Each year, more than 185,000 people in the U.S. lose a limb to injury or pathological conditions. Currently, the only replacement for amputated limbs is the fitting of a prosthetic device. While artificial limbs greatly enhance the lives of amputees, these devices have their limitations. Prosthetics tend to have reduced (or no) function and limited sensation. These devices require charging and maintenance, are expensive, and have a shorter life than a natural limb. Additionally, artificial limbs are not able to completely replace the biological roles of natural bone such as calcium-ion exchange and hematopoiesis. To overcome the limitations of prosthetics, the ideal replacement for an amputated limb would be the regrowth of a patient's own biological limb. Mammals have the ability to regenerate the distal portion of the third phalangeal element (P3). This regeneration response progresses through several distinct phases which are defined in this thesis. The initial phases - inflammation, histolysis, and epidermal closure - are not unique to the P3 amputation response, but are seen following injury to almost every tissue. For mammals, the most common response to injury is a repair process that starts with inflammation, histolysis and wound closure, but produces aberrant collagen deposition and loss of original structure. A mammalian P3 amputation is exceptional in that the initial stages following injury lead to a regeneration event. We aim to understand the initial stages of P3 regeneration and to determine if these stages play a role in creating (or inhibiting) a regeneration-permissive environment. We also examine what factors comprise a regeneration-permissive environment, specifically, how tissue oxygen tensions influence regeneration. We find that regeneration is dependent upon both temporal oxygen fluctuations and the initial influx of inflammatory cells. Future goals, based on this work, are to determine how we can manipulate both oxygen tensions and inflammation to augment the regeneration capabilities of the body. / acase@tulane.edu

Research using the adult mammalian model shows that regeneration in the limb is limited to the distal most portion of the terminal phalanx. Recent studies suggest that the cellular contributions made to the regenerating system are lineage restricted and that the niche bone marrow hematopoietic stem cell population’s contributions are minimal. These studies however, do not address other residing populations within the bone marrow, specifically the mesenchymal and endothelial stem cell populations. One of the residing populations, the reputed pericyte or perivascular cells, possesses the ability to differentiate into multiple other cell types. To assess the potential contribution of perivascular cells to the regeneration competency of the terminal phalanges, we began by identifying perivascular cells within the terminal phalanx by using two accepted pericyte markers: nerve-glial antigen 2 (NG2) and endosialin (TEM1). Using NG2 and TEM1 in conjunction with vascular marker Tie2 in the Tie2-EGFP murine reporter line, we confirm a large number of perivascular cells in the bone marrow’s unusually well-developed and organized vasculature and a lower density within the connective tissue microvasculature; implicating a great potential contribution from the bone marrow. Post-amputation, we observe a large population of NG2+ and TEM1+ cells within the regenerating blastema region. Co-immunohistochemical studies reveal the blastema have cells that co-express osteogenic and pericyte markers; strongly suggestive of a transdifferentiation event. We attempt to confirm our hypotheses made in our initial assessment by utilizing two independent cell tracing studies: a DiI labeling of the bone marrow of the terminal phalanx to identify a marrow derived cellular contribution to the regenerate and a genetic fate tracing study using transgenic NG2CreERTamR26REYFP mice to confirm a transdifferentiation event. Using a novel in vivo method , we DiI-label the bone marrow content before amputation and trace DiI labeled bone marrow derived cellular contributions to the regenerate. DiI labeled cells were observed within the blastema expressing either endothelial, perivascular, or osteogenic markers, confirming the bone marrow contributes multiple cell types during the regeneration process. Using a similar experimental design, we genetically label the terminal phalanx NG2 expressing cells using systemic tamoxifen induction of NG2CreERTamR26REYFP mice. We fate trace the initially labeled population during blastema formation and re-differentiation and observed transdifferentiation events of the perivascular cells into two distinctive lineages, endothelial and osteoprogenitor cells. Establishing a direct correlation between peri-vasculature and re-differentiation, we address NG2/perivascular necessity with a series of temporal loss of function studies using a blocking antibody (iNG2). We implant iNG2 soaked microcarrier beads into various regions of the terminal phalanx and during different stages of the regeneration process. The experiments confirm the necessity of NG2 expression for distal bone elongation, as well as ascertain the temporal nature of the NG2 expression in different microenvironments. These results establish the importance of NG2+ cells in the bone marrow during early stages of regeneration, with early iNG2 bone marrow implantation resulting in a complete failure of the regeneration process. In an attempt to rescue this iNG2 failed regeneration we employ an established position-specific fibroblast cell line that displays a surprising plasticity as a cell-based therapeutic. Through a series of RNAi lentiviral transfection of inhibitors of the TGFβ-BMP pathway we induce osteogenic plasticity in the line. These results reveal regeneration competency associated with the mammalian terminal phalanx is in part due to the ability to recruit local perivascular multipotent populations, which has great translational relevancy. / acase@tulane.edu

acase@tulane.edu

The stress response is an evolutionarily conserved mechanism critical for survival that requires orchestration of different systems in the body. Corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) represent the final common pathway leading to HPA axis activation in response to stress. Noradrenergic inputs to CRH neurons in the PVN provide a powerful drive to activate the HPA axis. Previous anatomical studies have shown that noradrenergic afferents synapse directly on CRH neurons, but electrophysiological analyses indicate that the noradrenergic activation of CRH neurons is mediated primarily by the stimulation of presynaptic glutamatergic neurons. Here, using whole cell patch clamp recordings in identified CRH neurons, I demonstrate that norepinephrine (NE) stimulates excitatory synaptic inputs by activating postsynaptic α1 adrenergic receptors in CRH neurons and inducing the release of the retrograde messenger nitric oxide, which drives upstream glutamate neurons to elicit spike-dependent synaptic glutamate release onto the CRH neurons. Notably, the NE effect is dependent on ATP transmission and astrocytic function, suggesting that astrocytes serve as an intermediary in the retrograde activation of glutamateregic synaptic inputs to the CRH neurons. In addition, I also show that the NE-induced excitation of CRH neurons is stress-status sensitive and corticosterone dependent, in that stress-induced corticosterone causes internalization of membrane α1 adrenergic receptors to desensitize the CRH neurons to NE. Taken together, my findings provide evidence that NE excites CRH neurons in a stress state-dependent manner by a retrograde NO stimulation of local glutamate circuits that is dependent on glial activation. This retrograde trans-neuronal-glial regulation of excitatory synaptic inputs to CRH neurons by NE provides a mechanism for the NE activation of the HPA axis in the early stage of stress response. The stress-/corticosterone-induced desensitization of CRH neurons to NE modulation by the internalization of α1 adrenergic receptors confers a stress state-dependent resistance of the CRH neurons to repeated noradrenergic activation, which provides a mechanism for the negative feedback regulation of the CRH neurons and the HPA axis by stress and glucocorticoids, and a means to restore neuroendocrine homeostasis after stress exposure. / acase@tulane.edu

Schizophrenia is a prevalent neurological disorder characterized by disrupted neuronal circuitry. Antipsychotic drugs (APDs) are capable of ameliorating the symptoms of schizophrenia with varying efficacy. Clozapine, the "gold-standard" for antipsychotic drug treatment, has been shown by this lab to induce the outgrowth of mediodorsal thalamic (MDT) dendritic arbor in rodents, a brain region which has altered function and decreased regional volume in schizophrenic patients. These studies further explored the ability of APD treatment to restructure dendrite arbor and the mechanisms of clozapine's ability to elaborate MDT arbor. Additionally, glutamate hypofunction is thought to contribute to the schizophrenic disease state. Using a novel model of perinatal glutamate hypofunction, we examined the long-term effects on dendritic architecture of developmental glutamate signaling disruption. MDT dysfunction is hypothesized to contribute to cognitive symptoms of schizophrenia. Clozapine has increased efficacy in ameliorating these symptoms. To further understand clozapine’s actions to remodel MDT dendritic architecture, we examined whether clozapine-induced morphological alterations are limited to the thalamus or if they also occur in additional regions associated with cognitive schizophrenic pathology, the hippocampus and striatum. We found that clozapine can induce dendritic remodeling in the hippocampus, but the not to the amplitude of remodeling seen in the thalamus, indicating that the MDT is uniquely altered by clozapine treatment and may be an important locus of clozapine's action. The mechanisms of clozapine's remodeling of MDT arbor, we examined changes to mRNA and miRNA expression and calcium dynamics in the MDT in response to APD treatment. Clozapine-treatment altered the expression of genes involved in cytoskeletal remodeling, external membrane receptors, and calcium dynamics, as well as increased the rate of calcium influx into thalamic neurons. Disruption to glutamate signaling has been hypothesized to contribute to schizophrenic pathology. Disruption to perinatal vesicular glutamate packaging along the corticolimbic axis has long term effects for neuronal morphology and function. Interestingly, we find that disruption along the corticolimbic axis also has downstream effects on MDT dendritic architecture. These studies show that the MDT is an important locus of action for clozapine and is capable of remodeling dendritic architecture in response to afferent circuitry dysfunction. / acase@tulane.edu

In 2005, 1.6 million Americans lived with a debilitating amputation and this figure is predicted to double by 2050. But the ability of a mammal to recapitulate a complex limb structure is not impossible. Evidence of children and mice re-growing digit tips following amputation midway through the terminal phalanx (P3) exists. The hallmark of this phenomenon is development of a blastema housing undifferentiated cells capable of being re-programmed to replicate the missing part. Our central goal is to understand specific components of this process for application into pro-scarring injuries. The mouse digit anatomy is prominently outlined by microfilaments containing ER-TR7, and antigen derived from fibroblast reticular cells (FRCs) of the thymus shown to facilitate intercellular communication to promote lymphoid organogenesis. A unique blastema characteristic is the upregulation of an ER-TR7+ scaffold stemming from half of the blastema population which reverts to its pre-existing pattern after regenerate differentiation concludes. We measured a correlation between ER-TR7 and type III collagen (COL3) at the transcriptional and protein levels both in vitro during induction of ER-TR7 in primary P3 cells and throughout digit regeneration. Co-expression with COL3 sheds light on ER-TR7 identity and allows testing various approaches to manipulation of the scaffold through the better understood mechanism of COL3 regulation. Furthermore, we aimed at determining the origin of ER-TR7+ blastema FRCs. Using bone marrow (BM) transplantation, we generated eGFP+ BM chimeras to study the fate of BM-derived cells (BMDCs) after amputation based on the hypothesis that in the regenerate, multipotent BMDCs contribute to various cellular phenotypes including FRCs. So we tested co-immunolocalization of eGFP with antigens particular to fibroblastic, hematopoietic, endothelial, osteoblastic, and mural cells. Many BMDCs homed to the injury throughout regeneration. But hematopoietic BMDCs were limited to inflammation whereas mesenchymal BMDCs expanded and were primed as ER-TR7+ FRCs in the P3 BM niche prior to homing to the blastema site, where they amounted to nearly 50% of cells. Moreover, BMDCs differentiated into endothelial, osteoblastic, and smooth muscle subpopulations and although diluted by pre-existing progenitors by the endpoint of regeneration, BMDCs persisted as part of various structures thus contributing to long-term function. / acase@tulane.edu

Humans and mice lack the broad regenerative capacity of Urodele amphibians, capable only of partial regeneration of the terminal phalanx (P3), i.e., amputation mid-way through P3 results in essentially complete regeneration of the digit tip mediated via blastema formation and subsequent direct bone formation, culminating in distal bone growth, patterning, and function. Conversely, amputation injuries occurring proximal to the mid-point of P3 result in scar formation. Here, in part, we studied the endochondral bone healing response following amputation of the middle phalanx (P2). We showed the endochondral ossification healing response post amputation of P2 is analogous to the fracture healing response of P2 and other long bones of the body, ultimately proving useful in yielding insight into effectively inducing regeneration of the amputated digit. We showed the periosteal-derived chondrocytes of P2 play an integral role in the bone healing process in that they provide a template for subsequent bone formation following amputation injury. We also showed the periosteal-derived cells can be targeted through the temporal application of BMP2 to accumulate and proliferate at the distal digit tip and thus induce regeneration of the amputated bone. Our studies indicated that P2 amputation injuries of various time points, i.e. previously healed injuries, can be induced to regenerate via re-wounding of the periosteal tissue and subsequent BMP2 application, and thus is immeasurably promising from a translational therapeutic perspective. Lastly, we studied the fracture healing response in conjunction with the intramembranous regeneration response of P3. Following fracture of the digit, we showed the relative lack of periosteal callus formation, the lack of periosteal chondrogenesis, and a novel endosteal/marrow chondrogenic response. Unlike P2, the periosteal tissue of the fractured P3 bone does not respond to BMP2-treatment via endochondral bone growth, instead the bone heals via intramembranous ossification, possibly via intrinsic differentiation limitations and extrinsic factors. Notably, we showed that in the absence of the periosteal tissue of the amputated P3 bone, the regeneration response was greatly attenuated. Taken together, our work blending regeneration and fracture repair may prove useful in enhancing regeneration studies with methods and ideas not previously considered. / acase@tulane.edu

<p>Evidence has shown that hypoxic hearts have greater heart/fetus mass ratio. However, it is still unclear if either hyperplasia or hypertrophy causes the relatively increased heart mass. Furthermore, the genes that might be involved in the process have not yet been identified. In the present study, the cardiac transcriptome was analyzed to identify differentially expressed genes related to hypoxia. Eggs were incubated for 15 and 19 days in two different environments, normoxic and hypoxic. Normalized microarray results were analyzed to isolate differentially expressed probes using the Affymetrix chip. Total RNA was also isolated from another set of fetuses incubated in the same conditions and used to perform a qPCR in order to confirm the microarray results. In the four groups (15N, 15H, 19N, 19H), some probes were differentially expressed. From the eggs incubated for 15 days, the microarray revealed five probes that were differentially expressed according to the criteria (p<0.01 and absolute fold change FC>2) in the two programs (PLIER & RMA) used to normalize the data. From the eggs incubated up to 19 days, eight probes were differentially expressed in both programs. No further tests were performed on the 19 days fetuses since there was no significant difference in that group after incubation for the heart/fetus mass ratio. Apolipoprotein-A1, p22, similar to ENS-1 and b2 adrenergic receptor were further tested in qPCR (15 days sample). The differently expressed genes are linked to cell division and should be further studied to identify their function, especially the similar to ENS-1.</p>

<p>Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex.</p><p>In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs.</p><p>The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.</p>

<p>Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions.</p><p>The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes.</p><p>Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins.</p><p>To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained.</p><p>Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface.</p><p>β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.</p>