<p>Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. </p><p>In order to study the BMP role for catecholaminergic neurons <i>in vivo</i>, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. </p><p>Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.</p><p>The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.</p><p>Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. </p><p>These results demonstrate a positive role of BMPs for MDN fate<i> in vivo</i>. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.</p>

<p>Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP).</p><p>Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis.</p><p>Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake.</p><p>To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (<i>creER</i><i>T2</i>) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreER^T2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.</p>

Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia. The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor. Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP). Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis. Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake. To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (creERT2) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreERT2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-01T13:51:16Z No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T16:01:04Z (GMT) No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) / Made available in DSpace on 2016-07-13T16:01:04Z (GMT). No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) Previous issue date: 2012-08-27 / O laser de baixa intensidade e o ultrassom terapêutico têm se mostrado opções para modulação da cicatrização, porém, os mecanismos de ação destas técnicas não são bem esclarecidos. Para avaliar os efeitos da laserterapia e do ultrassom terapêutico em feridas cutâneas cirúrgicas em ratos Wistar (n=24), foram utilizados quatro grupos: I (controle), II (LLLT), III (ultrassom) e IV (laserterapia e ultrassom). No décimo dia, as lesões foram fotografadas e medidas e, após removidas excisionalmente no momento da eutanásia, foram processadas para avaliação histopatológica para avaliação da densidade e organização das fibras colágenas; avaliação histomorfométrica para quantificação da angiogênese e infiltrado inflamatório; e imunoistoquímica, para expressão de TGFβ1. As amostras dos grupos tratados exibiram aspecto macroscópico mais maduro em relação ao grupo não tratado, sem diferença significativa no fechamento das lesões; microscopicamente, os resultados sugeriram que a laserterapia exerceu melhor efeito imunomodulador quando utilizado isoladamente e que o ultrassom terapêutico mostrou maior potencial angiogênico. A avaliação imunoistoquímica revelou que a maioria das células inflamatórias na área cicatricial não expressava TGFβ1. Ainda, embora a laserterapia e a aplicação do ultrassom atuem diretamente na redução do infiltrado, as terapias concomitantes não potencializam o efeito observado quando aplicadas isoladamente; apesar do tempo de fechamento das feridas não ter sido influenciado pelas terapias isoladas ou associadas, todos os tratamentos favoreceram a organização da matriz extracelular colagenosa. A LLLT isoladamente e a combinação de ambas as técnicas possibilitaram a reepitelização das feridas submetidas a estas modalidades terapêuticas. A maioria das células que migraram ou entraram em proliferação na área cicatricial não expressavam o TGFβ1, sugerindo que o controle do infiltrado inflamatório exercido pela LLLT e pelo UST não é modulado por esta citocina. / Low level laser therapy (LLLT) and therapeutic ultrasound have been demonstrated to be options for healing modulation, but the mechanisms of action involved in these processes are not clear. The effects of laser therapy and therapeutic ultrasound on surgical skin wounds in Wistar rats (n=24) were evaluated using four groups: I (control), II (LLLT), III (ultrasound) and IV (laser therapy and ultrasound). On the tenth day, the wounds were photographed and measured, and after excision at the moment of euthanasia, they were processed for histopathological evaluation to assess the density and organization of collagen fibers. In addition, histomorphometric evaluations were conducted to quantify angiogenesis and inflammatory infiltrates, and immunohistochemistry was performed to assess TGF1 expression. The samples from the treated group had a more mature macroscopic appearance compared to the untreated group, with no significant difference in wound closure. Microscopically, the results suggested that the laser therapy had a better immunomodulatory effect when used alone and that the therapeutic ultrasound showed a higher angiogenic potential. The immunohistochemical evaluation revealed that most of the inflammatory cells in the scar area did not express TGF1. Still, although the application of laser therapy and ultrasound act directly in the reduction of infiltration, the concomitant therapies do not potentiate the effect observed when applied in isolation, despite the time of wound closure was not influenced by therapy alone or associated, all treatments favored collagenous extracellular matrix organization. The LLLT alone and the combination of both techniques allowed the re-epithelialization of wounds under these treatment modalities. The majority of cells that migrated in the proliferation or entered in the cicatricial area is not expressed TGFβ1, suggesting that the control exercised by the inflammatory infiltrate and the LLLT UST is not modulated by this cytokine.

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance. Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors. Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors. Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

No description available.

Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.

<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Gemcitabine is a nucleoside pyrimidine analog that has long been the backbone of chemotherapy for PDAC, both as a single agent, and more recently, in combination with nab-paclitaxel. Since gemcitabine is hydrophilic, it must be transported through the hydrophobic cell membrane by transmembrane nucleoside transporters. Human equilibrative nucleoside transporter-1 (hENT1) and human concentrative nucleoside transporter-3 (hCNT3) both have important roles in the cellular uptake of the nucleoside analog gemcitabine. While low expression of hENT1 and hCNT3 has been linked to gemcitabine resistance clinically, mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. We identified that the matricellular protein Cysteine-Rich Angiogenic Inducer 61 (CYR61) negatively regulates expression of hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 significantly increased expression of hENT1 and hCNT3 and cellular uptake of gemcitabine. CRSIPR-mediated knockout of CYR61 sensitized PDAC cells to gemcitabine-induced apoptosis. Conversely, adenovirus-mediated overexpression of CYR61 decreased hENT1 expression and reduced gemcitabine-induced apoptosis. We demonstrate that CYR61 is expressed primarily by stromal pancreatic stellate cells (PSCs) within the PDAC tumor microenvironment, with Transforming Growth Factor- β (TGF-β) inducing the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in an in vitro co-culture assay with PDAC cells. Our results identify CYR61 as a TGF-β induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.</p> / Dissertation