Doctorado en Ciencias Biomédicashttps://repository.urosario.edu.co/handle/10336/4997https://repository.urosario.edu.co/retrieve/21c80270-9eea-4f69-986e-59584145808f/2024-03-28T19:16:50Z2024-03-28T19:16:50Z221A "Trojan horse" strategy to reverse drug-resistance in brain tumorsPinzon-Daza, Martha L.https://repository.urosario.edu.co/handle/10336/89922021-06-03T05:47:44Z2014-01-01T00:00:00Zdc.title: A "Trojan horse" strategy to reverse drug-resistance in brain tumors
dc.description.abstract: Malignant gliomas represent one of the most aggressive forms of Central Nervous System
(CNS) tumors. According to the WHO classification of brain tumors, astrocytomas have been
categorized into four grades, determined by the underlying pathology. Malignant (or highgrade)
gliomas include anaplastic glioma (WHO grade III) as well as glioblastoma multiforme
(GBM; WHO grade IV). These are the most aggressive brain tumors with the worst prognosis
(1). The therapeutic management of CNS tumors is based on surgery, radiotherapy and
chemotherapy, depending on the characteristics of the tumor, the clinical stage and age (2),
(3), however none of the standard treatments is completely safe and compatible with an
acceptable quality of life (3), (4). Chemotherapy is the first choice in disseminated tumors,
like invasive glioblastoma, high-risk medulloblastoma or multiple metastasis, but the
prognosis in these patients is very poor (2),(3). New targeted therapies (2), anti-angiogenic
therapies (3), (4) or gene therapies show a real benefit only in limited groups of patients with
known specific molecular defects (4). Thereby, the development of new pharmacological
therapies for brain tumors is mandatory. Malignant gliomas are frequently chemoresistant
and this resistance seems to depend on at least two mechanisms: first, the poor penetration
of many anticancer drugs across the blood-brain barrier (BBB), the blood-cerebrospinal fluid
barrier (BCSFB) and the blood-tumor barrier (BTB), due to their interaction with several
ATP-binding cassette (ABC) drug efflux transporters that are overexpressed by the
endothelial or epithelial cells of these barriers. Second, ABC drug efflux transporters in tumor
cells confer multidrug resistance (MDR) on several other solid tumors; they are present on
CNS tumors too and their role in gliomas is under investigation (5). Drug delivery across the
blood-brain barrier (BBB) is one of the vital problems in targeted therapy treatments. Recent
studies have shown that some small molecules used in these therapies are substrates of Pglycoprotein
(Pgp), as well as other efflux pumps like multidrug resistance-related proteins
(MRPs) and breast-cancer resistance related protein (BCRP), which extrude several
anticancer drugs and will not allow drugs to reach the tumor (1).
DOXOrubicin (DOXO), a drug widely used in anti-cancer therapy, is a substrate of Pgp and
BCRP, and it is very effective to attack the vitro brain tumor cells, but has a limited clinical use
for its low delivery across BBB and the resistance of tumors. On the other hand BBB cells and
brain tumor cells also have surface proteins, such as Low Density Lipoprotein Receptor
(LDLR), which could be used as a therapeutic target. The importance of this study is based on
the generation of therapeutical strategies to promote the passage of drugs through the BBB
and the intratumoral accumulation, and at the same time, on the analysis of cellular
mechanisms that induce increased expression of ABC transporters, to be used as therapeutic
targets.
In this work we demonstrated that the use of a new strategy based on the "Trojan horse",
which combines DOXOrubicin introduced into a liposome, could safeguards the drug to
prevent its recognition by the ABC transporters in both the BBB and the tumor cells. The
construction of liposome allowed using the LDLR receptor cells as docking receptor, ensuring
the entrance through the BBB and into the tumor cells through a process of endocytosis. This
mechanism was associated with the use of statins, anti-cholesterol drugs which favoured the
expression of LDLR and decreased the activity of ABC transporters, increasing the efficiency
of our Trojan horse. Accordingly, I demonstrated that the use of a new DOXOrubicin
liposomal formulation mimicking LDLs, called ApolipoDOXO, further favors drug delivery through the BBB, overcoming the resistance of tumor and reducing the side effects of
DOXOrubicin dose. In addition this strategy can be considered as a new strategy to increase
the effectiveness of different drugs in several brain tumors and ensures high efficiency even
in a hypoxic environment, characteristic of cancer cells, where the expression of Pgp
transporter was increased.
Taking advantage of another signaling pathway recognized as a modulator of Pgp activity this
study presents not only the strategy of the Trojan horse, but also a second therapeutic
proposal related to the use of Temozolomide plus DOXOrubicin. This strategy showed that
temozolomide (TMZ) penetrated the BBB in a way involved the Wnt/GSK3/β-catenin
signaling pathway, which modulates the expression of Pgp transporter. It was demonstrated
that the TMZ reduces Wnt3 protein and mRNA allowing raising the hypothesis that this drug
decreases Wnt3 gene transcription in BBB cells, decreasing β-catenin pathway activation by
its phosphorylation, reducing β-catenin nuclear translocation and binding to the promoter of
the mdr1 gene. Taking together the results of this study allowed the recognition of three basic
mechanisms related to the down-regulation of Pgp and associated strategies: the first was the
use of statins, which led to the transporter nitration decreasing its activity by NFκB pathway;
the second one was based on the use of temozolomide, which by methylating Wnt3 gene
reduces the activity of the β-catenin signaling pathway, decreasing the expression of Pgp
transporter; the third one consisted on the cross-talk between the Wnt/GSK3/β-catenin axis
and the Wnt/RhoA/RhoA kinase as a modulator of mdr1 transcription: we demonstrated that
RhoA protein kinase promoted the activation of the protein PTB1, which by phosphorylating
GSK3 induced phosphorylation of β-catenin, priming it for destruction by the proteasome,
avoiding the binding to the promoter of the mdr1 gene and therefore reducing Pgp
expression. In conclusion, the therapeutic startegies proposed in this work increased the
cytotoxicity of tumour cells by increasing permeability not only in the BBB, but also in tumor
barrier. Also, the "Trojan horse" strategy could be useful for the therapy of other diseases
associated with the central nervous system. On the other hand, these studies indicate that
recognition of mechanisms associated with the expression of ABC transporters could be a key
tool in the development of new anti-cancer therapies.
dc.description: Los gliomas malignos representan una de las formas más agresivas de los tumores del
sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad
de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias
farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas
malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al
menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre
expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las
células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a
través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia
del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina.
Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y
asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de
mecanismos asociados a la expresión de los transportadores ABC podría constituir una
herramienta clave en el desarrollo de nuevas terapias anticáncer.
2014-01-01T00:00:00ZPinzon-Daza, Martha L.Malignant gliomas represent one of the most aggressive forms of Central Nervous System
(CNS) tumors. According to the WHO classification of brain tumors, astrocytomas have been
categorized into four grades, determined by the underlying pathology. Malignant (or highgrade)
gliomas include anaplastic glioma (WHO grade III) as well as glioblastoma multiforme
(GBM; WHO grade IV). These are the most aggressive brain tumors with the worst prognosis
(1). The therapeutic management of CNS tumors is based on surgery, radiotherapy and
chemotherapy, depending on the characteristics of the tumor, the clinical stage and age (2),
(3), however none of the standard treatments is completely safe and compatible with an
acceptable quality of life (3), (4). Chemotherapy is the first choice in disseminated tumors,
like invasive glioblastoma, high-risk medulloblastoma or multiple metastasis, but the
prognosis in these patients is very poor (2),(3). New targeted therapies (2), anti-angiogenic
therapies (3), (4) or gene therapies show a real benefit only in limited groups of patients with
known specific molecular defects (4). Thereby, the development of new pharmacological
therapies for brain tumors is mandatory. Malignant gliomas are frequently chemoresistant
and this resistance seems to depend on at least two mechanisms: first, the poor penetration
of many anticancer drugs across the blood-brain barrier (BBB), the blood-cerebrospinal fluid
barrier (BCSFB) and the blood-tumor barrier (BTB), due to their interaction with several
ATP-binding cassette (ABC) drug efflux transporters that are overexpressed by the
endothelial or epithelial cells of these barriers. Second, ABC drug efflux transporters in tumor
cells confer multidrug resistance (MDR) on several other solid tumors; they are present on
CNS tumors too and their role in gliomas is under investigation (5). Drug delivery across the
blood-brain barrier (BBB) is one of the vital problems in targeted therapy treatments. Recent
studies have shown that some small molecules used in these therapies are substrates of Pglycoprotein
(Pgp), as well as other efflux pumps like multidrug resistance-related proteins
(MRPs) and breast-cancer resistance related protein (BCRP), which extrude several
anticancer drugs and will not allow drugs to reach the tumor (1).
DOXOrubicin (DOXO), a drug widely used in anti-cancer therapy, is a substrate of Pgp and
BCRP, and it is very effective to attack the vitro brain tumor cells, but has a limited clinical use
for its low delivery across BBB and the resistance of tumors. On the other hand BBB cells and
brain tumor cells also have surface proteins, such as Low Density Lipoprotein Receptor
(LDLR), which could be used as a therapeutic target. The importance of this study is based on
the generation of therapeutical strategies to promote the passage of drugs through the BBB
and the intratumoral accumulation, and at the same time, on the analysis of cellular
mechanisms that induce increased expression of ABC transporters, to be used as therapeutic
targets.
In this work we demonstrated that the use of a new strategy based on the "Trojan horse",
which combines DOXOrubicin introduced into a liposome, could safeguards the drug to
prevent its recognition by the ABC transporters in both the BBB and the tumor cells. The
construction of liposome allowed using the LDLR receptor cells as docking receptor, ensuring
the entrance through the BBB and into the tumor cells through a process of endocytosis. This
mechanism was associated with the use of statins, anti-cholesterol drugs which favoured the
expression of LDLR and decreased the activity of ABC transporters, increasing the efficiency
of our Trojan horse. Accordingly, I demonstrated that the use of a new DOXOrubicin
liposomal formulation mimicking LDLs, called ApolipoDOXO, further favors drug delivery through the BBB, overcoming the resistance of tumor and reducing the side effects of
DOXOrubicin dose. In addition this strategy can be considered as a new strategy to increase
the effectiveness of different drugs in several brain tumors and ensures high efficiency even
in a hypoxic environment, characteristic of cancer cells, where the expression of Pgp
transporter was increased.
Taking advantage of another signaling pathway recognized as a modulator of Pgp activity this
study presents not only the strategy of the Trojan horse, but also a second therapeutic
proposal related to the use of Temozolomide plus DOXOrubicin. This strategy showed that
temozolomide (TMZ) penetrated the BBB in a way involved the Wnt/GSK3/β-catenin
signaling pathway, which modulates the expression of Pgp transporter. It was demonstrated
that the TMZ reduces Wnt3 protein and mRNA allowing raising the hypothesis that this drug
decreases Wnt3 gene transcription in BBB cells, decreasing β-catenin pathway activation by
its phosphorylation, reducing β-catenin nuclear translocation and binding to the promoter of
the mdr1 gene. Taking together the results of this study allowed the recognition of three basic
mechanisms related to the down-regulation of Pgp and associated strategies: the first was the
use of statins, which led to the transporter nitration decreasing its activity by NFκB pathway;
the second one was based on the use of temozolomide, which by methylating Wnt3 gene
reduces the activity of the β-catenin signaling pathway, decreasing the expression of Pgp
transporter; the third one consisted on the cross-talk between the Wnt/GSK3/β-catenin axis
and the Wnt/RhoA/RhoA kinase as a modulator of mdr1 transcription: we demonstrated that
RhoA protein kinase promoted the activation of the protein PTB1, which by phosphorylating
GSK3 induced phosphorylation of β-catenin, priming it for destruction by the proteasome,
avoiding the binding to the promoter of the mdr1 gene and therefore reducing Pgp
expression. In conclusion, the therapeutic startegies proposed in this work increased the
cytotoxicity of tumour cells by increasing permeability not only in the BBB, but also in tumor
barrier. Also, the "Trojan horse" strategy could be useful for the therapy of other diseases
associated with the central nervous system. On the other hand, these studies indicate that
recognition of mechanisms associated with the expression of ABC transporters could be a key
tool in the development of new anti-cancer therapies.A trans-methylation mechanism between the two major H3K9 methyltransferases SETDB1 and SUV39H1 regulates heterochromatin establishmentCruz Tapias, Paola Andreahttps://repository.urosario.edu.co/handle/10336/198282021-03-04T00:40:47Z2018-01-01T00:00:00Zdc.title: A trans-methylation mechanism between the two major H3K9 methyltransferases SETDB1 and SUV39H1 regulates heterochromatin establishment
dc.description.abstract: Histone H3 lysine 9 trimethylation (H3K9me3) is a key epigenetic modification required for heterochromatin formation and maintenance, genome stability and silencing of transposable elements in embryonic stems cells (ESCs). The H3K9-specific methyltransferase (KMT) SETDB1 is vital for mammalian development as it regulates ESCs pluripotency in the early embryo. Here we unravel that SETDB1 undergoes automethylation on two lysines, embedded within its catalytic domain, both in vitro and in cells. Importantly, SETDB1 automethylation is required for mouse ESCs stemness, growth and viability. Hence, transcriptome-wide analyses (RNA-seq) show that the integrity of the two SETDB1 automethylated lysines is required for both coding genes and transposable elements silencing in mESCs. Indeed, our analyses of ChIP-seq show that automethylation-deficient SETDB1 expression leads to a lack of H3K9me3 establishment at target loci. Interestingly, our results point to a model in which SETDB1 auto-methylation paves the path to a subsequent trans-methylation by SUV39H1. This mechanism could regulate not only the SETDB1/SUV39H1 physical interaction (via the SUV39H1 chromodomain), but also cooperation in the establishment and maintenance of both heterochromatin blocks (large domains) and transposable elements silencing. Taken together, my findings uncover a novel mechanism regulating SETDB1 KMT key functions that are key in embryonic stems cells identity maintenance.
dc.description: La tri-metilación de la lisina 9 de la histona 3 (H3K9me3) es una modificación epigenética requerida para la formación y el mantenimiento de la heterocromatina, la estabilidad genómica y el silenciamiento de elementos transposables en células madre embrionarias (CMEs). SETDB1 es una metiltransferasa específica de la lisina 9 de la histona 3 crucial durante el desarrollo de los mamíferos debido a que regula la pluripotencia de las CMEs en el embrión. Los resultados de este trabajo sugieren que SETDB1 lleva a cabo un proceso de auto-metilación que es requerido para el mantenimiento de la pluripotencia, el crecimiento y la viabilidad de las CMEs murinas. Adicionalmente, análisis de transcriptoma completo (RNA-seq) mostraron que la integridad de las dos lisinas auto-metiladas es requerida para el silenciamiento tanto de genes codificantes como de elementos transposables. De hecho, análisis de ChIP-seq revelaron que una deficiencia en la auto-metilación conlleva a una disminución en el establecimiento de H3K9me3 en loci blanco. Nuestros resultados sugieren que la auto-metilación de SETDB1 es un pre-requisito para la trans-metilación por SUV39H1. Este mecanismo podría regular no solamente la interacción física entre SETDB1 y SUV39H1 (vía el cromodominio de SUV39H1), sino también la cooperación para el establecimiento y el mantenimiento de la heterocromatina y el silenciamiento de los elementos transposables. Por todo lo anterior, los resultados de este trabajo revelan un nuevo mecanismo que regula las funciones de SETDB1, el cual es crucial para la identidad y el mantenimiento de las CMEs.
2018-01-01T00:00:00ZCruz Tapias, Paola AndreaHistone H3 lysine 9 trimethylation (H3K9me3) is a key epigenetic modification required for heterochromatin formation and maintenance, genome stability and silencing of transposable elements in embryonic stems cells (ESCs). The H3K9-specific methyltransferase (KMT) SETDB1 is vital for mammalian development as it regulates ESCs pluripotency in the early embryo. Here we unravel that SETDB1 undergoes automethylation on two lysines, embedded within its catalytic domain, both in vitro and in cells. Importantly, SETDB1 automethylation is required for mouse ESCs stemness, growth and viability. Hence, transcriptome-wide analyses (RNA-seq) show that the integrity of the two SETDB1 automethylated lysines is required for both coding genes and transposable elements silencing in mESCs. Indeed, our analyses of ChIP-seq show that automethylation-deficient SETDB1 expression leads to a lack of H3K9me3 establishment at target loci. Interestingly, our results point to a model in which SETDB1 auto-methylation paves the path to a subsequent trans-methylation by SUV39H1. This mechanism could regulate not only the SETDB1/SUV39H1 physical interaction (via the SUV39H1 chromodomain), but also cooperation in the establishment and maintenance of both heterochromatin blocks (large domains) and transposable elements silencing. Taken together, my findings uncover a novel mechanism regulating SETDB1 KMT key functions that are key in embryonic stems cells identity maintenance.Autoantibody and environmental damage to the brainArango, María-Teresahttps://repository.urosario.edu.co/handle/10336/128752021-06-03T05:45:21Z2016-01-01T00:00:00Zdc.title: Autoantibody and environmental damage to the brain
dc.description.abstract: Behavioral abnormalities and cognitive dysfunction may be present in patients with autoimmune diseases. The mechanisms that responsible for these neuropsychiatric manifestations are still largely unknown, however several pathogenic pathways have been identified such as antibody damage, cytokine-induced neurotoxicity, as well as external factors like toxics, and medications. To further investigate some of these mechanisms we evaluated the effect of autoantibodies in animal models of neuropsychiatric systemic lupus erythematosus and narcolepsy, and the effect of the human papillomavirus vaccine in naïve mice.
Using the passive immunization method through intracerebro-ventricular injection of the antibodies, we demonstrated histological and behavioral changes. Mice immunized with 16/6 idiotypic antibodies developed cognitive impairments while those immunized with anti-ribosomal-P antibodies developed depression. The mice that received total IgG from narcoleptic patients developed sleep disturbances and brain histological changes. Further analyses of the role of the human anti-ribosomal-P autoantibody revealed that it can cross-react with the neuronal protein Gap43, interfering with cellular processes.
Immunization with the human papillomavirus vaccine induced the production of brain antibodies. Moreover, the mice immunized with the vaccine or with its adjuvant developed cognitive and behavioral deficiencies, which were ameliorated with dietary phospholipid supplementation.
Overall, herein we demonstrate that the behavioral and cognitive abnormalities can be part of the wide spectrum of clinical autoimmune manifestations. In addition, they can be caused by collateral damage due to the immune dysregulation caused by autoimmune conditions as well as by vaccination. We also suggest that different autoantibodies cause different symptoms based on different interactions with brain tissue.
dc.description: Las anormalidades de comportamiento y disfunciones cognitivas pueden presentarse en pacientes con enfermedades autoinmunes. Estos síntomas pueden fluctuar de leves hasta eventos potencialmente mortales. En su gran mayoría los mecanismos responsables de estas manifestaciones neuropsiquiatricas siguen siendo desconocidas, sin embargo se han identificado varias vías patogénicas. Por ejemplo; neurotoxicidad mediada por anticuerpos, vasculopatía inducida por anticuerpos anti-fosfolípidos, neurotoxicidad inducida por citoquinas, así como factores externos que incluyen sustancias tóxicas y medicamentos. Para poder investigar algunos de estos mecanismos, evaluamos el efecto de auto-anticuerpos relacionados con el lupus eritematoso sistémico neuropsiquiátrico en modelos animales (Anticuerpo idiotípico 16/6 y el anticuerpo anti-ribosoma P) y narcolepsia (empleando IgG de pacientes narcolépticos), así como el efecto de la vacuna del virus del papiloma humano en ratones.
La inmunización pasiva por inyección intracerebro-ventricular de estos anticuerpos condujo a cambios histológicos, cognitivos y de comportamiento. En particular, los ratones inmunizados con el anticuerpo idiotípico 16/6 desarrollaron déficit cognitivo y los ratones inmunizados con el anticuerpo anti-ribosomal P desarrollaron depresión. Los ratones que recibieron IgG total de pacientes narcolépticos desarrollaron trastornos del sueño y cambios histológicos cerebrales consistentes con la enfermedad. Un análisis más profundo del papel del anticuerpo anti-ribosomal P humano reveló que reacciona de manera cruzada con la proteína neuronal “Gap43” interfiriendo con procesos celulares.
Por otra parte, la inmunización con la vacuna del virus del papiloma humano causó la producción de anticuerpos contra componentes del cerebro. Adicionalmente, los ratones inmunizados con la vacuna, al igual que los inmunizados con su adyuvante desarrollaron deficiencia cognitiva y conductual, que fue mejorada con la suplementación alimenticia basada en fosfolípidos.
En general, aquí se demuestra que anormalidades del comportamiento y disfunciones cognitiva pueden ser parte de la amplia gama de manifestaciones clínicas autoinmunes. Además, nuestros resultados sugieren que estas pueden ser causadas por daños colaterales debido a la desregulación inmune causada por autoinmunidad, así como por la vacunación. Adicionalmente, sugerimos que diferentes auto-anticuerpos causan diferentes síntomas basados en diferentes interacciones con el tejido cerebral.
2016-01-01T00:00:00ZArango, María-TeresaBehavioral abnormalities and cognitive dysfunction may be present in patients with autoimmune diseases. The mechanisms that responsible for these neuropsychiatric manifestations are still largely unknown, however several pathogenic pathways have been identified such as antibody damage, cytokine-induced neurotoxicity, as well as external factors like toxics, and medications. To further investigate some of these mechanisms we evaluated the effect of autoantibodies in animal models of neuropsychiatric systemic lupus erythematosus and narcolepsy, and the effect of the human papillomavirus vaccine in naïve mice.
Using the passive immunization method through intracerebro-ventricular injection of the antibodies, we demonstrated histological and behavioral changes. Mice immunized with 16/6 idiotypic antibodies developed cognitive impairments while those immunized with anti-ribosomal-P antibodies developed depression. The mice that received total IgG from narcoleptic patients developed sleep disturbances and brain histological changes. Further analyses of the role of the human anti-ribosomal-P autoantibody revealed that it can cross-react with the neuronal protein Gap43, interfering with cellular processes.
Immunization with the human papillomavirus vaccine induced the production of brain antibodies. Moreover, the mice immunized with the vaccine or with its adjuvant developed cognitive and behavioral deficiencies, which were ameliorated with dietary phospholipid supplementation.
Overall, herein we demonstrate that the behavioral and cognitive abnormalities can be part of the wide spectrum of clinical autoimmune manifestations. In addition, they can be caused by collateral damage due to the immune dysregulation caused by autoimmune conditions as well as by vaccination. We also suggest that different autoantibodies cause different symptoms based on different interactions with brain tissue.Caracterización del complejo mayor de histocompatibilidad clase II en primates del género AotusSuarez Martinez, Carlos Fernandohttps://repository.urosario.edu.co/handle/10336/138092021-06-03T05:48:21Z2017-01-01T00:00:00Zdc.title: Caracterización del complejo mayor de histocompatibilidad clase II en primates del género Aotus
dc.description.abstract: This work was aimed to contribute to increase our knowledge on the MHC class II in monkeys from the genus Aotus. Determining the sequences of MHC-DPA and MHC-DRA genes has allowed to complete the characterisation of the Aotus MHC, contributing towards validating the role of this primate as experimental model and increasing our knowledge regarding MHC gene evolution in primates. It also dealt with in–depth analysis of MHC-DR genes’ convergence and polymorphism in primates. The study involves computational modelling of MHC-peptide binding methodologies (based on quantum chemistry and neural networks) as necessary tools for understanding the mechanisms of MHC class II peptide presentation to T-lymphocytes. Studying peptide binding region polymorphism has enabled developing strategies (pocket profiles) for efficiently reducing the amount of systems to be considered when designing peptides to be used as candidates for an antimalarial vaccine. Data-mining regarding Ramachandran distribution led to developing an amino acid structural similarity scale for use in developing/designing peptides as vaccine candidates. It was found that protein secondary structure has a clear relationship with amino acid substitution and mutability evolutionary patterns. A conceptual framework thus emerged aimed at developing peptide-based vaccines as a basis for studying the mayor histocompatibility complex polymorphism, the physicochemical/structural restrictions shaping the molecular recognition involved in MHC-peptide interaction and using computational methodologies for quantifying MHC-peptide binding.
dc.description: El presente trabajo tiene como propósito contribuir al conocimiento del complejo mayor de histocompatibilidad clase II (CMH-II) de los monos Aotus, contribuyendo a la validación de este primate como modelo experimental, y aumentando el conocimiento en la evolución de los genes del CMH en primates. Además, se profundizó en el análisis de convergencia y polimorfismo de los genes del CMH-DR en primates.
Se implementaron metodologías de modelación computacional de la unión CMH-péptido, como herramientas necesarias para entender los mecanismos de presentación de péptidos por parte del CMH clase II a los linfocitos T. El estudio del polimorfismo de la región de unión al péptido, permitió el desarrollo de estrategias para reducir eficientemente el número de sistemas a considerar en el diseño de péptidos a ser usados como candidatos a vacuna contra la malaria.
Usando minería de datos sobre distribuciones de Ramachandran, se desarrolló una escala de similitud estructural de aminoácidos, con el fin de implementar su uso en el desarrollo de péptidos candidatos a vacunas. Adicionalmente, se encontró que la estructura secundaria de las proteínas tiene una relación clara con los patrones evolutivos de sustitución y la mutabilidad de los aminoácidos.
Así, se ha generado un marco de conceptual que contribuye al desarrollo de vacunas basadas en péptidos, que tiene como base el estudio del polimorfismo del complejo mayor de histocompatibilidad, las restricciones fisicoquímicas/estructurales que moldean el proceso de reconocimiento molecular involucrado en la interacción CMH-péptido y la aplicación de metodologías computacionales para cuantificar el proceso de unión CMH-péptido.
2017-01-01T00:00:00ZSuarez Martinez, Carlos FernandoThis work was aimed to contribute to increase our knowledge on the MHC class II in monkeys from the genus Aotus. Determining the sequences of MHC-DPA and MHC-DRA genes has allowed to complete the characterisation of the Aotus MHC, contributing towards validating the role of this primate as experimental model and increasing our knowledge regarding MHC gene evolution in primates. It also dealt with in–depth analysis of MHC-DR genes’ convergence and polymorphism in primates. The study involves computational modelling of MHC-peptide binding methodologies (based on quantum chemistry and neural networks) as necessary tools for understanding the mechanisms of MHC class II peptide presentation to T-lymphocytes. Studying peptide binding region polymorphism has enabled developing strategies (pocket profiles) for efficiently reducing the amount of systems to be considered when designing peptides to be used as candidates for an antimalarial vaccine. Data-mining regarding Ramachandran distribution led to developing an amino acid structural similarity scale for use in developing/designing peptides as vaccine candidates. It was found that protein secondary structure has a clear relationship with amino acid substitution and mutability evolutionary patterns. A conceptual framework thus emerged aimed at developing peptide-based vaccines as a basis for studying the mayor histocompatibility complex polymorphism, the physicochemical/structural restrictions shaping the molecular recognition involved in MHC-peptide interaction and using computational methodologies for quantifying MHC-peptide binding. Caracterización y dinámica de los patrones de infecciones únicas y múltiples para seis tipos del virus de papiloma humano de alto riesgoSoto de León, Sara Ceciliahttps://repository.urosario.edu.co/handle/10336/103472021-06-03T05:46:11Z2014-01-01T00:00:00Zdc.title: Caracterización y dinámica de los patrones de infecciones únicas y múltiples para seis tipos del virus de papiloma humano de alto riesgo
dc.description.abstract: Cervical cancer (CC) have been reported and continues being the second cause of cancer-related death in Colombia’s female population, with high incidence (32. 9-36. 4 cases/year/100, 000 women) and mortality rates (18. 7 cases/year/100, 000 women). The main risk factor for developing pre-neoplastic cervical lesions is persistent infection by certain types of human papilloma virus (HPV), known as high risk types (HR-HPV), associated with ~90% of CC around the world. The present work was aimed at identifying HPV infection characteristics in a socio-demographically heterogeneous population of females living in different parts of Colombia; 2, 109 women from the cities of Chaparral, Tumaco, Leticia, Bogotá and Girardot were thus included in the study as they were attending CC prevention programmes in their respective hospitals. Every female provided sociodemographic information, data regarding their sexual conduct and a cervical smear. HPV infection was determined by polymerase chain reaction (PCR) using three sets of generic primers; type-specific primers were also used for determining the frequency of six high-risk HPV types (HR-HPV-16, -18, -31, -33, -45 and -58) and two low-risk types (LR-HPV-6/-11). Additionally viral load was evaluated in cervical tissue samples taken from 219 HPV infected females who were seen during a minimum of 4 visits (6 month intervals: + 3 months). Quantitation was performed by real-time PCR (RT-PCR) and the data was correlated by follow-up lasting two years for determining the dynamics of the single and multiple infection patterns found in Colombia.
dc.description: El cáncer de cuello uterino (CCU) es la segunda causa de muerte por cáncer en la población femenina de Colombia con tasas de incidencia y mortalidad altas (32,9-36,4 y 18,7 casos/año/100.000 mujeres, respectivamente). El principal factor de riesgo para el desarrollo de lesiones cervicales pre-neoplásicas es la infección persistente por ciertos tipos de Virus de Papiloma Humano (VPH) conocidos como de alto riesgo (VPH-AR), asociados con ~90% de CCU a nivel mundial.
Este trabajo tuvo como objetivo identificar las características de la infección por VPH en una población de mujeres socio-demográficamente heterogénea, que habitan en diferentes regiones de Colombia. Para esto, fueron incluidas 2109 mujeres provenientes de las ciudades de Chaparral, Tumaco, Leticia, Bogotá y Girardot, quienes acudieron a los programas de promoción y prevención de CCU implementados en los respectivos hospitales; cada mujer proporcionó información sociodemográfica y de conductas sexuales, además de una muestra de raspado cervical.
Se determinó la presencia de VPH por la técnica de PCR, empleando tres juegos de cebadores genéricos, adicionalmente, se usaron cebadores tipo-específicos para determinar la frecuencia de seis tipos de VPH de alto riesgo (VPH-AR-16, -18, -31, -33, -45 y -58) y dos de bajo riesgo (VPH-BR-6/11). Se evaluó también la carga viral de los tipos de VPH-AR mediante PCR en tiempo real y se correlacionaron los datos de 219 mujeres a través de un seguimiento a dos años (cada 6 meses), con el fin de determinar la dinámica de los patrones de infecciones únicas y múltiples encontrados en nuestro país.
2014-01-01T00:00:00ZSoto de León, Sara CeciliaCervical cancer (CC) have been reported and continues being the second cause of cancer-related death in Colombia’s female population, with high incidence (32. 9-36. 4 cases/year/100, 000 women) and mortality rates (18. 7 cases/year/100, 000 women). The main risk factor for developing pre-neoplastic cervical lesions is persistent infection by certain types of human papilloma virus (HPV), known as high risk types (HR-HPV), associated with ~90% of CC around the world. The present work was aimed at identifying HPV infection characteristics in a socio-demographically heterogeneous population of females living in different parts of Colombia; 2, 109 women from the cities of Chaparral, Tumaco, Leticia, Bogotá and Girardot were thus included in the study as they were attending CC prevention programmes in their respective hospitals. Every female provided sociodemographic information, data regarding their sexual conduct and a cervical smear. HPV infection was determined by polymerase chain reaction (PCR) using three sets of generic primers; type-specific primers were also used for determining the frequency of six high-risk HPV types (HR-HPV-16, -18, -31, -33, -45 and -58) and two low-risk types (LR-HPV-6/-11). Additionally viral load was evaluated in cervical tissue samples taken from 219 HPV infected females who were seen during a minimum of 4 visits (6 month intervals: + 3 months). Quantitation was performed by real-time PCR (RT-PCR) and the data was correlated by follow-up lasting two years for determining the dynamics of the single and multiple infection patterns found in Colombia. Disección molecular de la etiología de la falla ovárica prematura por secuenciación directa y NGS: estudio de la implicación de los genes CITED2, CDKN1B, FOXO4, BMP15 y ADAMTS19Fonseca Mendoza, Dora Janethhttps://repository.urosario.edu.co/handle/10336/133882019-09-19T12:37:54Z2016-01-01T00:00:00Zdc.title: Disección molecular de la etiología de la falla ovárica prematura por secuenciación directa y NGS: estudio de la implicación de los genes CITED2, CDKN1B, FOXO4, BMP15 y ADAMTS19
dc.description.abstract: Premature ovarian failure (POF) is a frequent disease, affecting 1% of women under 40 years old (Coulam et al, 1986). Clinically, POF women are affected by primary or secondary amenorrhoea and increased plasma levels of gonadotrophins (e.g. FSH and LH). POF can be originated from a variety of mechanisms leading to a decrease in the number of follicles, to the increase of follicular atresia or to follicle maturation failure (Beck-Peccoz et al, 2006).
POF has been associated with different etiologies, but in the majority of the cases the cause remains unknown. The prevalence of idiopathic cases suggests the existence of genetic and environmental aetiological factors (Maclaran et al, 2011). The inherent molecular complexity of sex determination, folliculogenesis and ovulation underlines that hundreds of genes might be related to POF phenotype (Laissue, 2015). In this context, direct sequencing of single genes and large scale genomic approaches are complementary tools for identifying new POF molecular actors. The present PhD thesis focused on the use of direct sequencing, NGS and functional in vitro tests allowing the identification and validation of new POF causative sequence variants.
dc.description: Entre las causas femeninas de infertilidad, la Falla Ovárica Prematura (FOP) es frecuente, ya que afecta al 1% de las mujeres menores de 40 años de edad (Coulam et al, 1986). La FOP se define clínicamente por amenorrea de más de 6 meses de duración, niveles plasmáticos elevados de gonadotrofinas y deficiencia de esteroides sexuales antes de los 40 años de edad. La FOP puede ocurrir por una variedad de mecanismos que conducen a la disminución del número de folículos, al incremento de la atresia folicular o a la falla en la maduración de los folículos (Beck-Peccoz et al, 2006).
La FOP se ha asociado a varias etiologías, pero en más del 80% de los casos, no se conoce la causa de la enfermedad. Esta alta prevalencia de casos idiopáticos sugiere la implicación de factores genéticos y ambientales (Maclaran et al, 2011). La complejidad de los procesos fisiológicos, celulares y genéticos subyacentes a la determinación sexual, la foliculogénesis y la ovulación sugiere la participación de numerosos genes regulados de una manera precisa en cada etapa del desarrollo (Laissue, 2015). En este contexto, es fundamental integrar el alcance de las distintas técnicas para la búsqueda de factores genéticos etiológicos de la FOP. En efecto, tanto la secuenciación directa como las aproximaciones multigénicas para el análisis genómico a gran escala son aproximaciones complementarias para este ejercicio.
La iniciativa principal de este trabajo doctoral consistió en realizar aproximaciones monogénicas (secuenciación de Sanger) y multigénicas Next generation sequencing (NGS) para la identificación de nuevos genes asociados a FOP. La identificación de nuevos genes y de variantes potencialmente deletéreas, así como su validación a través de estudios funcionales y análisis poblacionales, permitió aportar nuevo conocimiento en la etiología molecular de la FOP
2016-01-01T00:00:00ZFonseca Mendoza, Dora JanethPremature ovarian failure (POF) is a frequent disease, affecting 1% of women under 40 years old (Coulam et al, 1986). Clinically, POF women are affected by primary or secondary amenorrhoea and increased plasma levels of gonadotrophins (e.g. FSH and LH). POF can be originated from a variety of mechanisms leading to a decrease in the number of follicles, to the increase of follicular atresia or to follicle maturation failure (Beck-Peccoz et al, 2006).
POF has been associated with different etiologies, but in the majority of the cases the cause remains unknown. The prevalence of idiopathic cases suggests the existence of genetic and environmental aetiological factors (Maclaran et al, 2011). The inherent molecular complexity of sex determination, folliculogenesis and ovulation underlines that hundreds of genes might be related to POF phenotype (Laissue, 2015). In this context, direct sequencing of single genes and large scale genomic approaches are complementary tools for identifying new POF molecular actors. The present PhD thesis focused on the use of direct sequencing, NGS and functional in vitro tests allowing the identification and validation of new POF causative sequence variants.Effects of 17β-estradiol and tamoxifen on the induction of chromosomal abnormalities and on the expression of her2 gene in breast cancer cell linesRondón Lagos, Sandra Milenahttps://repository.urosario.edu.co/handle/10336/102152021-06-03T05:47:37Z2014-01-01T00:00:00Zdc.title: Effects of 17β-estradiol and tamoxifen on the induction of chromosomal abnormalities and on the expression of her2 gene in breast cancer cell lines
dc.description.abstract: This study covers an area of great importance in the research of breast cancer, related to the study of the effects of both estrogens (E2) and anti-estrogens (Tamoxifen) on chromosomes and of modulation of gene expression. Considering that breast cancer is a very heterogeneous disease and that patients respond differently to treatment, the identification of chromosomal abnormalities as well as genes responsive to 17β-estradiol (E2) and Tamoxifen (TAM) could provide the necessary framework to understand the complex effects of this hormone in target cells and could explain, at least in part, the development of cellular resistance to TAM treatment and the subsequent best therapeutic option. In this order of ideas, we determined the effects of E2 and TAM on the chromosomes and on the modulation of gene expression in four breast cancer cell lines, which represent three of the five subtypes of breast cancer known at present. The results are presented in six chapters - each one has a group of the results achieved around the cytogenetic characteristics and gene expression profiles of four cell lines and the effects of E2 and TAM incubation on those. The first chapter describes the main features of breast cancer, furthering the use and effects of E2 and TAM treatment.
2014-01-01T00:00:00ZRondón Lagos, Sandra MilenaThis study covers an area of great importance in the research of breast cancer, related to the study of the effects of both estrogens (E2) and anti-estrogens (Tamoxifen) on chromosomes and of modulation of gene expression. Considering that breast cancer is a very heterogeneous disease and that patients respond differently to treatment, the identification of chromosomal abnormalities as well as genes responsive to 17β-estradiol (E2) and Tamoxifen (TAM) could provide the necessary framework to understand the complex effects of this hormone in target cells and could explain, at least in part, the development of cellular resistance to TAM treatment and the subsequent best therapeutic option. In this order of ideas, we determined the effects of E2 and TAM on the chromosomes and on the modulation of gene expression in four breast cancer cell lines, which represent three of the five subtypes of breast cancer known at present. The results are presented in six chapters - each one has a group of the results achieved around the cytogenetic characteristics and gene expression profiles of four cell lines and the effects of E2 and TAM incubation on those. The first chapter describes the main features of breast cancer, furthering the use and effects of E2 and TAM treatment. Estudio conformacional de péptidos modificados de las proteínas STARP, CelTOS, TRSP y SERA 5 de Plasmodium falciparum y su relación con una respuesta inmune en malariaBermudez, Adrianahttps://repository.urosario.edu.co/handle/10336/126912021-06-03T05:46:39Z2015-01-01T00:00:00Zdc.title: Estudio conformacional de péptidos modificados de las proteínas STARP, CelTOS, TRSP y SERA 5 de Plasmodium falciparum y su relación con una respuesta inmune en malaria
dc.description.abstract: Developing an anti-malarial vaccine represents a dynamic area for exploration, even though one having enormous challenges, especially due to the complexity of the parasite’s lifecycle. Plasmodium falciparum’s different invasion stages must thus be blocked and the greatest amount of information possible concerning the artillery to be used in its attack must be extracted from them. Peptides from (sporozoite-derived) STARP, CelTOS and TRSP and (merozoite-derived) SERA 5 proteins having high binding affinity for HepG2 cells and red blood cells, respectively (known as conserved HABPs), have thus been modified, synthesised and evaluated at immune response level in Aotus monkeys as well as having been studied regarding their structural conformation by 1H-NMR.
The results have shown that native peptides are not immunogenic, but can induce high antibody titres when their critical or neighbouring residues are replaced by others having similar volume and mass but different polarity. Conformational study has revealed that native peptide structures are different from that of their modified peptides, having shorter or longer structured regions or not having any compared to their highly immunogenic modified analogues. Particular stereo-chemical characteristics in the side-chains of some of these modified peptides’ amino acid residues, such as physical-chemical features, seem to play an important role in inducing an appropriate immune response when they have been immunised in groups of Aotus monkeys, thereby complementing the design of a totally effective vaccine against malaria.
dc.description: El desarrollo de una vacuna contra malaria es un área de exploración activa pero con enormes retos debido especialmente a la complejidad del ciclo del parásito. Así, es necesario bloquear las diferentes etapas de la invasión que tiene el Plasmodium falciparum y extraer de ellas la mayor información posible de la artillería que utiliza para su ataque. Para esto, péptidos de las proteínas STARP, CelTOS y TRSP (del esporozoito) y SERA 5 (del merozoito) que tienen alta afinidad de unión a células HepG2 y a glóbulos rojos respectivamente (conocidos como cHABPs), han sido modificados (conocidos como mHABPs), sintetizados y evaluados a nivel de respuesta inmune en monos Aotus así como estudiados en su conformación estructural por RMN de 1H.
Los resultados muestran que los péptidos nativos no son inmunogénicos, pero pueden inducir altos títulos de anticuerpos cuando sus residuos críticos o sus vecinos son reemplazados por otro con un volumen y masa similar, pero diferente polaridad. El estudio conformacional pone de manifiesto que las estructuras de los péptidos nativos son diferentes de sus péptidos modificados ya sea que muestren regiones estructuradas más cortas o más largas o que no presenten ninguna, en comparación con sus análogos modificados altamente inmunogénicos. Las características estereoquímicas particulares en las cadenas laterales de algunos residuos de aminoácidos de estos péptidos modificados así como los rasgos fisicoquímicos parecen jugar un rol importante en la respuesta inmune apropiada cuando estos fueron inmunizados en grupos de monos Aotus confiriendo un avance al diseño de una vacuna contra malaria totalmente eficaz.
2015-01-01T00:00:00ZBermudez, AdrianaDeveloping an anti-malarial vaccine represents a dynamic area for exploration, even though one having enormous challenges, especially due to the complexity of the parasite’s lifecycle. Plasmodium falciparum’s different invasion stages must thus be blocked and the greatest amount of information possible concerning the artillery to be used in its attack must be extracted from them. Peptides from (sporozoite-derived) STARP, CelTOS and TRSP and (merozoite-derived) SERA 5 proteins having high binding affinity for HepG2 cells and red blood cells, respectively (known as conserved HABPs), have thus been modified, synthesised and evaluated at immune response level in Aotus monkeys as well as having been studied regarding their structural conformation by 1H-NMR.
The results have shown that native peptides are not immunogenic, but can induce high antibody titres when their critical or neighbouring residues are replaced by others having similar volume and mass but different polarity. Conformational study has revealed that native peptide structures are different from that of their modified peptides, having shorter or longer structured regions or not having any compared to their highly immunogenic modified analogues. Particular stereo-chemical characteristics in the side-chains of some of these modified peptides’ amino acid residues, such as physical-chemical features, seem to play an important role in inducing an appropriate immune response when they have been immunised in groups of Aotus monkeys, thereby complementing the design of a totally effective vaccine against malaria.Estudio de interacciones hospedero-patógeno y proteína-proteína en Plasmodium Vivax : evaluación de las proteínas del cuello de optrias -2, -4 y -5 y del antígeno apical de membrana-1Arévalo-Pinzón, Gabrielahttps://repository.urosario.edu.co/handle/10336/190952021-02-19T06:01:01Z2018-01-01T00:00:00Zdc.title: Estudio de interacciones hospedero-patógeno y proteína-proteína en Plasmodium Vivax : evaluación de las proteínas del cuello de optrias -2, -4 y -5 y del antígeno apical de membrana-1
dc.description.abstract: Malaria is one of the most important tropical diseases transmitted by vectors worldwide; Plasmodium vivax represents one of the most widely distributed species (affecting ~ 13.8 million people worldwide per year). Despite this, the apparently slow progress of infection and low parasitaemia levels in humans compared to those reported in Plasmodium falciparum have erroneously led to P. vivax infection being classified as benign. Added to this, the experimental challenges involved in culturing this parasite greatly hinder accumulating the biological, cellular and molecular knowledge necessary for developing effective control methods against P. vivax. It is known nowadays that unsuitable diagnosis, poor therapeutic management and/or delayed treatment can lead to relapses and severe disease states similar to those reported for P. falciparum malaria, thereby imposing challenges regarding the search for new, specific, alternative approaches to tackling this species. The present work has been focused on studying receptor-ligand and protein-protein interactions of P. vivax molecules located in intra-erythrocyte schizonts’ apical organelles regarding the need for identifying therapeutic targets against P. vivax. Protein interaction with human reticulocytes was characterised in P. vivax and PvRON2 ability to establish interactions with PvRON4, PvRON5 and PvAMA1 was evaluated, based on previous P. falciparum and Toxoplasma gondii studies, describing the functional importance of rhoptry neck proteins. Work began by using bioinformatics and experimental tools for predicting pvron4 and pvron5 genes in the P. vivax VCG-1 (Vivax Colombia Guaviare 1) strain’s genome and schizonts transcriptome. These two genes encode high molecular weight proteins which are expressed at schizonts’ apical poles and co-localise with proteins in the rhoptries. Such proteins were produced recombinantly and purified by affinity chromatography for evaluating PvRON2, PvRON4, PvRON5 and PvAMA1 ability to interact with receptors on human reticulocyte membrane. Recombinant PvRON5 bound to both CD71+ normocytes and reticulocytes, having a marked preference for human reticulocytes. PvAMA1 domains I and II (PvAMA-DI-DII), PvRON2 central region (PvRON2-RI) and PvRON4 carboxy-terminal region specifically interacted with CD71+CD45- reticulocytes. Competition studies with synthetic peptides covering recombinant protein sequences showed that PvAMA1-derived peptide 21270, PvRON4-derived 40305 and PvRON2-RI-derived 40595, were capable of inhibiting recombinant protein binding to CD71+CD45- reticulocytes, suggesting that these peptide sequences contained some of the evaluated proteins’ binding properties. The three peptides bound specifically and with high affinity to erythrocytes having higher (2%) binding percentages (obtained from specific binding curves), thereby allowing their classification as high erythrocyte binding capacity peptides (HABPs). PvAMA1 and PvRON4 binding to human erythrocytes was sensitive to erythrocytes treatment with different enzymes (trypsin, chymotrypsin and/or neuraminidase), suggesting the receptors’ protein type nature. These results highlighted the adhesin function of the proteins evaluated and revealed minimum host cell interaction regions suggesting these molecules’ active participation during P. vivax merozoite invasion of human reticulocytes (along with these proteins’ expression in intra-erythrocytic schizonts and location in apical organelles). Surface plasmon resonance was used for characterising PvRON2 interactions with PvRON4, PvRON5 and PvAMA1. This revealed that PvRON2-RI and carboxy-terminal regions (PvRON2-RII) specifically interacted and with great affinity with PvAMA1 domain II and III (PvAMA-DII-DIII) but with less affinity with PvAMA-DI-DII, PvRON4 and PvRON5. No significant differences were found in interaction association (Kon) or dissociation (Koff) rates or dissociation constant (kD) values when modifying some PvAMA1 residues reported as being critical in the P. falciparum RON2-AMA-1 interaction, suggesting that although conserved interactions between these parasites (Pv-Pf) have been observed, each parasite uses different regions to interact, thereby highlighting their ability to specialise or restrict themselves to invading a specific cell type and the need for designing specific control measures against P. vivax.
dc.description: La malaria es una de las enfermedades tropicales transmitidas por vectores más importantes a nivel mundial, donde Plasmodium vivax representa una de las especies más ampliamente distribuidas, afectando ~13.8 millones de personas por año. Pese a ello, el progreso aparentemente lento de la infección y los niveles bajos de parasitemia en el humano, comparados a lo reportado en Plasmodium falciparum, han llevado a clasificar a la infección por P. vivax erróneamente como benigna. Esto, sumado a los retos experimentales que trae consigo el cultivo de este parásito, obstaculizan en gran medida el conocimiento a nivel biológico, celular y molecular, necesario para el desarrollo de métodos de control efectivos contra P. vivax. Hoy en día, se conoce que un inadecuado diagnóstico, el mal manejo terapéutico y/o el retraso en el tratamiento, pueden llevar a recaídas y estados de enfermedad grave, similares a los reportados para la malaria producida por P. falciparum, lo que impone retos en la búsqueda de nuevas alternativas específicas contra esta especie. Teniendo en cuenta la necesidad de identificar posibles blancos terapéuticos contra P. vivax, este trabajo se enfocó en estudiar interacciones del tipo receptor-ligando y proteína-proteína de moléculas de P. vivax localizadas en los organelos apicales de esquizontes intraeritrocíticos. Basados en estudios previos en P. falciparum y Toxoplasma gondii, donde se describe la importancia funcional de las proteínas localizadas en el cuello de las roptrias (RONs) -2, -4 y 5 y del antígeno apical de membrana 1 (AMA1), se caracterizó en P. vivax la unión de cada una de estas proteínas con reticulocitos humanos y se evaluó la capacidad de PvRON2 para establecer interacciones con las proteínas PvRON4, PvRON5 y PvAMA1. Para esto, inicialmente se caracterizó mediante herramientas bioinformáticas y experimentales la presencia de los genes pvron4 y pvron5 en el genoma y transcriptoma de esquizontes de la cepa Vivax Colombia Guaviare 1 (VCG-1) de P. vivax. Estos dos genes codifican proteínas de alto peso molecular que se expresan en el polo apical de esquizontes y co-localizan con proteínas presentes en las roptrias. Para evaluar la capacidad de interacción de las proteínas PvRON2, PvRON4, PvRON5 y PvAMA1 con receptores sobre la membrana de reticulocitos humanos, todas ellas fueron producidas de forma recombinante y purificadas mediante cromatografía de afinidad. Se encontró que la proteína recombinante PvRON5 se unió tanto a normocitos como a reticulocitos CD71+, con una marcada preferencia por reticulocitos humanos. Por su parte, las proteínas recombinantes que incluyen los dominios I y II de PvAMA-1 (PvAMA-DI-DII), la región central de la proteína PvRON2 (PvRON2-RI) y la región carboxi-terminal de PvRON4, interactúan específicamente con reticulocitos CD71+CD45-. Los estudios de competencia de unión con péptidos sintéticos que cubren las secuencias de las proteínas recombinantes mostraron que los péptidos 21270 (derivado del DI de PvAMA1), 40305 (de PvRON4) y 40595 (de PvRON2-RI) fueron capaces de inhibir la unión de las proteínas recombinantes a reticulocitos CD71+CD45-, lo que sugiere que estas secuencias peptídicas contienen parte de las propiedades de unión de cada una de las proteínas de las que derivan. Los tres péptidos se unen específicamente y con alta afinidad a eritrocitos con porcentajes de unión mayores al 2% (obtenidas de las curvas de unión específica), permitiendo catalogarlos como péptidos con alta capacidad de unión a eritrocitos (HABPs, del inglés High Activity Binding Peptides). La unión de las proteínas PvAMA1 y PvRON4 a eritrocitos humanos fue sensible al tratamiento de los eritrocitos con diferentes enzimas (tripsina, quimotripsina y/o neuraminidasa), sugiriendo que la naturaleza de los receptores para estas proteínas es de tipo proteico. Estos resultados destacan la función de adhesina de las proteínas evaluadas y revelan las regiones mínimas de interacción con la célula hospedera, que sumado a la expresión de estas proteínas en esquizontes intraeritrocíticos y su localización en organelos apicales, sugieren una fuerte participación de estas moléculas durante el proceso de invasión de los merozoitos de P. vivax a reticulocitos humanos. Finalmente, mediante resonancia de plasmones de superficie, se caracterizaron las interacciones entre la proteína PvRON2 con las proteínas PvRON4, PvRON5 y PvAMA1. Los análisis revelaron que la región carboxi-terminal de la proteína PvRON2 (PvRON2-RII) y PvRON2-RI interactúan específicamente y con alta afinidad con el dominio II y III de PvAMA1 (PvAMA-DII-DIII) y con una menor afinidad con las proteínas PvAMA-DI-DII, PvRON4 y PvRON5. Al modificar algunos residuos de la proteína PvAMA1, reportados en P. falciparum como críticos en la interacción RON2-AMA1, no se encontraron diferencias importantes en los valores de las velocidades de asociación (Kon), disociación (Koff) y en la constante de disociación de la interacción (kD). Esto sugiere que, si bien existen interacciones proteína-proteína (IPP) conservadas entre estos parásitos (Pv-Pf), cada parásito utiliza distintas regiones de las proteínas para interactuar, lo que resalta su capacidad para especializarse o restringirse para invadir un tipo de célula específica y pone de manifiesto aún más la necesidad de diseñar medidas de control específicas contra P. vivax.
2018-01-01T00:00:00ZArévalo-Pinzón, GabrielaMalaria is one of the most important tropical diseases transmitted by vectors worldwide; Plasmodium vivax represents one of the most widely distributed species (affecting ~ 13.8 million people worldwide per year). Despite this, the apparently slow progress of infection and low parasitaemia levels in humans compared to those reported in Plasmodium falciparum have erroneously led to P. vivax infection being classified as benign. Added to this, the experimental challenges involved in culturing this parasite greatly hinder accumulating the biological, cellular and molecular knowledge necessary for developing effective control methods against P. vivax. It is known nowadays that unsuitable diagnosis, poor therapeutic management and/or delayed treatment can lead to relapses and severe disease states similar to those reported for P. falciparum malaria, thereby imposing challenges regarding the search for new, specific, alternative approaches to tackling this species. The present work has been focused on studying receptor-ligand and protein-protein interactions of P. vivax molecules located in intra-erythrocyte schizonts’ apical organelles regarding the need for identifying therapeutic targets against P. vivax. Protein interaction with human reticulocytes was characterised in P. vivax and PvRON2 ability to establish interactions with PvRON4, PvRON5 and PvAMA1 was evaluated, based on previous P. falciparum and Toxoplasma gondii studies, describing the functional importance of rhoptry neck proteins. Work began by using bioinformatics and experimental tools for predicting pvron4 and pvron5 genes in the P. vivax VCG-1 (Vivax Colombia Guaviare 1) strain’s genome and schizonts transcriptome. These two genes encode high molecular weight proteins which are expressed at schizonts’ apical poles and co-localise with proteins in the rhoptries. Such proteins were produced recombinantly and purified by affinity chromatography for evaluating PvRON2, PvRON4, PvRON5 and PvAMA1 ability to interact with receptors on human reticulocyte membrane. Recombinant PvRON5 bound to both CD71+ normocytes and reticulocytes, having a marked preference for human reticulocytes. PvAMA1 domains I and II (PvAMA-DI-DII), PvRON2 central region (PvRON2-RI) and PvRON4 carboxy-terminal region specifically interacted with CD71+CD45- reticulocytes. Competition studies with synthetic peptides covering recombinant protein sequences showed that PvAMA1-derived peptide 21270, PvRON4-derived 40305 and PvRON2-RI-derived 40595, were capable of inhibiting recombinant protein binding to CD71+CD45- reticulocytes, suggesting that these peptide sequences contained some of the evaluated proteins’ binding properties. The three peptides bound specifically and with high affinity to erythrocytes having higher (2%) binding percentages (obtained from specific binding curves), thereby allowing their classification as high erythrocyte binding capacity peptides (HABPs). PvAMA1 and PvRON4 binding to human erythrocytes was sensitive to erythrocytes treatment with different enzymes (trypsin, chymotrypsin and/or neuraminidase), suggesting the receptors’ protein type nature. These results highlighted the adhesin function of the proteins evaluated and revealed minimum host cell interaction regions suggesting these molecules’ active participation during P. vivax merozoite invasion of human reticulocytes (along with these proteins’ expression in intra-erythrocytic schizonts and location in apical organelles). Surface plasmon resonance was used for characterising PvRON2 interactions with PvRON4, PvRON5 and PvAMA1. This revealed that PvRON2-RI and carboxy-terminal regions (PvRON2-RII) specifically interacted and with great affinity with PvAMA1 domain II and III (PvAMA-DII-DIII) but with less affinity with PvAMA-DI-DII, PvRON4 and PvRON5. No significant differences were found in interaction association (Kon) or dissociation (Koff) rates or dissociation constant (kD) values when modifying some PvAMA1 residues reported as being critical in the P. falciparum RON2-AMA-1 interaction, suggesting that although conserved interactions between these parasites (Pv-Pf) have been observed, each parasite uses different regions to interact, thereby highlighting their ability to specialise or restrict themselves to invading a specific cell type and the need for designing specific control measures against P. vivax.Estudio de las modificaciones conformacionales y su influencia en la actividad inmunológica de péptidos derivados de las familias de las proteínas spect-1 y -2, siap-1 y -2 de plasmodium falciparumAlba Sandoval, Marthahttps://repository.urosario.edu.co/handle/10336/124382021-06-03T05:48:02Z2015-01-01T00:00:00Zdc.title: Estudio de las modificaciones conformacionales y su influencia en la actividad inmunológica de péptidos derivados de las familias de las proteínas spect-1 y -2, siap-1 y -2 de plasmodium falciparum
dc.description.abstract: SPECT-1 and -2 and SIAP-1 and -2 are Plasmodium falciparum sporozoite proteins causing the most aggressive form of malaria in humans. These proteins are involved in the parasite’s human host cell traversal and the invasion of hepatocytes, making them attractive targets for study. Conserved peptides having high binding (cHABPs) affinity for HeLa and HepG2 cells derived from these molecules are not immunogenic because they cannot induce an immune response but are key for the parasite to play a significant role during the infection of a human host.
The present work reports the finding that some SPECT-1 and -2 protein cHABPs are possibly involved in binding and pore formation on host cell membrane, thereby helping sporozoites regarding host cell traversal.
New peptides were obtained by replacing critical amino acids by other residues having similar molecular mass but different polarity aimed at changing SPECT-1 and -2 and SIAP-1 and -2 derived-cHABPs’ immunological behaviour. CD or 1H-NMR determined that such modifications promoted changes in modified peptide (mHABP) secondary structure compared to that of native cHABPs. These mHABPs’ immunological behaviour also became inverted, making them become antibody-inducing, immunogenic peptides. FIDIC’s search for a multi-stage, multi-epitope, chemically-synthesised anti-malarial vaccine has led to establishing a relationship between mHABP structure and their immunological activity, meaning that some mHABPs studied here could be candidates for such vaccine.
dc.description: SPECT-1 y -2 y SIAP-1 y -2 son proteínas pertenecientes al esporozoíto de Plasmodium falciparum causante de la malaria más agresiva en los humanos. Estas proteínas están involucradas en el paso del parásito a través de las células del hospedero humano y en la invasión del hepatocito, haciéndolas blancos atractivos para ser estudiadas. Péptidos conservados de alta capacidad de unión (cHABPs) a células HeLa y HepG2 derivados de estas moléculas son no inmunogénicos porque son incapaces de generar una respuesta inmunitaria, pero son claves para el parásito porque cumplen una función importante durante la infección del hospedero humano. En este trabajo se encontró que algunos cHABPs pertenecientes a las proteínas SPECT-1 y -2, están posiblemente involucrados con la unión y formación de poros sobre la membrana de las células hospederas, ayudando al esporozoíto a abrirse paso través de las células del hospedero.
Por otro lado, con el fin de cambiar el comportamiento inmunológico de cHABPs derivados de SPECT-1 y -2 y SIAP-1 y -2, se obtuvieron nuevos péptidos mediante el reemplazo de aminoácidos críticos por otros residuos cuya masa molecular sea similar, pero diferente en su polaridad. En este trabajo se reporta que dichas modificaciones promovieron cambios en la estructura secundaria (determinada por DC o 1H-RMN) de los péptidos modificados (mHABPs) cuando se comparó con la estructura de los cHABPs nativos; adicionalmente, estos mHABPs invirtieron su comportamiento inmunológico convirtiéndose en péptidos inmunogénicos inductores de anticuerpos. Lo que permite establecer la existencia de una relación entre la estructura que adoptan estos mHABPs con su actividad inmunológica.
Además, algunos de los mHABPs estudiados aquí, pueden ser candidatos a ser incluidos en la vacuna contra la malaria químicamente sintetizada multi-epitope y multi-estadio que se está desarrollando en la Fundación Instituto de Inmunología de Colombia (FIDIC).
2015-01-01T00:00:00ZAlba Sandoval, MarthaSPECT-1 and -2 and SIAP-1 and -2 are Plasmodium falciparum sporozoite proteins causing the most aggressive form of malaria in humans. These proteins are involved in the parasite’s human host cell traversal and the invasion of hepatocytes, making them attractive targets for study. Conserved peptides having high binding (cHABPs) affinity for HeLa and HepG2 cells derived from these molecules are not immunogenic because they cannot induce an immune response but are key for the parasite to play a significant role during the infection of a human host.
The present work reports the finding that some SPECT-1 and -2 protein cHABPs are possibly involved in binding and pore formation on host cell membrane, thereby helping sporozoites regarding host cell traversal.
New peptides were obtained by replacing critical amino acids by other residues having similar molecular mass but different polarity aimed at changing SPECT-1 and -2 and SIAP-1 and -2 derived-cHABPs’ immunological behaviour. CD or 1H-NMR determined that such modifications promoted changes in modified peptide (mHABP) secondary structure compared to that of native cHABPs. These mHABPs’ immunological behaviour also became inverted, making them become antibody-inducing, immunogenic peptides. FIDIC’s search for a multi-stage, multi-epitope, chemically-synthesised anti-malarial vaccine has led to establishing a relationship between mHABP structure and their immunological activity, meaning that some mHABPs studied here could be candidates for such vaccine.