No description available.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

No description available.

Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

Cells must regulate their metabolism in order to grow, adapt to changes in nutrient availability and maintain homeostasis. Flux, or the turnover of metabolites, through the metabolic network can be regulated at the allosteric and transcriptional levels. While study of allosteric regulation is limited to biochemical examination of individual proteins, transcriptional control of metabolism can be explored at a systems level. We endeavored to elucidate transcriptional mechanisms of metabolic flux regulation in the model organism Caenorhabditis elegans (C. elegans). We also worked to create a visual tool to explore metabolic pathways that will support future efforts in the research of metabolic gene regulation. C. elegans is a small, free-living nematode that feeds on bacteria and experiences a high level of diversity in nutrient level and composition. Previously, we identified a mechanism by which the essential cofactor, vitamin B12, regulates the expression of genes involved in the degradation of propionate, referred to as B12‑mechanism‑I. This mechanism functions to prevent the toxic accumulation of propionate and requires the TFs NHR-10 and NHR-68. Using genetic screens as well as transcriptomic and metabolomic approaches, we discover a second mechanism by which vitamin B12 regulates metabolic gene expression: B12-mechanism-II. Unlike B12-mechanism-I, B12-mechanism-II is independent of propionate, requires the transcription factor NHR-114 and functions to maintain the metabolic activity of the Methionine/S-adenosylmethionine cycle in a tightly regulated regime. We also present WormPaths, an online resource that allows visualization of C. elegans metabolic pathways and enables metabolic pathway enrichment of user-uploaded transcriptomic data.

Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...

Οι μεταγραφικοί παράγοντες COUP-TF (Chicken Ovalbumin Upstream Promoter Transcription Factor) ανήκουν στην υπεροικογένεια των υποδοχέων στεροειδών/ θυρεοειδών ορμονών και θεωρούνται “ορφανοί”, αφού μέχρι στιγμής δεν έχει βρεθεί το υπεύθυνο πρόσδεμα για την ενεργοποίηση τους. Πειραματικές διαδικασίες στο εργαστήριο μας έχουν δείξει ότι στο πρωτογενές μετάγραφο των COUP-TFs συμβαίνει εναλλακτικό μάτισμα που έχει ως αποτέλεσμα την παραγωγή δύο mRNAs που κωδικοποιούν δύο πρωτεΐνες οι οποίες διαφέρουν ως προς το μέγεθος λόγω της εισαγωγής 21 επίπρόσθετων αμινοξέων στην καρβοξυτελική περιοχή (Carboxy Terminal Extension) της περιοχής πρόσδεσης στο DNA (DBD). Πειράματα EMSA με τη χρήση in vitro μεταφρασμένων πρωτεϊνών, αποκάλυψαν ότι η μεγάλη πρωτεΐνη δεν μπορεί να προσδεθεί σε κανένα COUP-TF στοιχείο απόκρισης. Επίσης παρατηρήθηκε ότι παρουσία της μεγάλης πρωτεΐνης, η ικανότητα της μικρής πρωτεΐνης να προσδένεται στο DNA μειώνεται με ανταγωνιστικό τρόπο, οδηγώντας στο συμπέρασμα ότι το ετεροδιμερές πρωτεϊνικό σύμπλοκο δεν μπορεί να προσδεθεί στο DNA. Σκοπός μας είναι να ερευνήσουμε το ρόλο της ένθεσης των 21 αμινοξέων στην μεγάλη πρωτεΐνη, ως προς την ικανότητα πρόσδεσης της στο DNA. Στον αχινό Paracentrotus lividus, η αμινοξική ένθεση στην καρβοξυτελική περιοχή (CTE) της μεγάλης πρωτεΐνης περιέχει δύο προλίνες. Η υπόθεση μας είναι ότι αυτές οι δύο προλίνες παίζουν σημαντικό ρόλο στην στερεοδιαμόρφωση της πρωτεΐνης, επηρεάζοντας την ικανότητα πρόσδεσης στο DNA. Για να ελέγξουμε την υπόθεση αυτή, προκαλέσαμε σημειακές μεταλλάξεις, μεταλλάσσοντας συγχρόνως τις δύο προλίνες σε αλανίνες αλλά κάθε μία προλίνη σε αλανίνη μεμονωμένα, καθώς επίσης μελετήσαμε και μια σειρά εσωτερικών αμινοξικών ελλειμάτων μέσα στην ένθεση των 21 αμινοξέων στην καρβοξυτελική περιοχή. Το σύνολο των μεταλλάξεων έδειξε ότι οι μεταλλαγμένες μεγάλες πρωτεΐνες δεν προσδένονται στο DNA καθώς επίσης ότι οι μεταλλαγμένες μεγάλες πρωτεΐνες ετεροδιμερίζονται πιο αποτελεσματικά με την μικρή ισομορφή πιθανότατα λόγω επαγόμενης αλλαγής της στερεοδιαμόρφωσης της μεταλλαγμένης πρωτεΐνης. Επίσης μελετήσαμε την εξάπλωση του εναλλακτικού ματίσματός στα Δευτεροστόμια, χρησιμοποιώντας ειδικούς εκφυλισμένους εκκινητές σε πειράματα PCR. Εναλλακτικό μάτισμα των μεταγράφων COUP-TF παρατηρήθηκε επίσης στους οργανισμούς Sphaerechinus granularis (Εχινόδερμο) και Saccoglossus kowalevskii (Ημιχορδωτό). / COUP-TFs (Chicken Ovalbumin Upstream Promoter- Transcription Factors) belong to the superfamily of steroid/thyroid hormone receptors and they are consider orphans since the proper ligand that activates them is not yet found. Experimental procedures in our laboratory have shown that in Echinoderms the alternative splicing of the COUP-TF primary transcript results in two mRNAs which encode two protein variants that differ by a 21 amino acid insertion in the Carboxy Terminal Extension (CTE) of the DNA Binding Domain (DBD). EMSA experiments with the use of in vitro translated proteins revealed that the large protein variant is incapable of binding any COUP-TF response elements. Furthermore, in the presence of the large variant, the small COUP-TF protein ability to bind DNA is diminished in an antagonistic way, suggesting that the heterodimeric protein is also incapable of DNA binding. Our aim is to investigate the role of the 21aa insertion in the large variant regarding the DNA binding affinity. In sea urchins the CTE insertion in the large variant contains two prolines. Our hypothesis is that these two prolines play an important role in the protein‟s conformation which in turn is responsible for the loss of DNA binding. To check this, we created point mutations by mutating both prolines to alanines simultaneously and then each proline to alanine separately. We also analyzed a series of internal amino acid deletions within the 21aa insertion of the CTE. All the mutations proved that the large mutated proteins are incapable of binding DNA and that they heterodimerize more effectively with the small protein variant possibly because of the changed conformation of the large protein variant. We also studied the alternative splicing among Deuterostomes, by using degenerate primers in PCR experiments. We observed that alternative splicing of COUP-TF transcripts occurs in the sea urchin Sphaerechinus granularis and in the hemichordate Saccoglossus kowalevskii.

Intestinal epithelial cells function, in part, to detect infection with pathogenic organisms and are key regulators of intestinal immune homeostasis. However, it is not fully understood how intestinal epithelial cells sense pathogen infection and coordinate the induction of protective immune defenses. Here, we define two new mechanisms of innate immune regulation in a metazoan host. First, we characterize the first bacterial pattern recognition receptor and its natural ligand in Caenorhabditis elegans. We show that the C. elegans nuclear hormone receptor NHR-86/HNF4 directly senses phenazine-1-carboxamide (PCN), a metabolite produced by pathogenic strains of Pseudomonas aeruginosa. PCN binds to the ligand-binding domain of NHR-86/HNF4, a ligand-gated transcription factor, and activates innate immunity in intestinal epithelial cells. In addition, we show that C. elegans NHR-86 senses PCN, and not other phenazine metabolites, as a marker of pathogen virulence to engage protective anti-pathogen defenses. Second, we show that a phase transition of the C. elegans Toll/interleukin-1 receptor domain protein (TIR-1) controls signaling by the C. elegans p38 PMK-1 MAPK pathway. Physiologic stress, both P. aeruginosa infection and sterol scarcity, induce multimerization of TIR-1 within intestinal epithelial cells. Like the mammalian homolog of TIR-1, SARM1, oligomerization and phase transition of C. elegans TIR-1 dramatically potentiate its NAD+ glycohydrolase activity. TIR-1/SARM1 multimerization and NAD+ glycohydrolase activity are required for activation of C. elegans p38 PMK-1 pathway signaling and pathogen resistance. These data uncover a mechanism by which nematodes interpret environmental conditions to prime innate immune defenses and promote survival in microbe rich environments. C. elegans animals augment these immune defenses by surveying for ligands specifically associated with toxigenic pathogens that are poised to cause disease. These findings define a new paradigm of intestinal immune control that informs the evolution of innate immunity in all metazoans.