Expresión y disección funcional in vitro de las hormonas lactogénica placentaria y del crecimiento humano

Tesis (Doctorado en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANLUANLhttp://www.uanl.mx

TATA-box Binding Protein interacts with Antp, Scr, Ubx and AbdB through their N-terminal domains

Background: Hox proteins are transcriptional factors (TFs) that define segment identity during embryonic development regulating specific target genes. These TFs interact with cofactors for DNA specificity and other TFs to regulate gene expression, which include basal transcriptional machinery members like BIP2, Med19, TFIIEβ, M1BP and TBP. Since TBP glutamine homopeptide (PolyQ) act as an interaction domain involved transcriptional regulation, we analyzed if TBP interact with Antp, Scr, Ubx and AbdB through its PolyQ region. Methods: We used Bimolecular Fluorescent Complementation (BiFC) to determine TBP interaction with Antp, Scr, Ubx and AbdB as well as the implication of their homeodomain (HD) and the TBP polyQ region. We used expression vectors carrying the sequences of TBP and its PolyQ lacking version (TBP∆Q) fused to the N-terminal half of Venus (VN) and Antp, Scr, Ubx and AbdB as well as their HDs fused to the C-terminal (VC). All VN and VC constructions were co-transfected with pCAGmCherry in HEK293 cells. Fluorescent cells were quantified and BiFC percentage was calculated as green cells per each 100 red cells. We also used UAS-GAL4 system to direct the expression of VNTBP and VCAntp or AntpHD in D. melanogaster embryos using a Ptc-GAL4 driver. BiFC fluorescent embryos were acquired by confocal microscopy. Results: The results showed that TBP interact with Antp (78%), Scr (72%), Ubx (70%) and AbdB (95%) in cell culture. The interaction with HD decreased interaction percentage as follows: AntpHD(50%), ScrHD(44%), UbxHD(41%) and AbdBHD(61%) indicating the implication of the N-terminal in these interactions. Also, the PolyQ deletion in TBP decreased the signal to 41%, 25%, 34% and 49%, respectively, confirming the PolyQ importance in these interactions. The combination of deletions both in TBP and homeoproteins showed an additive effect. Additionally, we corroborated the TBP-Antp interaction in D. melanogaster embryos by BiFC and the Antp N-terminal involvement in this interaction as well. Conclusions: TBP interaction with Antp, Scr, Ubx and AbdB is mediated by Hox N-terminal regions and the PolyQ domain of TBP as well. Furthermore, TBP-Antp interaction also occurs in vivo, suggesting that they could have similar transcriptional regulation during development of Drosophila melanogaster

Detección de microRNAs extracelulares y su potencial como biomarcadores moleculares

RESUMEN Los microRNAs (miRNAs) son moléculas de RNA de 18 a 24 nucleótidos de longitud involucrados en la regulación de la expresión génica a nivel postranscripcional en la célula. En este trabajo analizamos la expresión de miRNAs en muestras de tejido y suero de pacientes con cáncer mamario, y este perfil permite distinguir pacientes con cáncer de mama y mujeres sanas con alta sensibilidad, especificidad y eficiencia. La combinación de dos miRNAs para tejido y tres para suero, usados en conjunto como un solo grupo, eleva significativamente la eficiencia de la prueba, y demuestra que los miRNAs tienen gran potencial como nuevos biomarcadores no invasivos en cáncer de mama. Adicionalmente, se realizó la búsqueda de miRNAs en el protozoario Entamoeba histolytica, por secuenciación profunda, lo que permitió realizar la primera descripción de miRNAs en este parásito. Se identificaron 199 miRNAs exclusivos para este parásito, que servirán de base para el estudio de la regulación génica en E. histolytica y el establecimiento de nuevos biomarcadores para la amibiasis. ABSTRACT MicroRNAs (miRNAs) are 18 to 24 nucleotide-long RNA molecules responsible for the regulation of gene expression at the post-transcriptional level in the cell. In this work, we analyzed the expression of miRNAs in tissue samples and serum of patients with breast cancer and we found that miRNA expression patterns distinguish patients with breast cancer from healthy women with high sensitivity, specificity, and efficiency. The combination of 2 and 3 miRNAs used as a group in tissue and serum respectively, significantly increases the efficiency of the test and shows that miRNAs have potential as novel noninvasive biomarkers in breast cancer detection. Additionally, miRNAs were detected by deep sequence in the protozoan parasite Entamoeba histolytica, which allowed the first description of miRNAs in this parasite. We identified 199 new miRNAs in E. histolytica, which are the base for the study of gene regulation and the establishment of new biomarkers for amoebiasis

Antp transcriptional activity is modulated by the formation of the trimeric Antp-TBP complexes with TFIIEβ, Exd and BIP2

Homeoproteins are transcriptional factors that bind to DNA through a highly conserved binding domain known as the homeodomain (HD) which recognizes short regions rich in AT to control the development of the body appendages of organisms. However, their structural and recognition similarities make it difficult to explain how homeoproteins are capable of carrying out their function. Previous results have shown that Antp homeoprotein can establish dimeric interactions with TBP, TFIIEβ, Exd, BIP2 and more recently through BiFC-FRET we confirmed that Antp and TBP can form trimeric complexes with TFIIEβ/Exd/BIP2. Therefore, is important to show how these trimeric complexes modulate Antp transcriptional activity. The experimental approach selected for this project was to perform in vitro transactivation assays in HEK-293 cells transfected with the combinations of Antp and TBP-producing plasmids with TFIIEβ / Exd / BIP2 plasmids using a Luciferase reporter plasmid. Our results show that trimeric interaction of Antp-TBP-TFIIEβ induced a significant increase of 138% in the Antp transcriptional activity. By contrast, trimeric complexes of Antp-TBP-Exd and Antp-TBP-BIP2 modified the transactivating capacity of Antp, decreasing transcription by 20 and 26% respectively. According to this, we were able to confirm that the trimeric complexes Antp-TBP/TFIIEβ/Exd/BIP2 are involved in the modulation of Antp transcriptional activity. So, now it is interesting to analyze how these trimeric complexes are involved in the activation and/or repression of target genes of Antp during genetic control of development in Drosophila melanogaster

Prenatal cafeteria diet exposure promotes lymphocyte infiltration into the brain and autism-like behavior in the offspring of C57BL6 mice

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an array of etiological causes, including environmental, genetic, and immunological triggers. High-energy diets activate the immune system during prenatal stages favoring infiltration of peripheral immune cells and cytokines into the brain by the choroid plexus and circumventricular regions and altering microglial activity. Accumulation of immune entities and microglia activation in brain have been reported to disrupt social behavior. However, the interplay between prenatal exposure to high-energy diets, neuroinflammation and defective social behavior has not been reported. Methods: Female C57BL6 mice were exposed to cafeteria diet during pregnancy and lactation. The effect of diet on social, locomotor, repetitive-stereotyped, and anxiety-like behavior was evaluated in the male offspring two-month-old. We quantified the number of infiltrating natural killer (NK1.1+), dendritic (CD11c+), lymphocytes (CD11b--CD45+), macrophages (CD11b+-CD45high), and M1 (CD11b+-CD45low-CD86+) or M2 (CD11b+-CD45low-CD206+) microglia in the choroid plexus and cerebral cortex, hippocampus, and striatum of the male offspring by flow cytometry. Results: Our results demonstrated that exposure to cafeteria diet during prenatal stage primed defective social interaction and repetitive-stereotyped behaviors in male offspring. Flow cytometry analysis showed lymphocyte infiltration in the cerebral cortex and hippocampus compared to the choroid plexus in the offspring exposed to cafeteria diet. No significant changes were observed in the NK, dendritic or macrophage levels in the choroid plexus and the cerebral cortex, hippocampus, and striatum regions of those subjects. The effect of cafeteria diet exposure also did not affect microglial density or M1/M2 phenotypes. Conclusions: Our results indicate that exposure to a cafeteria diet during prenatal development promotes an increase of lymphocytes in brain regions of importance for ASD that could contribute to the behavioral defect in the offspring. Testing the contribution of lymphocyte infiltrates in the development of ASD-like behaviors could better explain the cellular mechanisms related to the disorder

Visualización de la expresión génica tejido-específica mediante microscopía confocal láser

En el presente trabajo mostramos la proteÌna GFP en diferentes discos imaginales de larvas del tercer estadio de D. melanogaster utilizando el sistema binario del factor de transcripciÛn de levadura GAL4. Este sistema consiste en la cruza de una lÌnea pro- ductora que expresa GAL4 con una lÌnea reportera que contiene el gen de la proteÌna verde fluorescen- te (GFP), bajo la regulaciÛn de un promotor con las secuencias de uniÛn al factor transactivador GAL4 (UAS). Las larvas resultantes de las cruzas se diseca- ron para obtener los discos imaginales que se ob- servaron mediante microscopÌa confocal l·ser (Olympus IX70). Se detectÛ la expresiÛn de GFP en los diferentes discos imaginales que originan a las patas, ojo-antena y alas en adultos, sobreponiendo las im·genes obtenidas con las registradas en luz transmitida. La localizaciÛn de la expresiÛn precisa de GFP dirigida por cada uno de las regiones potenciadoras fue para M2-576 una expresiÛn concÈntrica en los discos de pata y del ala en la regiÛn anterior. Dll en las regiones distales del disco de pata, en la zona media del ala y los segmentos distales del disco de antena. Estos patrones facilita- r·n realizar estudios de la expresiÛn de proteÌnas funcionales de interÈs en regiones tejido especÌficas bas·ndonos en los patrones de expresiÛn de GFP visualizados en este trabajo

Trimeric complex interactions of Antp-TBP with TFIIEB and Exd are involved in the genetic control of Drosophila melanogaster

Background: Homeoproteins are transcriptional factors (TFs) that shape animal body axes during development. These TFs are highly conserved and represent one of the most fascinating groups of regulatory molecules. Reports shown the multiplicity of interactions in hox proteins, as complexes trimeric involved to transcriptional activity. The study of trimeric complexes in Hox interactome will allow the better understanding of Hox genetic regulation during embryonic development. Methodology: Using a new combination BiFCFRET approach performed in HEK293, the quantification was performed by FRETTY of ImageJ. Fly crosses were incubated at 25ºC on standard yeast-agar-cornmeal medium. Embryo cuticle preparations were carried out according to (Gibson & Gehring 1998) and mounted on slides with Visicol. For adult imaging, the heads and antennae were dissected and directly transferred to microscopic slides without coverslips. The images were merged using the software HeliconFocus. Results: We found the presence of trimeric interactions of Antp-TBP-TFIIEβ and Antp-TBP-Exd in cell culture. Further were confirmed these trimeric interactions using Antp mutants. Interestingly, the trimeric Antp-TBP-TFIIEB shown diminish of homeotic effect caused by Antp in larvae. To analyze the effect in antenna-leg transformation we drive the trimeric complex and found inhibition of antenna-leg in head of D. melanogaster. Antp-TBP-TFIIEB shown homeotic reduction of 77% in larvae and found inhibition of 100% of antenna-leg transformation. Conclusions: Our results show that these trimeric interactions are involved in the genetic control of Drosophila melanogaster