Fifteen years ago, the Steroid receptor RNA activator (SRA) was identified as a functional non-coding RNA able to increase the activity of the estrogen receptor (ER), a critical player mediating the mitogenic role of estradiol in breast cancer. Interestingly, four years later, SRA appeared to be the first ever discovered functional RNA also able to encode a protein (SRAP). As such, the products of the SRA1 gene delineate a fascinating bi-faceted system involving both a functional RNA and a protein. Since its discovery, the non-coding aspect of this system has been widely investigated, with multiple groups gathering information on SRA structure and related functions. Overall, the non-coding SRA transcript is thought to act as a broad co-regulator modulating the activity of different transcription factors. Conversely, limited information has been obtained on the coding aspect (SRAP) of this system，even though SRA/SRAP is currently believed as a whole to be involved in several mechanisms including tumourigenesis, tumour progression, myogenesis and adipogenesis. In this body of work, I have attempted to define the clinical relevance of SRAP to breast cancer and extend the understanding of the cellular processes potentially regulated by this protein. I have first established that SRAP had the potential to become a new prognostic and predictive factor in specific groups of patients. Indeed, I have demonstrated, using tissue microarray analyses (TMAs), that SRAP expression was up-regulated in some breast tumours, with high levels associated with poor prognosis in Estrogen Receptor (ER) positive breast cancer patients. Using the same technique, I have further identified a positive association between a positive response to tamoxifen treatment and a high level of SRAP expression in a large cohort of ER-α negative cases. This highlights the potential for SRAP to become a new predictive factor of response to endocrine therapy in this specific group of patients. Using RNA-seq to define the transcriptomes of cervical Hela and breast MDA-MB-231 cancer cells upon depletion or overexpression of this protein, I further identified cellular movement amongst the potential cellular processes affected by changes in SRAP expression. Using classical trans-wells assays as well as an live-cell imaging assays, I have confirmed that SRAP indeed regulates individual cancer cell motility. Overall, my results provide critical new insights into the potential functions of the protein counterpart of the intriguing SRA/SRAP bi-faceted gene system. SRAP herein appears as a potential new therapeutic target in the fight against breast cancer that remains to be further investigated. / February 2017

A genetic linkage map of mustard (Brassica juncea) was developed using two double haploid populations produced from crosses between a low erucic cultivar “ZEM1” and two moderate erucic acid lines “Vniimk351” and “Vniimk405” with the use of SSR and SRAP markers. The linkage map of the ZEM1xVniimk351 population included 13 linkage groups with an overall length of 791 cM with an average marker interval of 5.7 cM. The linkage map of the ZEM1xVniimk405 population also contained 13 linkage groups with a distance of 623 cM and an average marker interval of 4.6 cM. Using the linkage maps for the two populations, QTLs were detected for seed oil, protein and fatty acids. QTL analysis for fatty acids indentified QTLs on LG1, 7 and 12 for the ZEM1xVniimk351 population and LG1, 3 and 4 for the ZEM1xVniimk405 population. Analysis for the seed oil and protein content in the ZEM1xVniimk351 population identified 2 QTLs on LG1 and LG4 and 1 QTL on LG1 respectively. The QTL analysis ZEM1xVniimk405 of oil and protein content identified 1 QTL for oil and protein on LG1. The variation of fatty acids was shown to be the result of monogenic inheritance of the FAE1 gene in both populations.

A genetic linkage map of mustard (Brassica juncea) was developed using two double haploid populations produced from crosses between a low erucic cultivar “ZEM1” and two moderate erucic acid lines “Vniimk351” and “Vniimk405” with the use of SSR and SRAP markers. The linkage map of the ZEM1xVniimk351 population included 13 linkage groups with an overall length of 791 cM with an average marker interval of 5.7 cM. The linkage map of the ZEM1xVniimk405 population also contained 13 linkage groups with a distance of 623 cM and an average marker interval of 4.6 cM. Using the linkage maps for the two populations, QTLs were detected for seed oil, protein and fatty acids. QTL analysis for fatty acids indentified QTLs on LG1, 7 and 12 for the ZEM1xVniimk351 population and LG1, 3 and 4 for the ZEM1xVniimk405 population. Analysis for the seed oil and protein content in the ZEM1xVniimk351 population identified 2 QTLs on LG1 and LG4 and 1 QTL on LG1 respectively. The QTL analysis ZEM1xVniimk405 of oil and protein content identified 1 QTL for oil and protein on LG1. The variation of fatty acids was shown to be the result of monogenic inheritance of the FAE1 gene in both populations.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection. / October 2007

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Submitted by ISABELA CRISTINA GOMES HONÓRIO null (isabelagomeshonorio@gmail.com) on 2016-05-25T19:12:00Z No. of bitstreams: 1 ESTUDO DA DIVERSIDADE GENÉTICA E QUÍMICA DE Uncaria tomentosa Willd. ex Roem. & Schult. E Uncaria guianensis Gmell DE POPULAÇÕES NATURAIS LOCALIZADAS NA AMAZÔNIA..pdf: 2336644 bytes, checksum: 05f34f338ce3e8cd544dea281a52873d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-30T17:46:08Z (GMT) No. of bitstreams: 1 honorio_icg_dr_bot.pdf: 2336644 bytes, checksum: 05f34f338ce3e8cd544dea281a52873d (MD5) / Made available in DSpace on 2016-05-30T17:46:08Z (GMT). No. of bitstreams: 1 honorio_icg_dr_bot.pdf: 2336644 bytes, checksum: 05f34f338ce3e8cd544dea281a52873d (MD5) Previous issue date: 2016-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Uncaria tomentosa Willd. ex Roem. & Schult. e Uncaria guianensis Gmell. são plantas medicinais nativas da Amazônia, utilizadas na medicina tradicional com ação anti-inflamatória, sofrem pressão antrópica como o desmatamento e extrativismo predatório para uso e necessitam de cuidados em relação à conservação. U. tomentosa é uma das plantas medicinais que compõe a lista da relação nacional de medicamentos essenciais (RENAME) disponibilizada pelo Ministério da Saúde para os municípios brasileiros, através do Sistema Único de Saúde (SUS) e a U. guianensis apesar de não estar nesta lista apresenta também alcaloides oxindólicos pentacíclicos, como marcadores químicos, sendo utilizada popularmente como anti-inflamatória e sua ocorrência na natureza é maior quando comparada à U. tomentosa. Os objetivos desse trabalho foram avaliar a diversidade genética e química entre os indivíduos de U. tomentosa e U. guianensis por marcador molecular SRAP (Sequence-Related Amplifield Polymosphism) e quantificar os alcaloides oxindólicos pentacíclicos mitrafilina e isomitrafilina em folhas por HPLC. A coleta do material foi feita nos estados do Acre, Amazonas, Amapá e Pará. Para ambas as espécies foram coletadas oito populações, em municípios distintos com 20 indivíduos cada. A genotipagem de U. tomentosa foi realizada utilizando três combinações de primers e os fragmentos submetidos à eletroforese em gel de poliacrilamida. A genotipagem de U. guianensis foi realizada usando quatro combinações de primers e os fragmentos analisados no equipamento 4300 DNA Analyser LI-COR®. A quantificação dos alcaloides oxindólicos pentacíclicos mitrafilina e isomitrafilina foi realizada de acordo com método já descrito na literatura com algumas modificações. Nas populações avaliadas de U. tomentosa a variabilidade genética foi maior dentro (75%) das populações do que entre elas (25%). O valor de Fst foi de 0,246, evidenciando que as populações estão estruturadas em Modelo de Ilhas. A maior porcentagem de locus polimórficos (95,68%) e a maior diversidade genética foram encontradas na população coletada no município de Mâncio Lima - AC. O fluxo gênico foi considerado restrito (Nm=1,57) e não houve correlação entre as distâncias geográfica e genética. Para a espécie U. guianensis, a variabilidade genética também foi maior dentro das populações (81%) do que entre elas (19%). O valor de Fst foi de 0,188, o que mostra que as populações estão se aproximando do Modelo de Ilhas. A maior porcentagem de locus polimórficos (90,21%) e a maior variabilidade genética foram verificadas na população coletada no município de Mazagão-AC. O fluxo gênico foi considerado baixo (Nm = 2,57) e houve fraca correlação entre as distâncias geográfica e genética, portanto considerada não significativa. A maior concentração dos alcaloides mitrafilina (11,17 mg.g-1.PS) e isomitrafilina (2,99 mg.g-1.PS) em U. tomentosa foram encontradas nos indivíduos da população de Tarauacá-AC e em U. guianensis foi de 1,09 mg.g-1.PS de mitrafilina e 0,29 mg.g-1.PS de isomitrafilina encontradas na população de Boca do Acre-AM. Além disso, para essa espécie, foi possível quantificar mitrafilina em apenas 24,8% dos indivíduos e a isomitrafilina em 20,4% dos indivíduos estudados. Conclui-se que a conservação de ambas as espécies deverá ser realizada com a coleta de um maior número de indivíduos nas populações com maiores variabilidades genética e química. / Uncaria tomentosa Willd. ex Roem. & Schult. and Uncaria guianensis Gmell. are medicinal plants natives from Amazonia state, used in the ocidental medicine for its anti-inflammatory effect, which are suffering from the actions of mankind by deforestation and predatory extraction, requesting care for their conservation. U. tomentosa is one of the medicinal plants that make up the national ratio of the list of essential drugs (RENAME) made available by the Ministry of Health to the municipalities through the Unified Health System (SUS) and U. guianensis although this is not list also features pentacyclic oxindole alkaloids, as markers, being popularly used as anti-inflammatory and its occurrence in nature is higher compared to U. tomentosa. The objectives of the present study were to evaluate the chemical and genetical diversity between individuals of U. tomentosa and U. guianensis by molecular marker SRAP (Sequence-Related Amplifield Polymosphism) and quantify pentacyclic oxindolic alkaloides mitraphylline and isomitraphylline in leaves via HPLC. Samples were collected in the states of Acre, Amazonas, Amapá and Pará. For both species, 20 samples were collected from each of the eight populations coming from different locations. The genotyping of U. tomentosa was realized by using the combination of three primers and the fractions submitted to an electrophoresis in acrylamide gel. The genotyping of U. guianensis was realized by using the combination of four primers and the fractions analyzed with a 4300 DNA Analyser LICOR®. The quantification of pentacyclic oxindole alkaloides mitraphylline and isomitraphylline were realized by using the existing method with some modifications. The U. tomentosa populations showed higher genetic variability inside the same population(75%) than between different populations (25%). The value of Fst was 0,246, showing that the populations are following the Island Model. The highest percentage of polymorphic locus (95,68%) and the highest genetic diversity were found in the population from Mâncio Lima - AC. The gene traffic was considered restrictive (Nm=1,57) and there was no correlation between geographic and genetic distances. For U. guianensis, the genetic variability was higher inside same populations (81%) than between different populations (19%). The value of Fst was 0,188, showing that the populations are coming close to the Island Model (modelo de ilhas). A higher percentage of polymorphic locus (90,21%) and a higher genetic variability were confirmed in the population colected in Mazagão - AC. The gene traffic was considered low (Nm = 2,57) and as having a weak correlation between geographic and genetic distances and, therefore, considered not significant. The highest alkaloid concentrations of mitraphylline (11,17 mg.g-1 .PS) and isomitraphylline (2,99 mg.g-1 .PS) in U. tomentosa were found in samples of the population from Tarauacá - AC and in U. guianensis samples showed alkaloid concentrations of 1,09 mg.g-1 .PS of Mitraphylline and 0,29 mg.g-1 .PS of Isomitraphylline in the population from Boca do Acre - AM. Besides that, to this species, it was possible to quantify mitraphylline in only 24.8% of individuals isomitraphylline and 20.4% of subjects studied. It’s concluded that the conservation of both species should be done with the collection of a greater number of individuals in populations with higher genetic and chemical variability.

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