Biochemical and single-molecule analysis of signaling molecules

Mammalian cells use a variety of molecules to receive, process, and transmit information from the extracellular environment. Proteins receive and transmit signals through direct protein-protein interaction at the membrane surface and within the cytoplasm. Similarly, membrane lipids facilitate signal transmission across the membrane by direct interaction with intracellular proteins. In this disertation, I have investigated protein-protein and lipid-protein interactions of important signaling molecules by employing biochemical methods and novel single-molecule approaches. Mammalian target of rapamycin (mTOR) is a master regulator of mammalian cell growth and proliferation. Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. However the mechanism by which PA activates mTORC1 signaling has remained unknown. In Chapter I of my thesis, I discovered a new mechanism by which the lipid second messenger phosphatidic acid (PA) regulates mTORC1 complex. PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Additionally, PA binding to mTORC1 leads to an increase in kinase activity by an allosteric effect, independently of FKBP38. In conclusion, a dual mechanism for PA activation of mTORC1 is proposed– PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. In chapter III of my thesis, in collaboration with Dr. Ankur Jain from Dr. Taekjip Ha laboratory, I used the single-molecule pulldown (SiMPull) assay to investigate the stoichiometry and assembly of mTORCs. After validating this novel approach with mTORC1, confirming a dimeric assembly as previously reported, I show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, the data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed the distinction of complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR–raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential. Signaling phospholipids are critical mediator of biological processes such as cell growth, proliferation, and metabolism. Recognition of signaling phospholipids by proteins is important for the targeting and initiation of many signaling cascades, but the mechanisms that regulate such interactions are not completely understood. The majority of the biophysical methods used to measure these interactions are performed with pure proteins in-vitro. In Chapter IV, in collaboration with graduate student Vasudha Aggarwal from Dr. Taekjip Ha laboratory, I have developed a single-molecule fluorescence approach to analyze lipid-protein interaction using crude cell extracts. The assay is applicable to a variety of lipid-binding domains (LBDs) expressed in cell lysates, and these LBDs are specifically pulled down by their target phospholipids. The single-molecule analysis quantitatively describes the interaction of LBDs on the lipids in real-time. These allowed the distinction of assembly features and kinetics for the different LBDs uncovering novel interaction behaviors. As an extension to cellular proteins, I determined the assembly properties of protein kinase Akt. Overall, this study demonstrates the strength of our assay to investigate protein-lipid interaction mechanisms in a new light

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways

Sistema de Inventario y Facturación en Tienda Variedades Camila en el municipio de Condega, segundo semestre 2018.

El presente trabajo de investigación está orientado a la implementación de un sistema de inventario y facturación en tienda variedades Camila en el municipio de Condega en el segundo semestre 2018, con el objetivo que permita la agilización automatizada en los procesos de información. Para la realización de este sistema se utilizó la metodología de desarrollo ágil Scrum creada por Schwaber y Sutherland, que permite trabajar en una serie de interacciones en equipo. Esta metodología consta de un sprint con 4 fases: planeación, desarrollo, revisión y retrospectiva. Como resultado de esta investigación se obtuvo un sistema automatizado que facilita la realización de procesos de inventario y facturación

Propuesta de balance de carga en los paneles eléctricos principales por Centro de transformación del Recinto Universitario Simón Bolívar de la Universidad Nacional de Ingeniería

Presenta una propuesta de balance de carga en los paneles eléctricos principales por centro de transformación con el fin de corregir las pérdidas de energía eléctrica y disminuir la facturación eléctrica anual en busca del buen uso de la energía eléctrica

Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to 300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m 2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Diagnóstico de enfermedades bacterianas y sensibilidad “in vitro” a bactericidas, en el cultivo de café(Coffea arabica L)

El café es considerado un producto de gran importancia en la economía mundial y es el segundo con más valor en el mercado después del petróleo. La producción de café está limitada por plagas, que reducen las cosechas y disminuyen su calidad. Los objetivos del estudio fueron, identificación de bacteriasfitopatógenas, descripción de síntomas en campo y sensibilidad in vitro a bactericidasde uso agrícola. Se seleccionaron cinco fincas cafetaleras de los departamentos de Jinotega y Matagalpa, con antecedentes de enfermedades bacterianas; las muestras de tejido colectadas consistieron en hojas, bandolas, frutos, y flores.La identificación de géneros de bacterias fitopatógenas se realizó a partir de características macromorfológicas de crecimiento bacteriano y presencia de pigmentos fluorescentes en medio agar Pseudomonas (King B). Laidentificación de especies se realizaron las pruebas de oxidas y pruebas de producción de ácidos.La patogenicidad de las bacterias se realizó mediante la prueba de hipersensibilidad en plántulas de tabaco. La sensibilidad de las bacterias a bactericidas se determinómediante el método de Kirby-Bauer, la inhibición al crecimiento bacteriano por los bactericidas, fue evaluado en diámetros(mm). Los bactericidas evaluadosfueron, Clorhidrato de oxitetraciclina (Farmacina 5 WP), Estreptomicina más oxitetraciclina (Agrymicin WP 16. 5), Ácido oxolínico (Starner 20 WP) ySulfato de cobre (Phyton 24 SC). Los datos se analizaron de manera estadística. Las especies de bacterias identificadas fueron Pseudomonas syringae, Pseudomonascorrugata y PseudomonascichoriiSwingle 1925, P. cichoriipredominó con mayor frecuencia en los diferentes aislados, P. syringae, se aisló en todas las variedades muestreadas. Las especies de bacterias aisladas resultaron positivas a la prueba de hipersensibilidad, que indica su patogenicidad en el cultivo de café.Los síntomas en hojas, bandolas y flores causados por Pseudomonas spson manchas de coloración pardo-oscura, de forma y tamaño irregular, rodeada, generalmente por halos amarillentosy traslucidos. El bactericida que mostró efecto con mayores diámetros en halos de inhibición de crecimiento bacteriano fue el ácido oxolínico

Leucyl-tRNA Synthetase Activates Vps34 in Amino Acid-Sensing mTORC1 Signaling

Amino acid availability activates signaling by the mammalian target of rapamycin (mTOR) complex 1, mTORC1, a master regulator of cell growth. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. Here, we identify leucyl-tRNA synthetase (LRS) as a leucine sensor for the activation of Vps34-PLD1 upstream of mTORC1. LRS is necessary for amino acid-induced Vps34 activation, cellular PI(3)P level increase, PLD1 activation, and PLD1 lysosomal translocation. Leucine binding, but not tRNA charging activity of LRS, is required for this regulation. Moreover, LRS physically interacts with Vps34 in amino acid-stimulatable non-autophagic complexes. Finally, purified LRS protein activates Vps34 kinase in vitro in a leucine-dependent manner. Collectively, our findings provide compelling evidence for a direct role of LRS in amino acid activation of Vps34 via a non-canonical mechanism and fill a gap in the amino acid-sensing mTORC1 signaling network

Probing Cellular Protein Complexes via Single Molecule Pull- down

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways. Dynamic interactions between proteins guide almost every aspect of cellular function1. Understanding how the macromolecules in living cells interact, holds the key to decipherin