Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

One the most daunting technical challenges in the realization of biosensors is functionalizing transducing surfaces for the detection of biomolecules. Functionalization is defined as the formation of a bio-compatible interface on the transducing surfaces of bio-chemical sensors for immobilizing and subsequent sensing of biomolecules. The kinetics of functionalization reactions is a particularly important issue, since conventional functionalization protocols are associated with lengthy process times, from hours to days. The objective of this thesis is the improvement of the functionalization protocols and their kinetics for biosensing applications. This objective is realized via modeling and experimental verification of novel functionalization techniques in microfluidic environments. The improved functionalization protocols using microfluidic environments enable in-situ functionalization, which reduces the processing times and the amount of reagents consumed, compared to conventional methods. The functionalization is performed using self-assembled monolayers (SAMs) of thiols. The thiols are organic compounds with a sulphur group that assists in the chemisorption of the thiol to the surface of metals like gold. The two reactions in the functionalization process examined in this thesis are the SAM formation and the SAM/probe molecule conjugation. SAM/probe molecule conjugation is the chemical treatment of the SAM followed by the binding of the probe molecule to the SAM. In general, the probe molecule is selective in binding with a given biomolecule, called the target molecule. Within this thesis, the probe molecule is an antibody and the target molecule is an antigen. The kinetics of the reaction between the probe (antibody) and the target biomolecule (antigen) is also studied. The reaction between an antigen and its antibody is called the immunoreaction. The biosensing technique that utilizes the immunoreaction is immunoassay. A numerical model is constructed using the finite element method (FEM), and is used to study the kinetics of the functionalization reactions. The aim of the kinetic studies is to achieve both minimal process times and reagents consumption. The impact of several important parameters on the kinetics of the reactions is investigated, and the trends observed are explained using kinetic descriptive dimensionless numbers, such as the Damköhler number and the Peclet number. Careful numerical modeling of the reactions contributes to a number of findings. A considerably faster than conventional SAM formation protocol is predicted. This fast-SAM protocol is capable of reducing the process times from the conventional 24-hours to 15 minutes. The numerical simulations also predict that conventional conjugation protocols result in the overexposure of the SAM and the probe molecule to the conjugation reagents. This overexposure consequently lowers conjugation efficiencies. The immunoreaction kinetics of a 70 kilo-Dalton heat shock protein (HSP70) with its antibody in a hypothetical microchannel is also investigated through the FEM simulations. Optimal reaction conditions are determined, including the flow velocity and the surface concentration of the immobilized probes (antibodies). Based on the numerical results and a series of experimental studies, the fast-SAM protocol application is successfully confirmed. Moreover, the optimum reagent concentration for a given one- hour conjugation process time is determined. This functionalization protocol is successfully applied to immobilize the HSP70 antibody on gold surfaces. The use of the fast-SAM protocol and the predicted optimum conjugation conditions result in binding of the HSP70 antibody on gold, with the same or superior immobilization quality, compared to the conventional protocols. Upon implementation of a 70 μm.s^(-1) flow velocity, the reaction is observed to complete in around 30-35 minutes, which is close to the numerically predicted 30 minutes and 16 seconds. This immunoreaction time is considerably less than conventional 4-12 hour processes. The modified in-situ functionalization techniques achieved here are promising for substantially reducing the preparation times and improving the performance of biosensors, in general, and immunoassays, in particular.

One the most daunting technical challenges in the realization of biosensors is functionalizing transducing surfaces for the detection of biomolecules. Functionalization is defined as the formation of a bio-compatible interface on the transducing surfaces of bio-chemical sensors for immobilizing and subsequent sensing of biomolecules. The kinetics of functionalization reactions is a particularly important issue, since conventional functionalization protocols are associated with lengthy process times, from hours to days. The objective of this thesis is the improvement of the functionalization protocols and their kinetics for biosensing applications. This objective is realized via modeling and experimental verification of novel functionalization techniques in microfluidic environments. The improved functionalization protocols using microfluidic environments enable in-situ functionalization, which reduces the processing times and the amount of reagents consumed, compared to conventional methods. The functionalization is performed using self-assembled monolayers (SAMs) of thiols. The thiols are organic compounds with a sulphur group that assists in the chemisorption of the thiol to the surface of metals like gold. The two reactions in the functionalization process examined in this thesis are the SAM formation and the SAM/probe molecule conjugation. SAM/probe molecule conjugation is the chemical treatment of the SAM followed by the binding of the probe molecule to the SAM. In general, the probe molecule is selective in binding with a given biomolecule, called the target molecule. Within this thesis, the probe molecule is an antibody and the target molecule is an antigen. The kinetics of the reaction between the probe (antibody) and the target biomolecule (antigen) is also studied. The reaction between an antigen and its antibody is called the immunoreaction. The biosensing technique that utilizes the immunoreaction is immunoassay. A numerical model is constructed using the finite element method (FEM), and is used to study the kinetics of the functionalization reactions. The aim of the kinetic studies is to achieve both minimal process times and reagents consumption. The impact of several important parameters on the kinetics of the reactions is investigated, and the trends observed are explained using kinetic descriptive dimensionless numbers, such as the Damköhler number and the Peclet number. Careful numerical modeling of the reactions contributes to a number of findings. A considerably faster than conventional SAM formation protocol is predicted. This fast-SAM protocol is capable of reducing the process times from the conventional 24-hours to 15 minutes. The numerical simulations also predict that conventional conjugation protocols result in the overexposure of the SAM and the probe molecule to the conjugation reagents. This overexposure consequently lowers conjugation efficiencies. The immunoreaction kinetics of a 70 kilo-Dalton heat shock protein (HSP70) with its antibody in a hypothetical microchannel is also investigated through the FEM simulations. Optimal reaction conditions are determined, including the flow velocity and the surface concentration of the immobilized probes (antibodies). Based on the numerical results and a series of experimental studies, the fast-SAM protocol application is successfully confirmed. Moreover, the optimum reagent concentration for a given one- hour conjugation process time is determined. This functionalization protocol is successfully applied to immobilize the HSP70 antibody on gold surfaces. The use of the fast-SAM protocol and the predicted optimum conjugation conditions result in binding of the HSP70 antibody on gold, with the same or superior immobilization quality, compared to the conventional protocols. Upon implementation of a 70 μm.s^(-1) flow velocity, the reaction is observed to complete in around 30-35 minutes, which is close to the numerically predicted 30 minutes and 16 seconds. This immunoreaction time is considerably less than conventional 4-12 hour processes. The modified in-situ functionalization techniques achieved here are promising for substantially reducing the preparation times and improving the performance of biosensors, in general, and immunoassays, in particular.

La polyprotéine de structure Gag du VIH-1 est responsable de l’assemblage des particules virales dans les cellules infectées. Au niveau moléculaire, cette protéine s’oligomérise en formant des complexes Gag-Gag autour de deux plates-formes moléculaires, d'une part l'ARN génomique via son domaine NCp7 (NucleoCapsid protein 7) et d'autre part, la membrane plasmique via son domaine MA (Matrice). De plus, lors du trafic de Gag dans la cellule, Gag détourne les protéines ESCRT comme TSG101 et ALIX de la machinerie cellulaire afin de bourgeonner et d’être libérées dans le milieu extracellulaire. Dans cette thèse, nous avons étudié le rôle du domaine NCp7 seul ou au sein de Gag (GagNC) dans les interactions Gag-Gag et Gag-TSG101 en utilisant des approches biochimiques et de la microscopie de fluorescence quantitative. Les résultats ont montré que l'absence du domaine NCp7 affecte l’oligomerisation de Gag qui s’accumule alors dans le cytoplasme sous forme d’agrégats de taille importante. Par ailleurs, le trafic intracellulaire de Gag est affecté par les mutations dans le domaine GagNC avec une augmentation importante de temps nécessaire à Gag pour arriver à la membrane plasmique. Enfin, nous avons montré que GagNC i) renforce l’interaction entre le domaine p6 de Gag et TSG101 et ii) par sa fonction dans le trafic de Gag, est responsable de la localisation de TSG101 à la PM. Sur la base de ces résultats, des études sont maintenant en cours pour développer des tests afin d’identifier des molécules possédant un potentiel anti virale. / The Gag structural polyprotein of HIV-1 orchestrates viral particle assembly in producer cells, in a process that requires two platforms, the genomic RNA on the one hand and a membrane with a lipid bilayer, on the other. During its transportation from translating ribosomes to plasma membrane, Gag hijacks cellular proteins of the cytoskeleton and the ESCRT proteins like TSG101, Alix, etc., to egress viral particles. However, a number of questions remain to be answered before they are clearly apprehended. In this thesis, , we studied the role of the NC domain alone or as part of Gag (GagNC) in Gag-Gag and Gag-TSG101 interactions, which are essential for the assembly and budding of HIV-1 particles using quantitative fluorescent microscopy and biochemical approach. Results, showed that the absence of NC domain lead to (1) an accumulation of Gag as large aggregates that are dispersed in the cytoplasm, (2) a decrease of Gag-Gag condensation and (3) a delay for Gag-Gag complexes in reaching the PM, (4) improved interaction between Gag and TSG101, and (5) by its virtue in Gag trafficking docks TSG101 to the PM. This regulatory effect of NCp7 domain in either TSG101 or Gag or both protein- regulated pathways during virus budding can be exploited to develop inhibitors targeting HIV-1.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...

Dizertační práce se zabývá mechanismy mazání v náhradách kyčelního kloubu. Byla provedena systematická studie formování proteinového filmu při zahrnutí různých materiálů a provozních podmínek. Hlavní pozornost je přitom věnována roli jednotlivých proteinů obsažených v synoviální kapalině při současné přítomnosti dalších proteinů. Jelikož metody aplikované v předchozích studiích neumožňovaly separovat jednotlivé složky maziva, byla vyvinuta optická měřící metoda na principu fluorescenční mikroskopie. Z důvodu verifikace metody byly provedeny dvě nezávislé studie zaměřené na měření tloušťky mazacího filmu a dělení maziva na výstupu mazaného kontaktu. Z důvodu určitých limitací fluorescenční mikroskopie byla dále využita i metoda optické interferometrie, jejíž využití je ilustrováno ve studii zabývající se formováním mazacího filmu v náhradách kyčelního klubu při uvažování reálné konformity třecích povrchů. Závěrečná část práce představuje nový metodologický přístup založený na in situ pozorování kontaktní oblasti umožňující popsat roli jednotlivých proteinů ve vztahu k vývoji tloušťky mazacího filmu. Práce obsahuje originální výsledky, které přináší nové poznání v oblasti biotribologie náhrad kyčelního kloubu vedoucí k dalšímu vývoji implantátů při snaze zabránit jejich selhání v důsledku omezené životnosti.

Indiana University-Purdue University Indianapolis (IUPUI) / A crucial task in computer vision and biomedical image applications is to represent images in a numerically compact form for understanding, evaluating and/or mining their content. The fundamental step of this task is the segmentation of images into regions, given some homogeneity criteria, prior appearance and/or shape information criteria. Specifically, segmentation of cells in microscopic images is the first step in analyzing many biomedical applications. This thesis is a part of the project entitled "Construction and profiling of biodegradable cardiac patches for the co-delivery of bFGF and G-CSF growth factors" funded by National Institutes of Health (NIH). We present a method that simultaneously segments the population of cells while partitioning the cell regions into cytoplasm and nucleus in order to evaluate the spatial coordination on the image plane, density and orientation of cells. Having static microscopic images, with no edge information of a cytoplasm boundary and no time sequence constraints, traditional cell segmentation methods would not perform well. The proposed method combines deformable models with a probabilistic framework in a simple graphical model such that it would capture the shape, structure and appearance of a cell. The process aims at the simultaneous cell partitioning into nucleus and cytoplasm. We considered the relative topology of the two distinct cell compartments to derive a better segmentation and compensate for the lack of edge information. The framework is applied to static fluorescent microscopy, where the cultured cells are stained with calcein AM.

L’adhésine impliquée dans l’adhérence diffuse (AIDA-I) est une adhésine bactérienne présente chez certaines souches d’Escherichia coli qui, associée aux toxines Stx2e ou STb, contribue à l’apparition de la maladie de l’œdème ou de la diarrhée post-sevrage chez les porcelets. AIDA-I est un autotransporteur qui confère des capacités d’autoaggrégation, de formation de biofilms et d’adhésion. L’objectif principal du projet de recherche consistait en la recherche de récepteur(s) potentiel(s) d’AIDA-I. Les bactéries pathogènes adhèrent aux cellules-cibles soit en liant directement des molécules à la surface cellulaire ou en utilisant des molécules intermédiaires qui permettent de diminuer la distance séparant la bactérie de la cellule-cible. Puisque le sérum est un fluide qui contient de nombreuses molécules, celui-ci a été utilisé comme matériel de départ pour l’isolement de récepteur(s) potentiels. Nous avons isolé un récepteur potentiel à partir du sérum porcin : l’apolipoprotéine A-I. L’interaction entre l’apolipoprotéine A-I et AIDA-I a été confirmée par ELISA et microscopie à fluorescence. La capacité à envahir les cellules épithéliales offre aux pathogènes la possibilité d’établir une niche intracellulaire qui les protègent contre les attaques du milieu extérieur. La présente étude a démontré que la présence d’AIDA-I en tant que seul facteur de virulence chez une souche de laboratoire permet de conférer la capacité d’envahir les cellules sans promouvoir la survie intracellulaire. L’étude de la souche sauvage 2787, exprimant AIDA-I en association avec d’autres facteurs de virulence, a démontré une différence significative pour les phénotypes d’invasion et de survie intracellulaire face à la souche de laboratoire exprimant AIDA-I. / The adhesin involved in diffuse adherence (AIDA-I) is a bacterial adhesin associated with some Escherichia coli strains that might, when associated with toxin Stx2e or STb, contribute to the development of edema disease or post-weaning diarrhea in piglets. AIDA-I is an autotransporter that mediates various phenotypes such as adhesion, autoaggregation and biofilm formation. The main aim of our project was to find potential receptor(s) for AIDA-I. Pathogens can either bind cell directly by targeting exposed cell surface molecules or use an intermediate molecule as a bridge to lessen the space separating them from their target cell. Serum is known to contain a wide range of molecules so it has been used as raw material for the isolation of a putative receptor for AIDA-I. We isolated a putative receptor for AIDA-I: the apolipoprotein A-I. The interaction between the apolipoprotein A-I and AIDA-I was confirmed by ELISA and fluorescent microscopy. The capacity to invade epithelial cell enables pathogens to create an intracellular niche that protects them against attacks from the extracellular environment. The present report has shown that the presence of AIDA-I as the sole virulence factor in a laboratory strain, enable bacteria to invade cultured cells but does not promote intracellular survival. Studies conducted on wild-type strain 2787, which express AIDA-I in association with other virulence factors, has shown a significant difference in invasion and intracellular survival phenotypes compared to the laboratory strain expressing AIDA-I.

Fosfolipases D (FLD) do veneno das aranhas do gênero Loxosceles são capazes de causar entre outros efeitos, uma forte agregação plaquetária cujo mecanismo ainda não foi elucidado. Portanto, para estudar o papel das FLDs nesta atividade, uma FLD recombinante de L. gaucho (LgRec1) foi fusionada com a proteína fluorescente verde (EGFP) e utilizada como uma sonda para detectar a interação de LgRec1 com plaquetas. Essa quimera, denominada EGFP-LgRec1, manteve as principais características da LgRec1. A microscopia confocal das plaquetas mostrou que LgRec1 não requer componentes plasmáticos para se ligar às plaquetas, embora estes sejam necessários para que a LgRec1 induza agregação. Além disso, foi observado que a ação da LgRec1 leva à exposição de fosfatidilserina. Contudo, esta exposição não está relacionada à morte celular. Portanto, este trabalho mostrou que uma FLD de Loxosceles se liga a plaquetas, promovendo a exposição de fosfatidilserina, possibilitando a ligação de fatores de coagulação e resultando na agregação plaquetária. / Phospholipases D (PLD) from spider venom of the genus Loxosceles are capable of causing, among other effects, a strong aggregation of platelets and its mechanism has not yet been elucidated. Therefore, to study the role of PLDs in this activity, a recombinant L. gaucho PLD (LgRec1) was fused with a green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 with platelets. This chimera, named EGFP-LgRec1, remained the main activities of LgRec1. Platelet confocal microscopy has shown that LgRec1 does not require plasma components to bind to platelets, although these are required for LgRec1 to induce aggregation. In addition, it has been observed that the action of LgRec1 leads to exposures of phosphatidylserine. However, this exposure is not related to cell death. Therefore, this work showed that a Loxosceles PLD binds to platelets, promoting an exposure of phosphatidylserine, that may act as a scaffold for coagulation factors, resulting in platelet aggregation.