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Call number: LD2668 .T4 1984 L43 / Master of Science

Understanding the behavior of a cell requires that its molecular constituents, such as mRNA or protein levels, be profiled quantitatively. Typically, these measurements are performed in bulk and represent values aggregated from thousands of cells. Insights from such data can be very useful, but the loss of single-cell resolution can prove misleading for heterogeneous tissues and in diseases like cancer. Recently, technological advances have allowed us to profile multiple cellular parameters simultaneously at single-cell resolution, for thousands to millions of cells. While this provides an unprecedented opportunity to learn new biology, analyzing such massive and high-dimensional data requires efficient and accurate computational tools to extract the underlying biological phenomena. Such methods must take into account biological properties such as non-linear dependencies between measured parameters. In this dissertation, I contribute to the development of tools from harmonic analysis and computational geometry to study the shape and geometry of single-cell data collected using mass cytometry and single-cell RNA sequencing (scRNA-seq). In particular, I focus on diffusion maps, which can learn the underlying structure of the data by modeling cells as lying on a low-dimensional phenotype manifold embedded in high dimensions. Diffusion maps allow non-linear transformation of the data into a low-dimensional Euclidean space, in which pairwise distances robustly represent distances in the high-dimensional space. In addition to the underlying geometry, this work also attempts to study the shape of the data using archetype analysis. Archetype analysis characterizes extreme states in the data and complements traditional approaches such as clustering. It facilitates analysis at the boundary of the data enabling potentially novel insights about the system. I use these tools to study how the negative costimulatory molecules Ctla4 and Pdcd1 affect T-cell differentiation. Negative costimulatory molecules play a vital role in attenuating T-cell activation, in order to maintain activity within a desired physiological range and prevent autoimmunity. However, their potential role in T cell differentiation remains unknown. In this work, I analyze mass cytometry data profiling T cells in control and Ctla4- or Pdcd1-deficient mice and analyze differences using the tools above. I find that genetic loss of Ctla4 constrains CD4+ T-cell differentiation states, whereas loss of Pdcd1 subtly constrains CD8+ T-cell differentiation states. I propose that negative costimulatory molecules place limits on maximal protein expression levels to restrain differentiation states. I use similar approaches to study breast cancer cells, which are profiled using scRNA-seq as they undergo the pathological epithelial-to-mesenchymal transition (EMT). For this work, I introduce Markov Affinity based Graph Imputation of Cells (MAGIC), a novel algorithm designed in our lab to denoise and impute sparse single-cell data. The mRNA content of each cell is currently massively undersampled by scRNA-seq, resulting in 'zero' expression values for the majority of genes in a large fraction of cells. MAGIC circumvents this problem by using a diffusion process along the data to share information between similar cells and thereby denoise and impute expression values. In addition to MAGIC, I apply archetype analysis to study various cellular stages during EMT, and I find novel biological processes in the previously unstudied intermediate states. The work presented here introduces a mathematical modeling framework and advanced geometric tools to analyze single-cell data. These ideas can be generally applied to various biological systems. Here, I apply them to answer important biological questions in T cell differentiation and EMT. The obtained knowledge has applications in our basic understanding of the process of EMT, T cell biology and in cancer treatment.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 136-140).

Pour compléter leur cycle de vie, les virus interagissent avec de nombreux facteurs de la cellule-hôte. Le virus Herpès simplex de type 1 (HSV-1) ne fait pas exception. Une récente étude protéomique du virus effectuée par notre laboratoire a permis d’identifier 49protéines cellulaires potentiellement incorporées dans les virions matures d’HSV-1 [1]. Étant donné que certaines de ces protéines peuvent jouer des rôles importants au cours du cycle de vie du virus, elles constituent des cibles de choix pour identifier et caractériser de nouvelles interactions hôte-pathogène dans le contexte d’HSV-1. D’ailleurs le laboratoire a été effectué un criblage aux petits ARN d’interférence qui a démontré qu'au moins 15 des protéines incorporées sont impliqués dans le cycle de réplication de HSV-1 en culture cellulaire (Annexe 1). Des nombreuses études rapportent l'incorporation des protéines de l'hôte dans les virions matures mais très peu abordent l'importance de la fraction des protéines cellulaires incorporée dans les virions pour le cycle virale. Pour vérifier ça, nous avons déplété ces protéines des virions matures extracellulaires en utilisant des petits ARN d’interférence. Par la suite, nous avons utilisé ces virus déplétés pour réinfecter des cellules déplétées ou normales. Cette méthode nous a permis d'identifier pour la première fois 8 protéines (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 et 14-3-3ζ) dont l'absence dans les virions réduit la production virale d'au moins 50%. Pour mieux comprendre à quelle étape du cycle viral ces protéines sont nécessaires, nous avons aussi quantifié les virus intracellulaires, produits des cellules déplétées individuellement des quinze protéines cellulaires. Ainsi, nous avons trouvé que dans nos conditions 7 de ces 8 protéines cellulaires (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A et Rab10) semblent impliquées dans la production des virus intracellulaires, ce qui nous a stimulés à débuter une série de tests plus approfondis de l’entrée d’HSV-1. Les résultats préliminaires, démontrent l’implication dans l’entrée d’HSV-1 d’au moins 3 à 4 de ces protéines (HSPA8, KRT10, Rab5A et Rab10). / To complete their life cycle viruses interact with many factors of the host cell. Herpes simplex virus type 1 (HSV-1) is no exception. A recent proteomic study of the virus carried by our laboratory has identified up to 49 cellular proteins potentially incorporated into the mature virions of HSV-1[1]. Since some of these proteins may play important roles during the viral life cycle, they are interesting targets for identification and characterization of new host-pathogen interactions in the context of HSV-1. To target the proteins that are relevant to the viral life cycle of Herpes, the laboratory performed a screening with small interfering RNAs (siRNAs), which showed that at least 15 incorporated proteins are involved in the replication cycle of HSV- 1 in cell culture (Appendix 1). Numerous studies report the incorporation of host proteins in mature virions but few addresses the importance for the viral infectivity of the fractions of cellular proteins incorporated into the virions. To verify this, we depleted these proteins from the mature extracellular virions using siRNAs. Subsequently, we used these viruses to re-infect depleted or normal cells. This method allowed us to identify for the first time eight proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 and 14-3-3ζ) whose absence in virions reduced viral production by at least 50%. As part of understanding at what stage of the life cycle these proteins are necessary for HSV-1, we tested the infectivity of intracellular depleted viruses. Thus, we found at least seven cellular proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A and Rab10) to have a pronounced effect on the replication of herpes virus, which has stimulated us to begin a series of more in-depth tests of the entry of HSV-1. Preliminary results demonstrate the involvement in the entry of HSV-1 of at least three to four proteins (HSPA8, KRT10, Rab5A and Rab10).

Pour compléter leur cycle de vie, les virus interagissent avec de nombreux facteurs de la cellule-hôte. Le virus Herpès simplex de type 1 (HSV-1) ne fait pas exception. Une récente étude protéomique du virus effectuée par notre laboratoire a permis d’identifier 49protéines cellulaires potentiellement incorporées dans les virions matures d’HSV-1 [1]. Étant donné que certaines de ces protéines peuvent jouer des rôles importants au cours du cycle de vie du virus, elles constituent des cibles de choix pour identifier et caractériser de nouvelles interactions hôte-pathogène dans le contexte d’HSV-1. D’ailleurs le laboratoire a été effectué un criblage aux petits ARN d’interférence qui a démontré qu'au moins 15 des protéines incorporées sont impliqués dans le cycle de réplication de HSV-1 en culture cellulaire (Annexe 1). Des nombreuses études rapportent l'incorporation des protéines de l'hôte dans les virions matures mais très peu abordent l'importance de la fraction des protéines cellulaires incorporée dans les virions pour le cycle virale. Pour vérifier ça, nous avons déplété ces protéines des virions matures extracellulaires en utilisant des petits ARN d’interférence. Par la suite, nous avons utilisé ces virus déplétés pour réinfecter des cellules déplétées ou normales. Cette méthode nous a permis d'identifier pour la première fois 8 protéines (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 et 14-3-3ζ) dont l'absence dans les virions réduit la production virale d'au moins 50%. Pour mieux comprendre à quelle étape du cycle viral ces protéines sont nécessaires, nous avons aussi quantifié les virus intracellulaires, produits des cellules déplétées individuellement des quinze protéines cellulaires. Ainsi, nous avons trouvé que dans nos conditions 7 de ces 8 protéines cellulaires (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A et Rab10) semblent impliquées dans la production des virus intracellulaires, ce qui nous a stimulés à débuter une série de tests plus approfondis de l’entrée d’HSV-1. Les résultats préliminaires, démontrent l’implication dans l’entrée d’HSV-1 d’au moins 3 à 4 de ces protéines (HSPA8, KRT10, Rab5A et Rab10). / To complete their life cycle viruses interact with many factors of the host cell. Herpes simplex virus type 1 (HSV-1) is no exception. A recent proteomic study of the virus carried by our laboratory has identified up to 49 cellular proteins potentially incorporated into the mature virions of HSV-1[1]. Since some of these proteins may play important roles during the viral life cycle, they are interesting targets for identification and characterization of new host-pathogen interactions in the context of HSV-1. To target the proteins that are relevant to the viral life cycle of Herpes, the laboratory performed a screening with small interfering RNAs (siRNAs), which showed that at least 15 incorporated proteins are involved in the replication cycle of HSV- 1 in cell culture (Appendix 1). Numerous studies report the incorporation of host proteins in mature virions but few addresses the importance for the viral infectivity of the fractions of cellular proteins incorporated into the virions. To verify this, we depleted these proteins from the mature extracellular virions using siRNAs. Subsequently, we used these viruses to re-infect depleted or normal cells. This method allowed us to identify for the first time eight proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 and 14-3-3ζ) whose absence in virions reduced viral production by at least 50%. As part of understanding at what stage of the life cycle these proteins are necessary for HSV-1, we tested the infectivity of intracellular depleted viruses. Thus, we found at least seven cellular proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A and Rab10) to have a pronounced effect on the replication of herpes virus, which has stimulated us to begin a series of more in-depth tests of the entry of HSV-1. Preliminary results demonstrate the involvement in the entry of HSV-1 of at least three to four proteins (HSPA8, KRT10, Rab5A and Rab10).

Les récents progrès instrumentaux en spectrométrie de masse, notamment en terme de- rapidité de balayage et de résolution, ont permis l'émergence de l'approche « data independent acquisition» (DIA). Cette approche promet de combiner les points forts des approches « shotgun » et ciblées,mais aujourd'hui l'analyse des données DIA reste compliquée. L'objectif de cette thèse a été de développer des méthodes innovantes de spectrométrie de masse, et en particulier d'améliorer l'analyse des données DIA. De plus, nous avons développé une approche originale Top 3-ID-DIA, permettant à la fois un profilage complet des protéines de la cellule hôte (HCP) ainsi qu'une quantification absolue d'HCP clés dans les échantillons d'anticorps monoclonaux (mAb), au sein d'une même analyse.Cette méthode est prête à être implémentée en industrie, et pourrait fournir un support en temps réel aux développements du procédé de production de mAb, ainsi que pour évaluer la pureté des biomédicaments. / Recent instrumental developments in mass spectrometry, notably in terms of scan speed and resolution, allowed the emergence of “data independent acquisition” (DIA) approach. This approach promises to combine the strengths of both shotgun and targeted proteomics, but today DIA data analysis remains challenging. The objective of my PhD was to develop innovative mass spectrometry approaches, and in particular to improve DIA data analysis. Moreover, we developed an original Top 3-ID-DIA approach, allowing both a global profiling of host cell proteins (HCP) and an absolute quantification of key HCP in monoclonal antibodies samples, within a single analysis. This method is ready to be transferred to industry, and could provide a real time support for mAb manufacturing process development, as well as for product purity assessment.

This work is the result of evaluating a novel strategy for the purification of recombinant His-tagged proteins. Proteins purified in this study were the E. coli translational proteins IF-3, RF-1, and RFF. The study aimed to analyse the potential of using Bio-Works WorkBeads™40 TREN, a multimodal anion ion exchange chromatography resin, as a pretreatment step upstream an immobilized metal ion chromatography (IMAC) resin to enhance performance efficiency of His-tagged protein purification. The method demonstrated here shows potential for anyone seeking to increase the purity of His-tagged protein purification or to introduce an effective purification procedure by replacing a polishing step downstream IMAC with WorkBeads 40 TREN upstream IMAC. The latter contributing to guard the IMAC column from heavy bioburden. This study showed that running WorkBeads 40 TREN prior IMAC captures impurities and removes 97-98 % more dsDNA compared to direct IMAC. WorkBeads 40 TREN is therefore highly advantageous to include early in a purification process to remove protein binding DNA fragments. Moreover, WorkBeads 40 TREN increases purity in the final product by capturing more host cell proteins than when running direct IMAC. This concept is general and WorkBeads 40 TREN could be used upstream a variety of resins such as Protein A and RPC.

O vírus sincicial respiratório humano (HRSV) é o mais frequente agente patogênico da família Paramyxoviridae. Apesar de sua grande importância e impacto em saúde pública, alguns aspectos demandam elucidação. Entre eles, estão os mecanismos de tráfego intracelular de proteínas virais para o sitio de montagem. Baseado nisso, fizemos um estudo de imunofluorescência tentando contribuir para o entendimento da participação da via secretória no tráfego de proteínas estruturais de HRSV que não são glicosiladas: proteínas de matriz (M) e de nucleocapsídeo (N). Pudemos observar que essas proteínas seguem rota similar àquelas que são glicosiladas no Golgi, como a proteína de fusão (F). Ademais, as proteínas M e N, além de colocalizarem com proteínas celulares da via secretória, tais como trans-Golgi network-46 (TGN46) e sorting nexin-2 (SNX2), também influem no recrutamento de proteínas celulares para os corpos de inclusão virais, como mostrado no caso da proteína Glut1. Os dados indicam que proteínas M e N de HRSV seguem pela via endocítica inicial, acumulam-se em corpos de inclusão que seriam fábricas virais e, no caso de TGN46, podem ser incorporadas aos vírus em brotamento / Human respiratory syncytial virus (HRSV) is the most relevant cause of respiratory infection in children worldwide. Despite its importance in public health, some aspects of the mechanisms of the trafficking of viral structural proteins remain unclear. In the present study, immunofluorescence was used to understand how the virus matrix (M) and nucleocapsid (N) proteins, which are non-glycosylated , are addressed to inclusion bodies in Hep-2 cells (MOI=3). M and N proteins followed similar intracellular trafficking routes as compared to the glycosylated fusion (F) viral protein. Moreover, M and N proteins colocalized with two key elements of the secretory pathway: trans-Golgi network- 46 (TGN46) and sorting nexin-2 (SNX2). Viral proteins M and N appear to be involved in the recruitment of cell proteins at the formation of virus inclusion bodies, as shown for Glucose Transporter Type 1 (Glut1). The data suggest that HRSV M and N proteins follow the secretory pathway, initiating in early endosomes, as indicated by the co-localization with TGN46 and SNX2. In addition, these host cell proteins accumulate in inclusion bodies that are viral factories, and can be part of budding viral progeny. Therefore, HRSV M and N proteins, even though they are not glycosylated, take advantage of the secretory pathway to reach virus inclusion bodies. Confocal images suggest that SNX2, which is known for its membrane-deforming properties, could play a pivotal role in HRSV budding

O vírus sincicial respiratório humano (HRSV) é o mais frequente agente patogênico da família Paramyxoviridae. Apesar de sua grande importância e impacto em saúde pública, alguns aspectos demandam elucidação. Entre eles, estão os mecanismos de tráfego intracelular de proteínas virais para o sitio de montagem. Baseado nisso, fizemos um estudo de imunofluorescência tentando contribuir para o entendimento da participação da via secretória no tráfego de proteínas estruturais de HRSV que não são glicosiladas: proteínas de matriz (M) e de nucleocapsídeo (N). Pudemos observar que essas proteínas seguem rota similar àquelas que são glicosiladas no Golgi, como a proteína de fusão (F). Ademais, as proteínas M e N, além de colocalizarem com proteínas celulares da via secretória, tais como trans-Golgi network-46 (TGN46) e sorting nexin-2 (SNX2), também influem no recrutamento de proteínas celulares para os corpos de inclusão virais, como mostrado no caso da proteína Glut1. Os dados indicam que proteínas M e N de HRSV seguem pela via endocítica inicial, acumulam-se em corpos de inclusão que seriam fábricas virais e, no caso de TGN46, podem ser incorporadas aos vírus em brotamento / Human respiratory syncytial virus (HRSV) is the most relevant cause of respiratory infection in children worldwide. Despite its importance in public health, some aspects of the mechanisms of the trafficking of viral structural proteins remain unclear. In the present study, immunofluorescence was used to understand how the virus matrix (M) and nucleocapsid (N) proteins, which are non-glycosylated , are addressed to inclusion bodies in Hep-2 cells (MOI=3). M and N proteins followed similar intracellular trafficking routes as compared to the glycosylated fusion (F) viral protein. Moreover, M and N proteins colocalized with two key elements of the secretory pathway: trans-Golgi network- 46 (TGN46) and sorting nexin-2 (SNX2). Viral proteins M and N appear to be involved in the recruitment of cell proteins at the formation of virus inclusion bodies, as shown for Glucose Transporter Type 1 (Glut1). The data suggest that HRSV M and N proteins follow the secretory pathway, initiating in early endosomes, as indicated by the co-localization with TGN46 and SNX2. In addition, these host cell proteins accumulate in inclusion bodies that are viral factories, and can be part of budding viral progeny. Therefore, HRSV M and N proteins, even though they are not glycosylated, take advantage of the secretory pathway to reach virus inclusion bodies. Confocal images suggest that SNX2, which is known for its membrane-deforming properties, could play a pivotal role in HRSV budding