Analýza lysosomů Trichomonas vaginalis

(CZECH) Lysozomy představují centrální degradační kompartment eukaryotních buněk. Tyto organely s kyselým pH a řadou kyselých hydroláz jsou určeny k degradaci a recyklaci materiálu pro buněčnou homeostázu a výživu. Rozsáhlé studie savčích lysozomů odhalily dlouhý seznam lysozomálních proteinů. Jak lze očekávat, největší podskupinu tvoří hydrolázy, avšak funkce většiny z nich zůstává nejasná. O biogenezi a funkci lysozomů parazitických protistů je známo jen málo a ještě méně o jejich roli v sekreci. Cílem této studie bylo objasnit složení (fago-)lysozomálního proteomu lidského parazita Trichomonas vaginalis a jeho zapojení do sekrece hydroláz. Naše studie odhalili, že lysozomální proteom zahrnuje 462 proteinů ve 21 funkčních třídách. Hydrolázy představovaly největší funkční třídu a zahrnovaly proteázy, lipázy, fosfatázy a glykosidázy. Identifikace velkého souboru proteinů zapojených do vezikulárního transportu a přestavbě cytoskeletu ukazuje, že fagolysozomální kompartment je velmi dynamickou strukturou. Několik hydroláz jsme také identifikovali v sekretomu T. vaginalis, včetně cysteinové proteázy TvCP2, avšak způsob jejich sekrece byl nejasný. Studium lysosomů odhalilo, že k sekreci TvCP2 dochází spíše prostřednictvím lysozomů než klasickou sekreční cestou. Dále jsme ukázali, že cysteinová proteáza...Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode...Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Roundtable: Affordances, Diversity, and Inclusion on Dating Apps - A Dialogue between Sociologists and Media Studies Researchers about ‘Hinge’

This roundtable paper is part of the project ‘Digitized Love and Intimacy on Hinge.’ It aims to investigate how digital dating apps reconfigure cultural attitudes to love and intimacy and, conversely, how said attitudes influence digital dating practices. The conversation is informed by (n)ethnographic usage of the app. As algorithms and affordances of dating applications can implicitly or explicitly privilege certain groups of users and exclude others, this conversation mainly aims to make sense of how Hinge’s interface – or ‘affordances’– facilitates the dating process and how inclusive and diverse the application’s affordances are. We discuss that there is a contradiction between what Hinge portrays itself to be and what it practically ends up being, partly because of its affordances. This roundtable highlights the need to study affordances as relational technologies and to take the perceptions, ideas, and interpretations of users seriously alongside the actual features and designs offered by applications

Revisiting regulation of potassium homeostasis in Escherichia coli:the connection to phosphate limitation

Two-component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K+ homeostasis. In Escherichia coli, the histidine kinase KdpD senses K+ availability, whereas the response regulator KdpE activates synthesis of the high-affinity K+ uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate-responsive two-component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two-hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross-regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K+ and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K+ and phosphate homeostasis and provides a mechanism for fine-tuning of the balance between positively and negatively charged ions in the bacterial cell

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

Analysis of lysosomes of Trichomonas vaginalis

Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode..

Turning the Noose that Binds into a Rope to Climb: A Textual Search for Rhetorical and Linguistic Gender-markings in Speech Samples of Three Contemporary Female Orators

Feminine communication styles have been the subject of active investigation since Robin Tolmach Lakoff’s 1975 publication of Language and Woman’s Place. This dissertation adds to that body of research, using both Lakoff’s linguistic markers and the rhetorical concept of the double-bind as discussed by Kathleen Hall Jamieson in “Beyond the Double Bind.” The women selected for this study include Nancy Pelosi; Speaker of the United States House of Representatives, Katharine Jefferts Schori; who serves as Presiding Bishop of the Episcopalian Church-USA; and Ruth Bader Ginsburg, a Justice of the United States Supreme Court. Three genre-specific speech samples from each of the rhetors (the acceptance speech, the standard professional speech, and the interview) are analyzed using Lakoff’s markers and Jamieson’s concept of the double-bind. Using this research structure, the speeches are examined for both stylistic and substantive language use which might be understood as “feminine.” The dissertation concludes with a summary of the findings, including tables which demonstrate the extent of the usage of Lakoff’s markers by genre and by rhetor, and commentary on the way that genre influenced the rhetors’ usage of both linguistic and rhetorical strategies. Interestingly, genre appeared to be more of a deciding factor in the study than was originally anticipated, and this provides a powerful incentive for further research. Finally, I argue for the inclusion of these orators within the rhetorical canon, for we cannot attempt to re-gender the rhetorical tradition and claim to support inclusive rhetorical practices if we do not recognize the contemporary women whose voices are shaping and re-shaping the way rhetoric is applied in the public sphere

A Place Apart: The Role of Nostalgia in a Detached Community

This work investigated the power of nostalgia in perpetuating rituals and folkways in detached communities. The focus of this study was Camp Luther, a family camp operated under the auspices of Lutheran Outdoor Ministries. Over the summer of 2010, approximately sixty interviews were conducted at the camp, and qualitative data from these interviews was used to formulate ideas and theoretical underpinnings for a concept the author calls the nostalgic reflex. These interviews were transcribed and then coded to indicate the manner in which nostalgia was driving the thought processes of the interview subjects. Quotations from the interviews were used throughout the document to support the theory of the nostalgic reflex. The interview data was rich with language and notions that indicated the level to which adherents of the camp were under the influence of the nostalgic reflex insofar as their devotion to the ethos of the camp\u27s character was concerned. One of the most interesting data points was people\u27s willingness to raise a large sum of money to preserve the lakefront of the camp, giving credence to the theory of solastalgia as coined by Glenn Albrecht. The language of the interview subjects also dovetailed neatly into Diane Barthel\u27s tenets of the Staged Symbolic Community. Also, the work borrowed from Edwin Friedman\u27s study of homeostasis and emotional triangles in his germinal work Generation to Generation. The study also parsed the differences between Svetlana Boym\u27s restorative and reflective nostalgia, especially in maintenance of the hetero-normative male dominated status quo at the camp. The work concludes that the academy has a somewhat overt bias against nostalgia. It noted with irony how it is ultimately nostalgia that undergirds the structure of the academy itself in the postmodern era. Further studies were discussed in the conclusion, inviting more discourse on the topic, especially in gender and ethnic studies. Ultimately, the work added to the ongoing influences of Harper, Davis, Lowenthal, Boym, and Wilson on the power of nostalgia. From the conclusion: So the nostalgic reflex is there to remind us in times of joy and adversity of those things which matter most. Like any faculty, it can be used for good or ill, depending on the motives of one who wields it. It remains within all of us. It is triggered on a daily basis, and its presence in our cognitive processes is often unseen, yet influential. I know, on a purely cognitive level, that Camp Luther is a collection of mildewed cabins, bland food, with a panoramic vista that only hints at the greater grandeur of an ocean view. This is what the photographs show and the palate recalls. The narrative of camp as polished through the lens of nostalgia, however, breathes magic into my cognition, and my desire for its luminosity trumps my rationalism every time. I only hope that others feel this quickening as I do, not necessarily for camp per se, but for something that adds value to their existence—a sense of Harper\u27s presence, even if that translates into a temporary escape from ennui and malaise

A Place Apart: The Role of Nostalgia in a Detached Community

This work investigated the power of nostalgia in perpetuating rituals and folkways in detached communities. The focus of this study was Camp Luther, a family camp operated under the auspices of Lutheran Outdoor Ministries. Over the summer of 2010, approximately sixty interviews were conducted at the camp, and qualitative data from these interviews was used to formulate ideas and theoretical underpinnings for a concept the author calls the nostalgic reflex. These interviews were transcribed and then coded to indicate the manner in which nostalgia was driving the thought processes of the interview subjects. Quotations from the interviews were used throughout the document to support the theory of the nostalgic reflex. The interview data was rich with language and notions that indicated the level to which adherents of the camp were under the influence of the nostalgic reflex insofar as their devotion to the ethos of the camp\u27s character was concerned. One of the most interesting data points was people\u27s willingness to raise a large sum of money to preserve the lakefront of the camp, giving credence to the theory of solastalgia as coined by Glenn Albrecht. The language of the interview subjects also dovetailed neatly into Diane Barthel\u27s tenets of the Staged Symbolic Community. Also, the work borrowed from Edwin Friedman\u27s study of homeostasis and emotional triangles in his germinal work Generation to Generation. The study also parsed the differences between Svetlana Boym\u27s restorative and reflective nostalgia, especially in maintenance of the hetero-normative male dominated status quo at the camp. The work concludes that the academy has a somewhat overt bias against nostalgia. It noted with irony how it is ultimately nostalgia that undergirds the structure of the academy itself in the postmodern era. Further studies were discussed in the conclusion, inviting more discourse on the topic, especially in gender and ethnic studies. Ultimately, the work added to the ongoing influences of Harper, Davis, Lowenthal, Boym, and Wilson on the power of nostalgia. From the conclusion: So the nostalgic reflex is there to remind us in times of joy and adversity of those things which matter most. Like any faculty, it can be used for good or ill, depending on the motives of one who wields it. It remains within all of us. It is triggered on a daily basis, and its presence in our cognitive processes is often unseen, yet influential. I know, on a purely cognitive level, that Camp Luther is a collection of mildewed cabins, bland food, with a panoramic vista that only hints at the greater grandeur of an ocean view. This is what the photographs show and the palate recalls. The narrative of camp as polished through the lens of nostalgia, however, breathes magic into my cognition, and my desire for its luminosity trumps my rationalism every time. I only hope that others feel this quickening as I do, not necessarily for camp per se, but for something that adds value to their existence—a sense of Harper\u27s presence, even if that translates into a temporary escape from ennui and malaise

Reduction of Turgor Is Not the Stimulus for the Sensor Kinase KdpD of Escherichia coli▿

Stimulus perception by the KdpD/KdpE two-component system of Escherichia coli is still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting in kdpFABC expression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of the kdpFABC operon revealed that the system responds very effectively to K+-limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K+, Na+, glutamate, proline, glycine, trehalose, putrescine, and spermidine under K+-limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K+-limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K+ concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K+ concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD