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Within the last decade, neurodegenerative diseases such as Alzheimer’s and Parkinson’s have emerged as one of the top 5 leading causes of death globally, and there is currently no cure. All neurodegenerative diseases lead to loss of the functional cells in the nervous system, the neurons. One therapeutic approach is to replace the damaged and lost neurons with new, healthy neurons. Unfortunately, this is a difficult endeavor since mature neurons are not capable of cell division. Instead, researchers are turning to neural stem cells, which are able to self-renew and be rapidly expanded before being differentiated into functional cell phenotypes, such as neurons, allowing for large numbers of cells to be generated in vitro. Controlled differentiation of human neural stem cells into new neurons has been of interest due to the immense potential for improving clinical outcomes. Adult neural stem cell behavior, however, is not well understood and the transplanted stem cells are at risk for tumorigenesis. The focus of this dissertation is the development of engineered biomaterials as tools to study human neural stem cell behavior and neurogenesis (differentiation). A novel cell penetrating peptide was developed to enhance intracellular delivery of retinoic acid, a bioactive lipid known to induce differentiation. A hydrogel platform fabricated from hyaluronic acid, a naturally-occurring polysaccharide found in brain extracellular space, was designed to serve as a biomimetic soft substrate with similar mechanical properties to the brain. The biological behavior of the stem cells was characterized in response to chemical and physical cues. / Bioengineering

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue. / Ph. D.

Cell phenotypic transitions, or cell fate decision making processes, are regulated by complex regulatory networks composed of genes, RNAs, proteins and metabolites. The regulation can take place at the epigenetic, transcriptional, translational, and post-translational levels to name a few. Epigenetic histone modification plays an important role in cell phenotype maintenance and transitions. However, the underlying mechanism relating dynamical histone modifications to stable epigenetic cell memory remains elusive. Incorporating key pieces of molecular level experimental information, we built a statistical mechanics model for the inheritance of epigenetic histone modifications. The model reveals that enzyme selectivity of different histone substrates and cooperativity between neighboring nucleosomes are essential to generate bistability of the epigenetic memory. We then applied the epigenetic modeling framework to the differentiation process of olfactory sensory neurons (OSNs), where the observed 'one-neuron-one-allele' phenomenon has remained as a long-standing puzzle. Our model successfully explains this singular behavior in terms of epigenetic competition and enhancer cooperativity during the differentiation process. Epigenetic level events and transcriptional level events cooperate synergistically in the OSN differentiation process. The model also makes a list of testable experimental predictions. In general, the epigenetic modeling framework can be used to study phenotypic transitions when histone modification is a major regulatory element in the system. Post-transcriptional level regulation plays important roles in cell phenotype maintenance. Our integrated experimental and computational studies revealed such a motif regulating the differentiation of definitive endoderm. We identified two RNA binding proteins, hnRNPA1 and KSRP, which repress each other through microRNAs miR-375 and miR-135a. The motif can generate switch behavior and serve as a noise filter in the stem cell differentiation process. Manipulating the motif could enhance the differentiation efficiency toward a specific lineage one desires. Last we performed mathematical modeling on an epithelial-to-mesenchymal transition (EMT) process, which could be used by tumor cells for their migration. Our model predicts that the IL-6 induced EMT is a stepwise process with multiple intermediate states. In summary, our theoretical and computational analyses about cell phenotypic transitions provide novel insights on the underlying mechanism of cell fate decision. The modeling studies revealed general physical principles underlying complex regulatory networks. / Ph. D.

No / A solid-phase Mitsunobu reaction between a resin-bound 1-thiosugar and an N-Fmoc protected amino alcohol was successfully employed for thio-linked glycopeptide synthesis. Facile cleavage and deprotection in one step afforded the target glycopeptide in good yield and purity.

The phylum of Chlamydiota is composed of gram negative obligate intracellular bacteria that live as symbionts of diverse eukaryotes, from protists to animals and humans. Members of the phylum can be split into two groups: the environmental Chlamydia, which includes symbionts of amoeba, and the pathogenic Chlamydia, which includes species infecting animals, birds, and humans and includes Chlamydia trachomatis the leading cause of reportable, bacterial sexually transmitted infections and the ocular infection, trachoma. The characterized phylum members undergo a biphasic developmental cycle alternating between the infectious elementary body (EB) and the replicative reticulate body (RB), with each form having distinct morphological and physiological properties. Differentiation between these forms occurs within a host cell membrane-derived vacuole termed the inclusion. The molecular mechanisms governing and executing bacterial development and RB growth remain unclear. The essentiality and uniqueness of development makes it a prime target for the development of novel, chlamydial-specific therapeutics. Reductive evolution has resulted in the loss of or fragmentation of numerous metabolic pathways, particularly in the pathogenic Chlamydia (~1 Mbp genome) as compared to the environmental Chlamydia (~2.5 Mbp). We hypothesize that the bacterium senses environmental changes (host cytoplasm) to ensure that development and growth coincide with host cell energy and metabolite levels. We predict that an encoded partner switching mechanism (PSM) plays a key role in: 1) regulation of growth by acting as a molecular throttle through regulation of the housekeeping sigma factor, and 2) differentiation by impacting the composition of the sigma factor pool allowing for transcriptional changes needed for developmental transitions. We also predict that PSM regulation occurs through sensing of nucleotide triphosphates, TCA-cycle intermediates, metal concentrations, and redox. Canonical PSMs have a PP2C-type sensor phosphatase (SP), an anti-sigma factor (ASF, serine kinase), an anti-anti-sigma factor (AASF, substrate of the SP and ASF) and a stress-response related alternative sigma factor. The PSM in pathogenic Chlamydia is atypical, and despite its reduced genome, is comprised of two SPs (RsbU which responds to α-ketoglutarate and CTL0852), two AASFs (RsbV1 and RsbV2), one ASF (RsbW), and, unusually, the ASF regulates the availability of the “housekeeping” sigma factor, σ66. To test our hypotheses, we first constructed and purified a variety of amino acid point mutants of the two AASFs, ASF, and the SP for in vitro analyses. Kinase and phosphatase activity towards RsbV1/V2 was measured in the presence of different metals, phosphate donors, and pH and redox conditions. Phos-tag acrylamide gels were used to assess protein phosphorylation status. We discovered that metalation impacts enzyme activity and the substrate specificity of RsbU, and that RsbW can use multiple phosphate donors. Prior work, and our data, found that RsbW and RsbU have higher enzymatic activity towards RsbV1 than RsbV2, leading us to explore the importance of RsbV2 in chlamydial biology. Genome gazing revealed that environmental Chlamydia possess a single AASF, and bioinformatic analyses support that it is more similar to RsbV2 than RsbV1 suggesting that the pathogenic Chlamydia gained RsbV1. Comparing the biochemical features of the two AASFs provides potential reasons for the different enzyme affinities. To flesh out the in vivo importance of each AASF, we characterized bacterial growth, infectious progeny production, and the levels of RsbV1/V2 in a cell culture infection model using a collection of C. trachomatis L2 rsbV1 null or rsbV2 knockdown strains. We also overexpressed the AASFs in strains grown with different glucose levels. Note that C. trachomatis is an auxotroph for glucose-6-phosphate. In normal chlamydial culture glucose medium levels, the rsbV1 null strain showed an ~1 log reduction in infectious progeny numbers while the rsbV2 knockdown or AASF overexpression strains had no defects. We also observed that the rsbV1 null strain has a developmental delay and exhibits growth differences in response to glucose levels, i.e. a functional PSM seems to set a “growth cap” in response to different glucose availability. Immunoblotting analysis of RsbV1/V2 demonstrated the presence of both proteins throughout development, and protein levels remained the same in low or high glucose levels and in the wild type or rsbV1 null strains (measuring RsbV2 only for the RsbV1 null strain). These results tell us that the AASF levels have minimal impact on chlamydial biology, suggesting that phosphorylation status is key to regulation. To assess phosphorylation, we used protein pulldown assays and Phos-tag gels to assess RsbV1 and RsbV2 phosphorylation during development. Both RsbV1 and RsbV2 were phosphorylated during the EB stage, which is similar to our prior results using Chlamydia caviae. In conjunction with the in vivo phosphorylation data, we hypothesize that stage-dependent inhibition of AASF/RsbW interactions frees RsbW to sequester σ66. Reduced pools of σ66 would promote RB-EB conversion through increased RNAP binding to the late gene sigma factors σ54 and σ28. Supporting this model, overexpression of a non-phosphorylatable RsbV2 S55A mutant (an RsbW “trap”), but not overexpression of RsbV1 S56A, resulted in a 3 log reduction in infectious progeny production without gross changes in inclusion morphology or bacterial numbers, while causing a reduction in σ54 and σ28 regulated EB-specific proteins and inhibition of RB-EB transition shown via transmission electron microscopy. As an alternative approach to assess the consequence of reduced “free” RsbW, we used a CRISPRi knockdown system targeting rsbW and observed a reduction in infectious progeny production under some conditions, which is consistent with the RsbV2 S55A expression strain results. The rsbW CRISPRi-associated phenotype was weaker than the RsbV2 S55A phenotype. As bacterial redox status changes throughout development (RBs are reduced and EBs are oxidized), we also assessed whether the cysteine-rich proteins RsbV2 and RsbW were redox responsive. In parallel to the unique AASF expansion in the pathogenic Chlamydia, RsbV2 in the pathogenic Chlamydia has a CXCC motif that is not found in the RsbV homolog in the environmental Chlamydia. Our in vitro studies found that, under oxidizing conditions, RsbV2 is dimerized, and the dimer form inhibits phosphorylation of RsbV2 by RsbW. We predict that retention of RsbV2 after RsbV1 acquisition has been selected for, in part, owing to a unique redox-sensing role compared to RsbV1 and that the presence of two AASFs enables more sensitive tuning of growth and development in response to metabolite levels. The different phenotypes when overexpressing non-phosphorylatable RsbV1 and RsbV2 also hints at a potential non-PSM or expanded PSM role for RsbV1. The in vitro redox findings need to be further explored in an in vivo model. Collectively, we think the expansion of the PSM, in addition to other gene gain events, facilitated infection of multi-cellular organisms. Additionally, our data support that the PSM regulates growth/cell differentiation in response to energy/nutrients, and that redox levels and biochemical features of RsbV1 and RsbV2 govern PSM-component interactions. As disruption of normal PSM function significantly reduces production of infectious progeny, compounds targeting the PSM components could serve as novel, narrow spectrum inhibitors.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mervin C. Yoder, Jr., David A. Ingram, Jr., Lawrence A. Quilliam, Mark D. Pescovitz. Includes vitae. Includes bibliographical references (leaves 171-194).

Dans la première partie de cette thèse effectuée en collaboration avec le groupe expérimental de C. Chazaud (Clermont Université), nous avons étudié théoriquement un processus de différenciation cellulaire intervenant avant l’implantation de l’embryon dans l’utérus. Il s’agit de la spécification des cellules de la masse cellulaire interne (MCI) en épiblaste (EPI) et en endoderme primitif (EPr), processus dans lequel les facteurs de transcription Nanog et Gata6 jouent un rôle essentiel. En effet, en absence de Nanog, les cellules de la MCI acquièrent toutes une identité EPr, tandis qu’en absence de Gata6, elles se différencient toutes en EPI. De plus, la voie de signalisation Fgf/Erk active l’expression de Gata6 et inhibe celle de Nanog. Enfin, Nanog active la sécrétion dans le milieu extracellulaire de Fgf4, une molécule qui active la voie de signalisation Fgf/Erk en se liant au FgfR2. Nous avons développé un modèle mathématique pour ce réseau de régulations, fondé sur des équations différentielles ordinaires décrivant l’évolution temporelle des niveaux de protéines Nanog, Gata6, Fgf4 et Fgfr2 et de l’activité de la voie Fgf-Erk. Nous avons validé ce modèle en montrant qu’il récapitule les résultats expérimentaux obtenus in vivo, dans les embryons wild-type et dans les mutants Nanog-/- et Gata6-/-. De plus, l’analyse des résultats du modèle permet de proposer un nouveau mécanisme pour l’émergence d’une population mixte de cellules EPI et EPr au sein de la MCI. Ce mécanisme repose sur le fait que le système décrit par notre modèle peut présenter trois états stationnaires stables, dont les niveaux d’expression de Nanog et Gata6 correspondent à l’EPI, l’EPr et la MCI non-différenciée, respectivement. De plus, le modèle a été utilisé afin d’interpréter des résultats expérimentaux récents et contre-intuitifs, concernant les embryons hétérozygotes Gata6+/-. Enfin, nous avons établi des prédictions théoriques, dont certaines ont été ultérieurement vérifiées en laboratoire. <p>Dans la seconde partie de la thèse, effectuée dans le laboratoire d’O. Pourquié (Université de Strasbourg), nous avons étudié un processus de différenciation in vitro, par une approche expérimentale. Il s’agit de la différenciation des cellules souches embryonnaires (ES) en cellules de mésoderme paraxial, un tissu dont dérivent –au cours du développement embryonnaire– les cellules formant notamment les vertèbres, les côtes, la peau et les muscles squelettiques du dos.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished