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The role of sympathetic nervous system activity and inflammation in arterial remodeling in age-dependent hypertensionAmraei, Razie 02 November 2023 (has links)
Hypertension is a major public health issue impacting one in two adults in the United States and accounts for 10 million deaths each year worldwide. It is a leading risk factor for multiple diseases, including stroke, myocardial infarction, chronic kidney disease, and peripheral artery disease. The prevalence of hypertension increases with age in both sexes. In addition, aging is associated with significantly higher hypertension-related morbidity and mortality and insufficient control of high blood pressure. Critically, menopause is linked to a 2-fold increase in hypertension risk and premenopausal women have lower hypertension rates compared to men of similar age.
Increased sympathetic nervous system activity, inflammation, and remodeling of large arteries like the aorta have been implicated in both normal aging and the pathophysiology of hypertension. Despite extensive hypertension research and the rapidly growing aging population, only 4% of hypertension studies focus on aging. Increased molecular insight into the mechanisms mediating arterial remodeling in age-dependent hypertension could uncover potential therapeutics for more targeted treatment of hypertension.
In this thesis, Sprague Dawley rats were used in the models of normal aging and norepinephrine-induced hypertension to investigate the complex interplay between aging, sympathetic nervous system activity, and inflammation in arterial remodeling in age-dependent hypertension and potential sex differences. Our key findings from the abdominal aorta and renal artery are (1) Sex-dependent changes in the phosphorylation of c-Src kinase and ERK1/2, and expression of caveolin-1, (2) Altered expression of alpha-SMA and MHY-11, (3) Partial recapitulation of aged hypertensive phenotype in the abdominal aorta of young male rats following NE-infusion. Our proteomic and phosphoproteomic analyses of the aorta of young normotensive and aged hypertensive male rats have identified 58 differentially expressed proteins and 39 differentially phosphorylated proteins. Proteome analysis further revealed that the proteins in extracellular matrix, actin cytoskeleton and inflammatory pathways were the top affected proteins or pathways. Moreover, in phosphoproteome analysis, major differences were found in neurons, synapse structures, vascular smooth muscle cells, and focal adhesions. Notably, approximately two-thirds of differentially phosphorylated proteins (22 out of 39) were found to be at neurons and synapses. In the assessment of inflammatory mediators, we found that increases in multiple homeostatic cytokines including, CCL21, MMP2, and osteopontin were associated with the aortic remodeling in age-dependent hypertension. Collectively, these results support a model in which aging, increased sympathetic nervous system activity, and inflammation contribute to arterial remodeling in age-dependent hypertension. / 2024-11-02T00:00:00Z
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Phosphoproteomic strategies for protein functional characterization of phosphatases and kinasesAndrew G. DeMarco (17103610) 06 April 2024 (has links)
<p dir="ltr">Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here we described two methods for the LC-MS/MS-based characterization of protein phosphatases and kinases. The first is an <i>in-vitro</i> method designed to probe the inherent substrate specificity of kinase or phosphatases. This method utilizes an enzyme reaction with synthetic peptides, serving served as substrate proxies, coupled with LC-MS/MS for rapid, accurate high-throughput quantification of the specificity constant (<i>k</i><sub><em>cat</em></sub><i>/K</i><sub><em>M</em></sub>) for each substrate in the reaction and amino acid preference in the enzyme active site, providing insight into their cellular roles. The second couple’s auxin-inducible degradation system (AID) with phosphoproteomics for protein functional characterization. AID is a surrogate for specific chemical inhibition, which minimizes non-specific effects associated with long-term target perturbation. Using this system, we demonstrate-PP2A in complex with its B-subunit Rox Three Suppressor 1 (PP2A<sup>Rts1</sup>) contributes to the phosphoregulation of a conserved fungal-specific membrane protein complex called the eisosome. By maintaining eisosomes in their hypophosphorylated state, PP2A<sup>Rts1</sup> aids fungal cells in preserving metabolic homeostasis. This work demonstrates the power of mass spectrometry as a critical tool for protein functional characterization.</p>
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Towards Curing an Alzheimer’s Mouse ModelBlasco Tavares Pereira Lopes, Filipa 27 January 2023 (has links)
No description available.
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Identification and Characterization of Interactors of Plasmodium falciparum PfPK6, An Atypical Protein KinaseCummins, Andi J 01 January 2016 (has links)
Plasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics that target the parasite, but show low toxicity in the human host. Protein kinases, because of their integral roles in cell signaling networks, are considered to be attractive drug targets. Cyclin dependent kinases, or CDKs, and Mitogen-Activated Protein kinases, or MAPKs, are common to eukaryotes and regulate cellular processes of growth and proliferation. Plasmodium falciparum Protein Kinase 6, or PfPK6, is an atypical protein kinase that shows similarities to both MAPKs and CDKs. PfPK6 is expected to have an important role in the intraerythrocytic cell cycle progression and growth in the malaria organism, as it has been found to be essential in the parasite.
In order to better understand the function of PfPK6 within Plasmodium, we have identified serveral potential substrates and interactors of the kinase using co-immunoprecipitation with an HA epitope-tagged cell line of PfPK6, as well as phosphoproteomic analysis. These methods resulted identification of 15 novel protein interactors, with 4 being studied for further investigation, and 45 putative substrates after strict peptide filtering, five of which are used in this study. In order to verify putative substrates and interactors, both in vitro and in vivo methods were used. In vitro kinase assays using GST-PfPK6 with 5 recombinant substrates confirmed direct phosphorylation of two novel substrates: MAL7P1.38, a regulator of chromosome condensation, and PF10_0047, a putative RNA binding protein. After attempts to generate bacterial constructs of several putative interactors and a global failure of a usable amount of protein to express under IPTG induction conditions, an alternative form of expression using a cell free Transcription and Translation reaction (TNT) with Wheat Germ Extract was used to generate radiolabeled PF11_0154, PFF0625w, and PF11_0305. Pull down analysis using GST-PfPK6 showed the kinase's ability to "pull" the interactors out of solution, confirming the interactions defined by the initial epitope tagged Co-Immunoprecipitation. Additionally, for in vivo analysis, parasites were transfected with RFP- PFF_0695w, an uncharacterized Plasmodium protein, in order to cellular localization of this interactors. Immunofluorescence assays of transfected lines showed punctate forms of PFF_0695w in the host erythrocyte in the late trophozoite and schizont stages of the parasite development, suggesting this interactor is a previously undiscovered protein in the Plasmodium secretome. The research presented here is an initial step to defining the interactome of PfPK6.
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The Cell Membrane Proteome of the SKBR3/HER2+ Cells and Implications for Cancer Targeted TherapiesKarcini, Arba 02 June 2023 (has links)
Breast cancer is the second most common type of cancer among women in the US and the second leading cause of cancer death. HER2+ breast cancers represent ~20% of all cancer types, are highly invasive, and can be treated by using targeted therapies against the HER2 receptor. However, these therapies are challenged by the development of drug resistance, often induced by the presence of mutations in the cell-membrane proteins and receptors and/or by alternative signaling pathways that cross-talk with- or transactivate HER2+ triggered signaling. This study was aimed at investigating the cell membrane proteome of SKBR3 cells, representative of HER2+ breast cancers, and the signaling landscape and cellular responses elicited by the cell membrane receptors when the cells are stimulated with either growth factors or therapeutic drugs. It was hypothesized that the identification of a broad range of cell membrane proteins with roles in cancer progression and signaling crosstalk will lead to a more comprehensive understanding of the biological processes that sustain the proliferation of cancer cells, and will guide the selection of more efficient drug targets. The project was conceptualized in three stages: (1) profiling the cell membrane proteins of SKBR3 cells, (2) determining the functional role of the detected cell membrane proteins in the context of cancer hallmarks and exploring their mutational profile, and (3) analyzing the cellular events that occur in response to treatment with a single therapeutic agent or a combination of drugs. Mass spectrometry technologies were used for performing proteomic and phosphoproteomic profiling of SKBR3 cells, detecting changes in the abundance of the detected proteins, and identifying the presence of mutations in the cell membrane proteins. Orthogonal enrichment methods were developed for profiling the low-abundance cell membrane proteins, for generating a rich landscape of cell membrane receptors with various functional roles and relevance to the cancer hallmarks, and for enabling the detection of potentially new drivers of aberrant proliferation. The analysis of serum-starved, stimulated (with growth factors), or inhibited (with kinase inhibitors) cells revealed alternative protein players and crosstalk activities that determine the fate of cells, and that may fuel the development of resistance to treatment with drugs. The proteome profiles that were generated in this project expand the opportunities for targeting cancer-relevant processes beyond proliferation, which is commonly attempted, broadening the landscape to also include apoptosis, invasion, and metastasis. Altogether, the findings that emerged from this work will lay the ground for future studies that aim at developing more complex and effective targeted cancer treatment approaches. / Doctor of Philosophy / Breast cancer is one of the most common cancers among women in the US and the second major contributor to cancer-related deaths. Several therapies that have been developed for the treatment of cancer target the HER2 receptor, which is overexpressed in ~20% of breast cancers and results in a highly invasive cancer phenotype. However, most patients receiving these therapies observe cancer reoccurrence within a year due to the development of resistance to the therapeutic drug. The current challenge stands in identifying novel protein targets, and in developing new therapies that can be used in combination with the existing approaches to eradicate cancer. Research has indicated that proteins located at the cell membrane play crucial roles in cancer progression and invasion due to their involvement in cell response to stimuli and in initiating signaling cascades within the cell. Knowledge about the cell membrane proteins of HER2+ breast cancer cells is limited due to the challenges associated with their isolation. Therefore, this project was aimed at profiling the cell membrane proteins of HER2+ breast cancer cells, and their intra-cellular signaling activity, to provide insights into the behavior of these cells and to support the identification of potentially novel drug targets. The three objectives of the work were to (1) isolate the cell membrane proteins through various approaches using cell culture conditions that would encourage or discourage cancer cell growth, (2) identify the cancer-relevant signaling pathways and processes represented by the detected cell membrane proteins, and (3) investigate the behavior of cancer cells when treated with drugs. To approach these objectives, a powerful analytical technology, called mass spectrometry, was utilized. Mass spectrometry can accurately and simultaneously detect the presence of the proteins in a biological sample. Our study identified cell membrane proteins that are involved in cancer progression through various signaling pathways, and how these proteins interact with each other to drive the behavior of cells. The study also provided insights into how cancer cells respond when they are treated with various drugs, uncovering to the scientific community a variety of proteins with potential therapeutic value. Lastly, this study sheds light on the complex biology of breast cancer and highlights the importance of continued research to develop more effective treatments.
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Régulation des aquaporines et réponse des racines d'Arabidopsis thaliana à des stimuli abiotiques et nutritionnels. / Regulation of aquaporins and response of Arabidopsis thaliana roots to abiotic and nutritional stimuli.Di Pietro, Magali 13 December 2011 (has links)
La conductivité hydraulique racinaire (Lpr) traduit la facilité du passage de l'eau au travers des racines. Ce paramètre, majoritairement contrôlé par l'activité de canaux hydriques membranaires (aquaporines), est modulable par diverses contraintes environnementales. Ce travail a permis de caractériser, sur un même organisme (Arabidopsis), les effets d'un ensemble de contraintes abiotiques et biotiques, représentatives de situations environnementales, sur la Lpr. Alors que la flagelline n'affecte pas la Lpr, les contraintes osmotiques (NaCl, mannitol), oxydantes (H2O2, NO) et nutritionnelles (carence en phosphate, en nitrate, culture des plantes en nuit prolongée) inhibent la Lpr. Par contre, la réalimentation en phosphate ainsi que l'addition de saccharose à des plantes cultivées en nuit prolongée stimulent la Lpr. Une approche phosphoprotéomique quantitative, basée sur l'analyse par MS de protéines microsomales racinaires purifiées à partir de plantes cultivées dans trois de ces contextes (NaCl, NO, phosphate) a permis de quantifier les variations d'abondance de l'ensemble des aquaporines racinaires ainsi que de leur état de MPT. D'un point de vue qualitatif, 22 aquaporines ont été identifiées dans la racine ainsi que plusieurs types de MPTs, incluant des nouveaux sites de phosphorylation (7), de méthylation (13 à 15), de formylation (4) et de déamidation (25 à 26). D'un point de vue quantitatif, cette étude a permis de conclure que les observations réalisées au niveau de la Lpr sont la résultante de mécanismes multifactoriels incluant l'état de phosphorylation des trois sites de l'extrémité C terminale de PIP2;1/2;2/2;3, l'état de phosphorylation de l'extrémité N terminale de PIP1;1/1;2, ainsi que les aquaporines TIPs. Ce travail permet donc de proposer de nouveaux mécanismes moléculaires impliqués dans la régulation de la Lpr en réponse à des contraintes de l'environnement / The water uptake capacity of plant roots (root hydraulic conductivity, Lpr) is mainly determined by water channels (aquaporins) and is modulated by environmental constraints. The present work characterised, in a unique organism (Arabidopsis), effects on Lpr of abiotic and biotic constraints representative of environmental situations. Whereas flagelline does not affect Lpr, osmotic (NaCl or mannitol), oxidative (H2O2 or NO) and nutritional (phosphate or nitrate starvation, prolonged night) stimuli inhibit Lpr. However, phosphate and sucrose resupply stimulate Lpr. A phosphoproteomics approach based on MS analysis of microsomal proteins extracted from roots of plants cultivated in different environmental constraints (NaCl, NO,phosphate starvation and resupply) allowed to quantify variations of abundance of roots aquaporins and of their PTMs. As a qualitative point of view, 22 aquaporins were identified in roots as well as several post-translational modifications including new sites of phosphorylation (7), methylation (13 to 15), formylation (4) and deamidation (25 to 26). From a quantitative point of view, the present work drove to the conclusion that the modulations of Lpr result from multifactorial mechanisms including the phosphorylation status of the C terminal part of PIP2;1/2;2/2;3 and of the N-terminal part of PIP1;1/1;2 and TIP aquaporins. This study proposes new molecular mechanisms implicated in Lpr regulation in response to various environmental situations.
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Integrated strategies to develop post-translationally modified proteins in extracellular vesicles as candidate disease markersHillary Andaluz Aguilar (9745967) 15 December 2020 (has links)
Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing proteins and
nucleic acid cargo. These vesicles are released by almost all cell types and provide an effective
and ubiquitous path for intercellular communication and transmission of pathogenic and signaling
molecules among cells. Research into potential biomarkers isolated from EV has been propelled
by the development of methods and tools to acquire them by minimally and non-invasive means,
which reinforces their great diagnostic potential. In the context of cancer, this opens the door to
apply EV based liquid biopsy for early detection prior to alternate, more prevailing diagnostic tools
like imaging studies. In autoimmune diseases, EVs play a crucial role in immune responses and as
immunomodulatory agents as they can modulate the function of a wide variety of immune cells,
especially in antigen-presenting cells (APCs). Several efforts have been made to study EVs and
their cargo in numerous disease models, but very few in autoimmunity. Autoimmune diseases are
chronic, have been underexplored especially in the omics area, and their diagnosis and treatment
rely on traditional therapy. Therefore, there is a need for efficient methods to elucidate biomarkers
that could provide additional layers of information for treatment, diagnosis, and prognosis.
Additionally, protein post-translational modifications (PTMs), such as phosphorylation,
glycosylation, and acetylation, are involved in multiple essential cellular processes and represent
an important mechanism of regulation for cellular physiological functions, leading to the
development of effective and targeted therapeutics. Discovery and profiling PTMs have
established the relevance of PTMs in EVs and associated EV functions and novel applications.
This dissertation proposes integrated proteomic strategies to efficiently isolate and analyze
EVs in human plasma from different types of pathologies like cancer and autoimmune diseases.
The main focus is the development of the platforms, to not only isolate the proteome from EVs,
but also PTMs including phosphorylation, glycosylation and acetylation, simultaneously. Chapter
one, which is the core of this dissertation, describes the platform to sequentially isolate and analyze
the EV proteome, phosphoproteome and glycoproteome from human plasma. Chapters two and
three focus on the ongoing application of this platform with slight modifications into different
disease models, in this case breast cancer subtypes and autoimmune diseases.
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Phosphoproteomics analysis of normal and malignant granulocyte-colony stimulating factor receptor signalingDwivedi, Pankaj 02 October 2018 (has links)
No description available.
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Úloha fosforylace proteinů v progamické fázi vývoje samčího gametofytu tabáku / The role of protein phosphorylation during progamic phase of tobacco male gametophyte developmentFíla, Jan January 2016 (has links)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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Caracterização do papel das proteínas quinases C (PKCs) na proliferação e auto-renovação das células tronco embrionárias murinas / Characterization of the role of protein kinases C (PKC) in proliferation and self-renewal of murine embryonic stem cellsGaravello, Nicole Milaré 04 August 2011 (has links)
Células tronco embrionárias (CTE) são capazes de proliferar indefinidamente mantendo a sua pluripotência, isto é, a capacidade de se diferenciar em diversos tipos celulares perante estímulos adequados. Esse potencial tem sido intensamente estudado, de modo a permitir a utilização dessas células em terapias de reposição celular. Trabalhos anteriores demonstraram que as proteínas kinases C (PKC) são importantes moduladores moleculares de cascatas de sinalização que levam ao processo de proliferação e auto-renovação das CTE. Porém o papel exato das diferentes isoenzimas das PKCs ainda não foi elucidado. Isso ocorre porque a família das PKCs é composta por pelo menos dez isoenzimas e apenas, recentemente, desenvolveram-se moduladores específicos para as diferentes isoenzimas, o que permitirá estudar o papel específico dessas quinases. No presente trabalho verificamos que a ativação da PKCδ induziu a proliferação de CTE indiferenciadas sem induzir a diferenciação das mesmas. Para tentar elucidar as vias de sinalização mediadas pela PKCδ que levam à proliferação das CTE indiferenciadas realizamos estudos de fosfoproteômica o que possibilitou a identificação de potenciais alvos diretos e indiretos da PKCδ. Dentre os alvos identificados foram encontradas diversas proteínas relacionadas com proliferação, transcrição, tradução e resposta ao stress (chaperonas), contribuindo para a hipótese de que a ativação da PKCδ leva à proliferação das CTE indiferenciadas. Em diversos sistemas, a ativação da PKCδ leva à ativação da MAPK, em particular das ERK1/ 2, sendo essa via capaz de induzir a proliferação de diversas linhagens celulares. Identificamos diversas proteínas alvos da PKCδ, que interagem também com componentes da via das MAPKs. Desta forma, verificamos a influência da ativação da PKCδ na via das MAPKs. De fato, a ativação da PKCδ na linhagem de CTE murinas indiferenciadas, E14TG2a, ativou a MEK, ERK1/ 2 e o fator de transcrição ELK-1. Como estudos anteriores demonstraram que a inibição da ERK1/ 2 mantém CTE indiferenciadas e que a ativação desta via poderia levar à diferenciação de CTE, investigamos a cinética de ativação da ERK pela PKCδ. Demonstramos que a ativação da ERK pela PKCδ se da de modo transiente e que apesar da PKCδ não translocar para o núcleo, sua ativação induz a fosforilação e translocação nuclear da ERK, que atuará na fosforilação do fator de transcrição ELK-1. Desta forma, concluímos que a PKCδ induz a proliferação das CTE murinas indiferenciadas ativando transitoriamente a via das ERK1/ 2, que translocam para o núcleo fosforilando fatores de transcrição como a ELK1 e levando possivelmente ao aumento de proliferação dessas células. A ativação transiente das ERK1/ 2 pela PKCδ é importante para a auto-renovação das CTE. / Embryonic stem cells (ESC) are able of proliferating indefinitely maintaining their pluripotency, which is the capability to differentiate in different cell types upon appropriate stimuli. Pluripotency has been intensely investigated in order to allow the use of these cells in cellular replacement therapies. Previous work has demonstrated that the serine/ threonine kinases, such as, Protein kinases C (PKC) are important modulators of signaling cascades that lead to the process of proliferation and self-renewal of ESC. However, the exact role of the different PKC isoenzymes still remains to be elucidated. Due to the fact that the PKC family is composed of at least ten different isoenzymes and only recently isoenzyme specific modulators have been developed, which now allows the elucidation of these kinases roles. In the present work we verified that activation of PKCδ induced undifferentiated ESC have their proliferation rate increased. Trying to elucidate the signaling pathways mediated by PKCδ that lead to the proliferation increase we performed phosphoproteomic studies to identify potential PKCδ targets. Between the targets identified we found several proteins related with proliferation, protein transcription, translation and stress response (chaperones). These targets contributed to the hypothesis that PKCδ activation leads to undifferentiated ESC proliferation. In different cell lines, PKCδ activation leads to MAPK activation, through ERK1/ 2 activation, which are frequently involved with cellular proliferation. We also identified several targets of PKCδ that Interact with several components of MAPK`s signaling cascade. PKCδ activation in murine undifferentiated ESC line, E14TG2a, led to MEK, ERK1/ 2 and the transcription factor Elk-1 activation. Some articles demonstrate that the inhibition of ERK1/2 are responsible to maintains ESC undifferentiated and that it`s activation could lead to ESC differentiation. Analysing the kinetics of ERK activation in the ESC by PKCδ, we show that ERK activation was transient and despite the fact that PKCδ does not translocated to the nucleus upon activation, but induces ERK activation and it`s nuclear translocation, where ERK could phosphorylate the transcription factor Elk-1. In conclusion PKCδ induces undifferentiated murine ESC proliferation increase by a transient ERK activation and it`s nuclear translocation.
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