECD 415 protokol (1983) vir een generasie reproduktiewe toksologie toets was aangepas om een kontrole en drie eksperimentele groepe te huisves. Die eksperimentele groepe rotte is oraal gedoseer met 'n mengsel van zilpaterol, dietielstilbestrol (DES) en α-zearalanol (Groep 2); β-trenboloon en metieltestosteroon (Groep 3); 'n kombinasie van al bogenoemde (Groep 4); en 'n kontrole groep wat katoensaad olie VGS ontvang het nie. Estrogeniese aktiwiteit en sitotoksisiteit was teenwoordig in die water vanaf die voerkrale. Die verkorte anogenitale afstand (AGD) (Groep 3), kleiner seminale vesikel (SV) massa (Groep 4), kleiner prostaat massa (Groep 4), groter lumen deursneë (Groep 3 en 4), laer spermtelling (Groep 3), verhoogde T4 (Groep 2 en 3), het almal statisties-betekenisvol van die kontrole groep verskil. In die F2 generasie het die liggaamsmassas van die mannetjies in Groep 2 en liggaamsmassas van die wyfies in Groepe 2, 3 , 4, almal statisties-betekenisvol laer as die kontrole Groep. Die verkorte AGD, kleiner SV en verhoogde T4 kan moonlik wees as gevolg van 'n estrogeniese effek en die verlaagde sperm konsentrasie weens 'n in utero en laktasie blootstelling. Die biologiese seltoetse het die teenwoordigheid van estrogeniese aktiwiteit in voerkrale se water bevestig. Die gevolge van die blootstelling van EOC mengsels op voortplantings-parameters bevestig die moontlikheid van EOC effek geassosieer met VGS. Verdere navorsing is nodig om die dosisresponsverhoudings van verskillende VGS te ondersoek. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / School of Health Systems and Public Health (SHSPH) / Unrestricted