El deterioro del espacio público y su impacto en las áreas destinadas a la socialización y al desarrollo de la accesibilidad en las ciudades medias mexicanas : caso Culiacán, Sinaloa /

Departament responsable de la tesi: Departament de Geografia.El siglo veinte transformó las calles de las ciudades. Aquellas que conformaban el espacio público continuo fueron convertidas en arterias distribuidoras de automóviles y otros vehículos motorizados -más individuales que colectivos- con espacios muy limitados para el resto de las funciones que originalmente tenían. La socialización entre las personas se trasladó a los parques y a las plazas con todo y las reducidas condiciones que ahí existe para que suceda. La consecuencia ha sido el paulatino abandono de estos espacios y el traslado de la actividad social del ciudadano a espacios cerrados, de carácter privado como los grandes centros comerciales que hoy proliferan en la mayoría de las ciudades medias latinoamericanas. En la práctica, la calle dejó de ser espacio público. Solo parques y plazas se conciben en el uso común de las personas como los espacios públicos, los espacios donde todos tienen iguales condiciones de uso. Y estos, en las ciudades, cada día están más vulnerables. Este proceso ha impactado negativamente en el desarrollo de las ciudades. Las calles dejaron de ser el espacio de encuentro de las personas donde había eventuales posibilidad de circulación de algunos vehículos motorizados para convertirse hoy en el espacio vial con alta jerarquía para la circulación de los vehículos motorizados con disminuidos espacios para las personas de a pie. La presente investigación se enfoca en los procesos de crecimiento de las ciudades medias mexicanas en el periodo de 1980 a 2010 y cómo este proceso transformó su espacio público. Concretamente, la socialización de las personas analizado en el caso de la ciudad de Culiacán y tres vialidades prototipo seleccionadas estratégicamente. La evaluación del espacio público se refuerza con un proceso de investigación donde se sustrajo la opinión de hombres y mujeres de diversos estratos sociales mediante entrevista y grupos focales, así como entrevistas con actores claves, para luego analizarlo de una manera transversal con una exploración etnográfica realizada por el autor donde se detallaron las condiciones percibidas por él mismo en un recorrido a lo largo de tres calles de la ciudad que reúnen las diferentes características que engloban el conjunto de prototipos de calle existentes en la ciudad de Culiacán. Es así como la presente investigación reúne, mediante una mezcla de metodologías, una interpretación de los cambios que ha sufrido el espacio público en la ciudad de Culiacán desde la percepción en sitio por el autor en sus recorridos, así como de la opinión de sus habitantes con la finalidad de evaluar el cambio de uso que en ellos se ha realizado y cómo la socialización de la gente que antes existía en ellas hoy es limitada en función de las características, uso y destino que estos espacios tienen en la ciudad.The twentieth century transformed the streets of the cities which defined the continuing public space into distributive arteries for automobiles and other motorized vehicles-- more the individual than the collective-- with very limited spaces for the rest of the functions for which the streets were originally designed. Social interaction among people transitioned to parks and plazas and reduced conditions soon followed. The consequence was the gradual abandonment of these spaces and the activities of social citizen transitioned into closed spaces from whose private domains came the great commercial centers which today proliferate the majority of the Latin American cities. In practice, the street is no longer a public space, so only parks and plazas are available for the common use under equal conditions. Yet day by day, these are becoming more vulnerable in cities. This process has negatively impacted the development of cities. Therefore, streets have left the public space where individuals can meet and interact and will possibly continue to transform into an exclusive space with a greater hierarchy given to motor vehicles and gradually diminished space for pedestrians. The present research focuses on the growth process of average Mexican cities in the period from 1980 to 2010, how this process has transformed public space and how it has impacted the socialization of people in the case study of Culiácan. This study has analyzes three previously selected prototypes as valid alternatives. The analysis of this thesis is presented in two stages: the first will lay out the current situation in Mexican cities with an emphasis on the twenty preselected medium-sized cities (between 500,000 and one million inhabitants), and the second will address Culiacán and the transformative process of its public space within the last three decades. The evaluation of the public space supports a research process where I took the opinion of men and women from diverse social classes by means of focus groups and interviews with key actors, in order to create through a variety of methods an ethnographic study, which details the conditions along the three main streets of the city, and combines the diverse characteristics including the combination of prototypes for existing streets in Culiacán. As such this research through a variety of means presents a mix of methodologies, it interprets the changes which the public space has suffered in the city of Culiacán from the viewpoint by the author through his tours and from the opinion of the inhabitants. The latter's view derives from their experience. and how the socialization of the people, which before existed in the streets, is now limited in function, use and spac

Identification and Structure–Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors

Type II Inhibitors Targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg²⁺ binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in–out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target

Type II Inhibitors Targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg²⁺ binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in–out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target

Discovery of a Selective Allosteric Inhibitor Targeting Macrodomain 2 of Polyadenosine-Diphosphate-Ribose Polymerase 14

Macrodomains are conserved protein interaction modules that can be found in all domains of life including in certain viruses. Macrodomains mediate recognition of sequence motifs harboring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterization of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallization of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localization to sites of DNA damage

Human Cyclin-dependent kinase 12 (CDK12), kinase domain; A Target Enabling Package

<p>Cyclin-dependent kinase 12 (CDK12) phosphorylates RNA Pol II C-terminal domain (CTD) to promote transcriptional elongation of large DNA damage response genes. CDK12 is frequently mutated or amplified in cancer and its loss sensitises cells to DNA damage. Here we present 3 crystal structures of the human CDK12/CycK complex including apo, AMP-PNP and covalent inhibitor complexes. Kinase assays compare domain truncations and report the Km values for substrate. THZ531 is presented as a potent and selective inhibitor of CDK12 with nanomolar activity in leukemic cell lines.</p

Human Cyclin-dependent kinase 12 (CDK12), kinase domain; A Target Enabling Package

<p>Cyclin-dependent kinase 12 (CDK12) phosphorylates RNA Pol II C-terminal domain (CTD) to promote transcriptional elongation of large DNA damage response genes. CDK12 is frequently mutated or amplified in cancer and its loss sensitises cells to DNA damage. Here we present 3 crystal structures of the human CDK12/CycK complex including apo, AMP-PNP and covalent inhibitor complexes. Kinase assays compare domain truncations and report the Km values for substrate. THZ531 is presented as a potent and selective inhibitor of CDK12 with nanomolar activity in leukemic cell lines.</p

Representation of the kinome coverage of the virtual set of 457 narrow spectrum inhibitors.

<p>Red background shows the human protein kinases ranked by number of citations. Blue bars show the protein kinases for which an assay is available at one of 10 commercial vendors. Black bars identify protein kinases that are covered by the virtual set of inhibitors (subset of PKIS, PKIS2, and literature) described in the text.</p

Schematic: Utilization of the KCGS.

<p>(<b>a</b>) Disease relevant phenotypic screen highlights active compounds (<b>b</b>) Because all the targets are annotated, active molecules points to potential targets of interest. (<b>c</b>) Follow up experiments can be used to help confirm importance of these highlighted targets (<b>d</b>) Body of evidence implicates kinases that can be inhibited to impact disease.</p

The screenable protein kinome.

<p>Using assays from 10 vendors a total of 436 unique non-mutant human protein kinases can be readily screened. (<b>a</b>) Subfamily representation of the protein kinases that were not available through the 10 vendors. Nearly half of the unscreenable human kinome is composed of pseudokinases (<b>b</b>) Histogram illustrating how many vendors can be used to screen across the kinome. For example, there are 43 kinases that all 10 vendors have screens for (“all” bar). There are another 109 kinases screened by 9 out of 10 vendors (“9/10” bar). There are 38 kinases that only 1 out of the 10 vendors has assays for (“1/10” bar). <i>Note</i>: <i>Our definition of the “screenable kinome” is those kinases for which there is a commercial assay that can be accessed</i>. <i>It is likely that assays could indeed be configured for many of the kinases not currently on this list</i>.</p