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Síntesis y caracterización de receptores basados en calix[4]areno modificados en el borde superior a través de puentes con grupos piridina y tiosemicarbazona flexibles
(Universidad de Chile, 2019)
Los calixarenos son macrociclos compuestos por unidades fenólicas conectadas por puentes de metileno, formando una cavidad hidrofóbica que permite la generación de complejos de inclusión con una variedad de moléculas. Sus principales características...
Explorando la interacción entre el péptido señal de translocación reticular y el translocón Sec61 mediante espectroscopía de fuerza a nivel de molécula individual
(Universidad de Chile, 2020)
mutación Ala13Glu puede ser multifactorial. Una menor distancia al estado de transición implica una mayor rigidez de la interacción, mientras que una menor adhesividad de las esferas apunta a un efecto negativo sobre la asociación con el translocón, lo que...
Sec61 is a protein transporter channel, known as the translocon, found in the membrane of the endoplasmic reticulum in eukaryotes. Its function is to allow the passage of proteins from the cytosol to the reticular lumen in a process known as translocation, which is the first step in the protein secretory pathway. In humans, about 38% of the proteins synthesized by the cell translocate to the reticular lumen. This is because they possess an amino acid sequence, at its N-terminus, known as a “signal peptide”, which plays a role of recruiting the protein to the reticulum membrane and, later, of interaction with the translocon. This last function represents the first step in the translocation process, making it particularly interesting to study. The signal peptide has a poorly conserved sequence between proteins, however, three common regions are distinguished in all of them: an N-terminal region of positively charged residues (n region), a hydrophobic region in the form of an α helix (h region) and a C-terminal region of short-chain polar residues (c region). The h region is essential for the translocation process, as mutations in hydrophobic residues in this sequence dramatically decrease translocation and secretion rates. The h region of the signal peptide acts by inserting it into a "lateral gate" of the Sec61 channel, causing a conformational change that opens the pore and leaves the translocon in an active conformation. To study the translocation process, the S. cerevisiae protein Prepro-alpha-factor (PpαF), which was the first protein described to present a post-translational translocation mechanism in eukaryotes, was used as a model. The Ala13Glu point mutation, in the h region of the PpαF signal peptide, has been shown to decrease the translocation rate by up to 50 times. One hypothesis that would explain this phenomenon is that the Ala13Glu mutation decreases the free energy of the transition state of the dissociation process, while increasing the dissociation kinetic constant, in the interaction between the signal peptide and the Sec61 channel, compared to the protein wild type. This hypothesis was studied by force spectroscopy using optical tweezers, a tool that allows the interaction between proteins to be measured at the single molecule level (in singulo). As a first target, the PpαF protein and its Ala13Glu mutant were prepared for manipulation with optical tweezers. To this end, PCR-directed site mutagenesis was carried out to incorporate a cysteine residue, necessary to covalently bind to proteins, at position 165 (Tyr165Cys mutation). The Tyr165Cys and Ala13Glu/Tyr165Cys mutants were then expressed and purified, both with a 6xHis-tag at their C-terminus, by affinity chromatography. A second vital component of the experiment are the DNA handles, which serve as a spacer and as a force standard, due to their characteristic deformation at 67 pN. The handles were synthesized by PCR from a plasmid, using a biotinylated and a thiolated primer, to obtain a final product that forms a disulfide bond to PpαF and binds to the microsphere coated with streptavidin thanks to the incorporated biotin. Third, the yeast Sec61 translocon was purified. For this, microsomal fractions were separated and the translocon was purified from them using digitonin as a detergent. The translocon was prepared for the experiment by incubating with microspheres coated with anti-Sec61 antibodies. The force spectroscopy experiment was performed to obtain the rupture force of the interaction between the PpαF-DNA chimeras and the translocon. The rupture force histograms were filtered from the histogram of the negative control without PpαF, corresponding to the nonspecific interactions that are not under study. The data were fitted to the Dudko-Hummer-Szabo model to obtain the parameters of lifetime (τ0), distance to the transition state (Δx‡) and free energy to the transition state (ΔG‡) of the dissociation process. The curve of best fit was to an exponential decay model (Bell equation), which does not allow to obtain data from ΔG‡. The parameters obtained for wild PpαF were τ0 = 10 ± 4 s and Δx‡ = 1×10-2 ± 6×10-2 nm, and for the Ala13Glu mutant, τ0 = 7 ± 3 s and Δx‡ = 4×10-3 ± 2×10-3 nm were obtained. Δx‡ has a significant difference with p <0.05, but not τ0. The dissociation kinetic constants were calculated as the multiplicative inverse of τ0: koff = 1×10-1 ± 4×10-2 s-1 and 1.4×10-1 ± 5×10-2 s-1 respectively, without significant difference. On the other hand, at the same protein concentrations, spheres with PpαF Ala13Glu interact a significantly lower percentage (31%) with the translocon than those with PpαF with wild-signal peptide (49%). These results would indicate that the translocation defect of the Ala13Glu mutation can be multifactorial. A smaller distance to the transition state implies a greater rigidity of the interaction, while a lower adhesiveness of the spheres points to a negative effect on the association with the translocon. This rejects the original hypothesis...
Sec61 is a protein transporter channel, known as the translocon, found in the membrane of the endoplasmic reticulum in eukaryotes. Its function is to allow the passage of proteins from the cytosol to the reticular lumen in a process known as translocation, which is the first step in the protein secretory pathway. In humans, about 38% of the proteins synthesized by the cell translocate to the reticular lumen. This is because they possess an amino acid sequence, at its N-terminus, known as a “signal peptide”, which plays a role of recruiting the protein to the reticulum membrane and, later, of interaction with the translocon. This last function represents the first step in the translocation process, making it particularly interesting to study. The signal peptide has a poorly conserved sequence between proteins, however, three common regions are distinguished in all of them: an N-terminal region of positively charged residues (n region), a hydrophobic region in the form of an α helix (h region) and a C-terminal region of short-chain polar residues (c region). The h region is essential for the translocation process, as mutations in hydrophobic residues in this sequence dramatically decrease translocation and secretion rates. The h region of the signal peptide acts by inserting it into a "lateral gate" of the Sec61 channel, causing a conformational change that opens the pore and leaves the translocon in an active conformation. To study the translocation process, the S. cerevisiae protein Prepro-alpha-factor (PpαF), which was the first protein described to present a post-translational translocation mechanism in eukaryotes, was used as a model. The Ala13Glu point mutation, in the h region of the PpαF signal peptide, has been shown to decrease the translocation rate by up to 50 times. One hypothesis that would explain this phenomenon is that the Ala13Glu mutation decreases the free energy of the transition state of the dissociation process, while increasing the dissociation kinetic constant, in the interaction between the signal peptide and the Sec61 channel, compared to the protein wild type. This hypothesis was studied by force spectroscopy using optical tweezers, a tool that allows the interaction between proteins to be measured at the single molecule level (in singulo). As a first target, the PpαF protein and its Ala13Glu mutant were prepared for manipulation with optical tweezers. To this end, PCR-directed site mutagenesis was carried out to incorporate a cysteine residue, necessary to covalently bind to proteins, at position 165 (Tyr165Cys mutation). The Tyr165Cys and Ala13Glu/Tyr165Cys mutants were then expressed and purified, both with a 6xHis-tag at their C-terminus, by affinity chromatography. A second vital component of the experiment are the DNA handles, which serve as a spacer and as a force standard, due to their characteristic deformation at 67 pN. The handles were synthesized by PCR from a plasmid, using a biotinylated and a thiolated primer, to obtain a final product that forms a disulfide bond to PpαF and binds to the microsphere coated with streptavidin thanks to the incorporated biotin. Third, the yeast Sec61 translocon was purified. For this, microsomal fractions were separated and the translocon was purified from them using digitonin as a detergent. The translocon was prepared for the experiment by incubating with microspheres coated with anti-Sec61 antibodies. The force spectroscopy experiment was performed to obtain the rupture force of the interaction between the PpαF-DNA chimeras and the translocon. The rupture force histograms were filtered from the histogram of the negative control without PpαF, corresponding to the nonspecific interactions that are not under study. The data were fitted to the Dudko-Hummer-Szabo model to obtain the parameters of lifetime (τ0), distance to the transition state (Δx‡) and free energy to the transition state (ΔG‡) of the dissociation process. The curve of best fit was to an exponential decay model (Bell equation), which does not allow to obtain data from ΔG‡. The parameters obtained for wild PpαF were τ0 = 10 ± 4 s and Δx‡ = 1×10-2 ± 6×10-2 nm, and for the Ala13Glu mutant, τ0 = 7 ± 3 s and Δx‡ = 4×10-3 ± 2×10-3 nm were obtained. Δx‡ has a significant difference with p <0.05, but not τ0. The dissociation kinetic constants were calculated as the multiplicative inverse of τ0: koff = 1×10-1 ± 4×10-2 s-1 and 1.4×10-1 ± 5×10-2 s-1 respectively, without significant difference. On the other hand, at the same protein concentrations, spheres with PpαF Ala13Glu interact a significantly lower percentage (31%) with the translocon than those with PpαF with wild-signal peptide (49%). These results would indicate that the translocation defect of the Ala13Glu mutation can be multifactorial. A smaller distance to the transition state implies a greater rigidity of the interaction, while a lower adhesiveness of the spheres points to a negative effect on the association with the translocon. This rejects the original hypothesis...
Estudio de factibilidad económica para proyecto de inversión turístico
(Universidad de Chile, 2003)
sociales y culturales en un determinado corte en el tiempo. En la actualidad, en el centro histórico de Santiago de Chile podemos apreciar que ha perdido el gran dinamismo que vivió en algún momento de la historia, sin embargo, paralelo a esto, comienzan a...
Riesgo volcánico a escala Nacional/Regional: Estudio comparado de variantes metodológicas, para su evaluación y adaptación al contexto volcánico de Chile
(Universidad de Chile, 2016)
El Servicio Nacional de Geología y Minería (SERNAGEOMIN), mediante la adaptación de la metodología National Volcano Early Warning System (NVEWS), desarrollada por el Servicio de Geología de los Estados Unidos, ha determinado el riesgo asociado a los...
Análisis de estrategias de despacho de una central fotovoltaica con almacenamiento a través de bombeo hidráulico con agua de mar
(Universidad de Chile, 2017)
En los últimos años, se ha incrementado la preocupación por incorporar e integrar de mejor forma las Energías Renovables No Convencionales (ERNC) a los sistemas eléctricos, para lo cual se han propuesto los sistemas de almacenamiento. En este...
The southeast pacific countries, the United Nations convention on the law of the sea and the exclusive economic zone
(Universidad de Chile, 2009)
Factibilidad estratégica, técnica y económica de un taller de reparación, mantención y calibración de materiales de pañol
(Universidad de Chile, 2017)
Esta tesis de grado evalúa la factibilidad estratégica, técnica y económica de la implementación de un taller de reparación y mantención de materiales de pañol, equipado con un laboratorio de calibración de instrumentos de medición, acreditado por...
Evaluación de los tiempos de rutina de trabajo y rendimientos de salas de ordeña espina de pescado, mediante un sistema computacional de análisis y simulación (PASS)
(Universidad de Chile, 2011)
(PASS, DeLaval), midiéndose el Tiempo de Rutina de Trabajo esencial (TRTe), y los de sus componentes. PASS permitió medir también los tiempos en Actividades Misceláneas (TM), Ocioso (TO), la proporción TO + TM/TRTe y el tiempo de postura de la primera...