211 research outputs found

    3D Architectural Analysis of Neurons, Astrocytes, Vasculature & Nuclei in the Motor and Somatosensory Murine Cortical Columns

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    Characterization of the complex cortical structure of the brain at a cellular level is a fundamental goal of neuroscience which can provide a better understanding of both normal function as well as disease state progression. Many challenges exist however when carrying out this form of analysis. Immunofluorescent staining is a key technique for revealing 3-dimensional structure, but subsequent fluorescence microscopy is limited by the quantity of simultaneous targets that can be labeled and intrinsic lateral and isotropic axial point-spread function (PSF) blurring during the imaging process in a spectral and depth-dependent manner. Even after successful staining, imaging and optical deconvolution, the sheer density of filamentous processes in the neuropil significantly complicates analysis due to the difficulty of separating individual cells in a highly interconnected network of tightly woven cellular arbors. In order to solve these problems, a variety of methodologies were developed and validated for improved analysis of cortical anatomy. An enhanced immunofluorescent staining and imaging protocol was utilized to precisely locate specific functional regions within brain slices at high magnification and collect four-channel, complete cortical columns. A powerful deconvolution routine was established which collected depth variant PSFs using an optical phantom for image restoration. Fractional volume analysis (FVA) was used to provide preliminary data of the proportions of each stained component in order to statistically characterize the variability within and between the functional regions in a depth-dependent and depth-independent manner. Finally, using machine learning techniques, a supervised learning model was developed that could automatically classify neuronal and astrocytic nuclei within the large cortical column datasets based on perinuclear fluorescence. These annotated nuclei were then used as seed points within their corresponding fluorescent channel for cell individualization in a highly interconnected network. For astrocytes, this technique provides the first method for characterization of complex morphology in an automated fashion over large areas without laborious dye filling or manual tracing

    Aerospace medicine and biology: A continuing bibliography with indexes (supplement 349)

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    This bibliography lists 149 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during April, 1991. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

    Novel methodologies and technologies for the multiscale and multimodal study of Autism Spectrum Disorders (ASDs)

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    The aim of this PhD thesis was the development of novel bioengineering tools and methodologies that provide a support in the study of ASDs. ASDs are very heterogeneous disturbs and their abnormalities are present both at local and global level. For this reason a multimodal and multiscale approach was followed. The analysis of microstructure was executed on single Purkinje neurons in culture and on organotypic slices extracted from cerebella of GFP wild-type mice and animal models of ASDs. A methodology for the non-invasive imaging of neurons during their growth was set up and a software called NEMO (NEuron MOrphological analysis tool) for the automatic analysis of morphology and connectivity was developed. Microstructure properties can be inferred also in vivo through the quite recent technique of Diffusion Tensor Imaging (DTI). DTI studies in ASDs are based on the hypothesis that the disorder involves aberrant brain connectivity and disruption of white matter tracts between regions implicated in social functioning. In this study DTI was used to investigate structural abnormalities in the white matter structure of young children with ASDs. Moreover the neurostructural bases of echolalia were investigated. The functionality of the brain was analyzed through Functional Magnetic Resonance Imaging (fMRI) using a novel task based on face processing of human, android and robotic faces. A case-control study was performed in order to study how the face processing network is altered in ASDs and how robots are differently processed in ASDs and control groups. Measurements characterizing physiology and behavior of ASD children were also collected using an innovative platform called FACE-T (FACE-Therapy). FACE-T consists of a specially equipped room in which the child, wearing unobtrusive devices for recording physiological and behavioral data as well as gaze information, can interact with an android (FACE, Facial Automaton for Conveying Emotions) and a therapist. The focus was on ECG, as from the analysis of power spectrum density of ECG it is possible to extract features related to the autonomic nervous system that is correlated with brain functionality. These studies give new insights in the study of ASDs exploring aspects not yet addressed. Moreover the methodologies and tools developed could help in the objective characterization of ASD subjects and in the definition of a personalized therapeutic protocol for each child

    Raman Microspectroscopy and Fluorescence Lifetime Imaging Microscopy based Data-Driven Tissue Discrimination and Diagnostics

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    The ultimate objective of any new interventional or surgical techniques is to achieve a balance of minimal invasiveness, optimal efficacy, and rapid treatment duration, all while minimizing the risk of complications. A pivotal component in the management of any disease is the distinction between the impacted target structures and the neighboring healthy tissue throughout all medical interventions, encompassing surgical procedures. Such differentiation is paramount in reducing harm to healthy tissue and augmenting the efficacy of the treatment. Within the current therapeutic procedures, innovations in the field of preoperative and postoperative diagnostics contribute to the improved differentiation of benign and malignant tissue structures. On the one hand, sophisticated imaging techniques support an improved surgical decision-making, while on the other hand, histopathological examination methods enable a precise classification of the tissue after surgery. In contrast, intraoperative tissue differentiation has been based on the time-consuming gold standard of frozen section diagnostics for many years. However, by incorporating the supplementary data provided by advanced imaging sensors during surgery, and integrating it with cutting-edge machine learning methodologies, it is feasible to augment the quality of information utilized for tissue differentiation, thereby increasing the precision of the overall process. Another important task of modern medicine is the patient-specific treatment of cancer, as it has been found that different patients do not respond in the same way to drug treatment due to developed resistance mechanisms. This thesis aimed to establish Raman microspectroscopy (RMS) as marker-independent, and non-destructive technique to monitor fibrotic and epigenetic modifications in malign and benign human tissue. Additionally, the potential of RMS and fluorescence lifetime imaging microscopy (FLIM) was evaluated to non-invasively monitor the drug efficacy on patient-derived organoids from cancer patients. Towards this aim, collagen type I (COL I) structures of formalin-fixed paraffin-embedded (FFPE) tissue sections of various fibrotic diseases were compared to respective control tissue sections. Incorporating Raman measurements into the experimental protocol, we conducted routine histological and immunofluorescence (IF) staining techniques to showcase the superior efficacy of RMS when paired with spectral deconvolution. This technique offers a time- and cost-efficient alternative to conventional procedures. For the differentiation of pathological tissue, the identification of epigenetic modes of action is a promising and potentially successful approach. In alignment with IF imaging of the most abundant epigenetic modification of 5-methylcytosine (5mC), Raman spectra of cell nuclei were evaluated using multivariate data analysis. Compared to invasive staining methods, non-invasive RMS showed promising results for the differentiation of pathological tissue changes in cardiac fibrosis and endometriosis. In addition, RMS and FLIM have been used on several bladder and colon cancer organoids to evaluate their potential to monitor patient-specific responses to drug treatment. The results showed both that organoids are generally suitable as a screening platform for drug treatments and that Raman and FLIM have the potential to assess drug sensitivity. This work highlights the potential of RMS for future applications in ex vivo tissue discrimination of fibrotic diseases and identification of epigenetic changes. In addition, this work demonstrates the proof of principle that RMS and FLIM are suitable for monitoring patient-specific responses to medications on organoid models

    Virulence aspects of Fonsecaea species associated to disseminated infection based on the comparative genomic analysis and in vivo assays

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    Orientadora: Vania Aparecida Vicente PhDCoorientadores: Sybren de Hoog PhD, Renata Rodrigues Gomes PhD, Mauro Antônio Alves Castro PhDTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 10/03/2020Inclui referênciasÁrea de concentração: MicrobiologiaResumo: Fungos melanizados são caracterizados por pigmentação escura na parede celular. Estima-se que mais de cem espécies de fungos pretos possam causar doenças, que podem ocorrer por via de inoculação traumática desses agentes nos tecidos. Este grupo de fungos são membros da ordem Chaetothyriales e são relatados como agentes de cromoblastomicose e feo-hifomicose. A cromoblastomicose é descrita como uma infecção subcutânea que apresenta lenta evolução de lesões verrucosas nos tecidos, que formam células septadas nos tecidos, denominadas células muriformes. Enquanto, a feo-hifomicose é definida pela presença de hifas nos tecidos de vários órgãos, como fígado, coração, rim, cérebro e outros. Mas o neurotropismo ocorre com frequência notável por esse grupo de fungos, possivelmente devido à capacidade de algumas espécies de metabolizar hidrocarbonetos aromáticos. As leveduras negras Fonsecaea monophora e Fonsecaea pugnacius compartilharam o mesmo perfil de patogenicidade, sendo relatadas como agente de cromoblastomicose e agente causador de infecção cerebral primária em humanos. Este trabalho teve como objetivo gerar dados de seqüenciamento de genoma e analisar comparativamente os genomas das espécies de F. monophora e F. pugnacius para entender os mecanismos de biologia e virulência desses agentes. Foi realizada uma análise genômica comparativa das linhagens de irmãos: F. monophora e F. pugnacius, incluindo a montagem do genoma de F. pugnacius. O tamanho dos genomas são 34,21 e 34,8 Mb respectivamente, e o core do genoma dessas espécies compreende quase 70% dos genes. A similaridade do repertório de enzimas associada à ocorrência de fatores de virulência sugeriu uma tolerância geral a condições extremas, o que pode explicar a tendência oportunista dessa espécie como as demais espécies do gênero Fonsecaea. A virulência foi testada nos modelos: Tenebrio molitor e Balb/C para apoiar essa hipótese. A capacidade de sobrevivência de ambos os fungos no interior do Tenebrio molitor foi confirmada por análise histopatológica e pela presença de melanina no tecido hospedeiro. Embora F. pugnacius tenha sido isolado do cérebro de BALB/C após infecção intraperitoneal, os níveis de citocinas não foram estatisticamente significativos, corroborando com a hipótese de ser agente oportunista. Uma dupla capacidade ecológica pode ser concluída a partir da presença de vias metabólicas para eliminação de nutrientes e extremotolerância, combinadas com a capacidade de infectar hospedeiros humanos.Abstract: Black yeasts-like are fungi characterized by dark pigmentation in the cell wall. It has been estimated that more than a hundred species of black fungi can cause disease, which can occur in the injured tissues by fungi. This group of fungi are members of Chaetothyriales order and are related like as agents of chromoblastomycosis and phaeohyphomycosis. Chromoblastomycosis is characterized as a subcutaneous infection present slow evolution of verrucous lesions in the tissues, which form septate cells in the tissues, called muriforms cells. While, phaeohyphomycosis is defined by the presence of hyphae in the tissues of several organs such as liver, heart, kidney, brain and others. But the neurotropism occurs with remarkable frequency in the fungal group, possibly due to the ability of some species to metabolize aromatic hydrocarbons. The black yeasts Fonsecaea monophora and Fonsecaea pugnacius shared the same pathogenicity profile, being reported as a chromoblastomycosis agent and causal agent of primary brain infection in humans. This work aimed generate genome sequencing data and comparative analyze the genomes of F. monophora and F. pugnacius species to understand the biology and virulence mechanisms these agents. A comparative genomic analysis of sibling strains: F. monophora and F. pugnacius was undertaken, including the genome assembly of F. pugnacius. The genome size of strains are 34,21 and 34,8 Mb and the core genomes of those species comprises almost 70% of the genes. The similarity of enzymes repertory associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of this species like the others Fonsecaea sibling species. Virulence was tested in the Tenebrio molitor model and Balb/C were performed in order to supportthis hypothesis. The capacity of both fungi to survive inside Tenebrio molitor was confirmed by histopathological analysis and by presence of melanin in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts

    Blocking Plasmodium development in the mosquitoes by human antibodies

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    Malaria ist eine Krankheit, die durch den Protozoen Plasmodium verursacht und von Anopheles Moskitos durch infektiöse Stiche übertragen wird. Diese ̈Übertragung kann durch verschiedene Interventionsstrategien blockiert werden. Eine relativ neue Strategie, die bisher nur im Labor getestet wurde, ist der Einsatz genetisch veränderter Moskitos, die den Parasiten nicht auf einen neuen menschlichen Wirt übertragen können. Ein Ansatz ist die Entwicklung von Moskitos, die mit murinen Antikoepern ausgestattet sind, die gegen relevante Oberflächenproteine des Parasiten, dem Circumsporozoite Protein (CSP) gerichtet sind. Es ist jedoch nach wie vor unklar, welches Entwicklungsstadium angegriffen werden soll und welche Antikörper für diesen Ansatz effizient sind. Hier zeige ich, dass in Stechmücken, die mit einem humanen Anti-CSP-Antikörper ausgestattet sind, die Sporogonie der Oozysten in Abhängigkeit von der Parasiten-dichte blockiert wird und somit die Sporozoitenlast in den Mücken signifikant verringern. Insbesondere Antikörper, die sich an die ’Repeat Region’ des CSP binden, können die Sporozoitenlast in der Stechmücke verringern. Des Weitern, zeigen diese Stechmücken kaum Defekte in der Entwicklung und im Überleben. Diese Ergebnisse bestätigen die zuvor beschriebene Bedeutung von CSP während der Sporogonie und unterstreichen die Effizienz von humanen, ’Repeat Region’ bindenden Anti-CSP-Antikörpern bei der Beeinträchtigung der Parasitenentwicklung in dem Vektor. Darüber hinaus ist in Stechmücken, die mit humanen Anti-CSP Antikörpern ausgestattet sind, die Entwicklung von Sporozoiten teils limitiert und teils komplett verhindert. Dies macht sie zu einem vielversprechenden Instrument für Maßnahmen zum Malaria Kontrolle. In dieser Arbeit habe ich weitere Einblicke in den Mechanismus , durch den Anti-CSP-Antikörper die Parasitenentwicklung in der Mücke stören, und gezeigt, dass Oozysten ein effizientes Ziel für diesen Ansatz sind.Malaria is a disease caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes trough infectious bites, these transmission events can potentially be blocked by different intervention strategies. A relatively new strategy which has been only tested in the laboratory is the use of genetically modified mosquitoes unable to transmit the parasite to a new human host. In the past, murine derived antibodies directed against relevant parasite surface proteins were used with limited success. It remains unclear which developmental stage is targeted, and which antibodies are useful. Here, I show that in mosquitoes equipped with a human derived anti-CSP repeat binding antibody, oocyst sporogony is blocked in a parasite density dependent manner. Only repeat binding antibodies could decrease sporozoite loads in the mosquito. These results confirm the previously described importance of CSP during sporogony and highlight the efficiency of human derived repeat binding anti-CSP antibodies in interfering with parasite development even in a different host. Additionally, mosquitoes equipped with human derived anti-CSP antibodies show little (in high density infections) to no (in low density infections) sporogonic development, making them a promising tool for malaria transmission blocking interventions. I provided additional insights into the mechanism by which anti-CSP antibodies interfere with parasite development in the mosquito showing that oocysts are an efficient target for this approach. Therefore, the mosquitoes used here are potentially resistant in a more natural setting. Additionally, I provided a new tool allowing a faster screening of antibodies in a mosquito context by injection of single chain Fabs into the mosquito hemolymph. Taken together, this approach could one day give rise to alternative strategies in tackling malaria transmissions
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