The role of auxin in cell specification during arabidopsis embryogenesis

Auxin is a structurally simple molecule, yet it elicits many different responses in plants. In Chapter 1 we have reviewed how specificity in the output of auxin signaling could be generated by distinct regulation and the unique properties of the members of the Aux/IAA and ARF transcription factor families. In Chapter 2 we further investigated the generation of specificity in auxin responses by generating a set of sensitive transcriptional reporter lines for all Arabidopsis ARFs. This facilitated a comprehensive identification of the ARF complement within a cell/tissue of interest. Our analysis of ARF expression in the root meristem revealed both ubiquitous and specific ARF expression patterns and ARF subsets that distinguished the actively dividing cells from those undergoing elongation. Moreover, a striking correlation between cell type and ARF expression patterns was revealed in the early Arabidopsis embryo, where each cell type expressed a unique ARF complement. In Chapter 3 we characterized a novel cell-autonomous auxin response is required for hypophysis specification and root meristem initiation, and identify Aux/IAA and ARF transcription factors that mediate this response. We show that auxin response components in the proembryo and the suspensor are intrinsically different, and their regulated, lineage-specific expression creates a prepattern enabling different developmental auxin responses. Surprisingly, we find that, in addition to mediating hypophysis specification, auxin response also acts to maintain suspensor cell identity. We show that auxin controlled maintenance of suspensor cell identity includes repression of the embryonic program. This finding gave us an experimental system in which to investigate suspensor cell identity and embryonic transformation. In Chapter 4 the targeted and specific inhibition of auxin response in the suspensor was coupled to new embryo dissection techniques and a microarray based approach was used to generate a unique dataset which was subsequently mined for cell identity regulators. Unexpectedly, inhibition of auxin response induced the misregulation of thousands of genes, prior to gross morphological changes, revealing a high degree of transcriptional plasticity in these cells. This complicated the identification of regulators. Moreover, the dataset also included secondary/indirect changes in embryo expressed genes, which were inevitable given the connectivity and developmental connectedness between the embryo and suspensor. One of the most striking findings from analysis of the dataset generated in Chapter 4 was the convergent regulation of members of many gene families involved in all facets of auxin homeostasis, as investigated in Chapter 5. It appears that transient auxin response inhibition is sensed as an auxin minimum and in general auxin homeostasis genes were activated or repressed in such a way that would increase cellular auxin levels (and response). Finally, many bHLH superfamily members were misregulated upon the inhibition of suspensor auxin response and subsequently found to have specific expression patterns in the embryo, the focus of in Chapter 6. Several bHLHs were shown to lose their lineage specific expression patterns upon inhibition of auxin response in the suspensor, validating further research to place these factors into the auxin response pathways controlling cell identity in the embryo. </p

Exploring the Potentials and Limitations of Solid Tumor Treatment by Thermosensitive Liposomes and Hyperthermia

Traditional chemotherapy for solid tumors is often associated with undesired side effects in treated patients. Furthermore, infused drugs are rapidly cleared from the circulation which largely hinders accumulation in the target tissue. An emerging therapeutic strategy consists in the encapsulation of chemotherapeutic drugs inside liposomes in order to prevent side effects and rapid clearance. Throughout the last decades of research, there has been an increasing interest in liposomes that are able to locally release their contents at the site of solid tumors. This has led to the initiation of clinical trials for the evaluation of thermosensitive liposomes (TSLs), which are drug-loaded nanoparticles that rapidly release the drug within mildly heated tissue (40-43 °C). However, these liposomes are relatively unstable in circulation during the tumor heating period, which may lead to sub-optimal drug delivery to the tumor. This thesis first describes the improved design of TSLs for enhanced cancer therapy, as well as a comprehensive characterization of such formulations in a pre-clinical setting. Subsequently, a comparative study was conducted to assess the influence of varying delivery strategies, which focused either on intravascular drug release of TSLs or their prior accumulation in the tumor tissue, in generating a therapeutic response. Furthermore, the relationship between variation in tumor biology, drug delivery kinetics, and therapeutic response was investigated. Lastly, a feasibility study on magnetic nanoparticle entrapment into liposomes was performed for potential applications in image-guided drug delivery. The present findings contribute to the improvement of existing therapies using TSLs, provide an improved understanding of the mechanism of their delivery, and of cancer types that are more likely to respond to TSL-based therapy

Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue

Abstract In an experimental model of unilateral pneumonia caused by Klebsiella pneumoniea in rats we investigated whether intravenous administration of liposomes with prolonged blood circulation time resulted in significant localization of liosomes in infected lung tissu. Liposomes (100 nm) composed of hydrogenated phosphatidylinositol:hydrogenated phosphatidylcholine:cholesterol (mola ratio, 1:10:5) radiolabeled with gallium-67-deferoxamine showed relatively long blood circulation time. The degree of localization of these long circulating liposomes in the infected lef lung was significantly higher compared to that of localization of 110 nm egg phosphatidylglycerol:egg phosphatidylcholine:cholesterol (molar ratio, 1:10:5) liposomes which exhibited relatively short blood circulation time. At 16 h after administration of the long circulating liposomes (when 10% of the injected dose was still present in the bloodstream) localization of liposomes in the infected left lung was increased up to 10-fold compared to the left lung of uninfected rats, and appeared to be highly correlated with the intensity of the infection. In the uninfected right lung the localization of long circulating liposomes was not increased. The degree of localization of liposomes in the infected tissue is dependent on the residence time of liposomes in the blood compartment. The extent of localization of long circulating liposomes in infected tissue appeared to be dependent on the liposomal dose administered

Investigation of particle accumulation, chemosensitivity and thermosensitivity for effective solid tumor therapy using thermosensitive liposomes and hyperthermia

Doxorubicin (Dox) loaded thermosensitive liposomes (TSLs) have shown promising results for hyperthermia-induced local drug delivery to solid tumors. Typically, the tumor is heated to hyperthermic temperatures (41-42 °C), which induced intravascular drug release from TSLs within the tumor tissue leading to high local drug concentrations (1-step delivery protocol). Next to providing a trigger for drug release, hyperthermia (HT) has been shown to be cytotoxic to tumor tissue, to enhance chemosensitivity and to increase particle extravasation from the vasculature into the tumor interstitial space. The latter can be exploited for a 2-step delivery protocol, where HT is applied prior to i.v. TSL injection to enhance tumor uptake, and after 4 hours waiting time for a second time to induce drug release. In this study, we compare the 1- and 2-step delivery protocols and investigate which factors are of importance for a therapeutic response. In murine B16 melanoma and BFS-1 sarcoma cell lines, HT induced an enhanced Dox uptake in 2D and 3D models, resulting in enhanced chemosensitivity. In vivo, therapeutic efficacy studies were performed for both tumor models, showing a therapeutic response for only the 1-step delivery protocol. SPECT/CT imaging allowed quantification of the liposomal accumulation in both tumor models at physiological temperatures and after a HT treatment. A simple two compartment model was used to derive respective rates for liposomal uptake, washout and retention, showing that the B16 model has a twofold higher liposomal uptake compared to the BFS-1 tumor. HT increases uptake and retention of liposomes in both tumors models by the same factor of 1.66 maintaining the absolute differences between the two models. Histology showed that HT induced apoptosis, blood vessel integrity and interstitial structures are important factors for TSL accumulation in the investigated tumor types. However, modeling data indicated that the intraliposomal Dox fraction did not reach therapeutic relevant concentrations in the tumor tissue in a 2-step delivery protocol due to the leaking of the drug from its liposomal carrier providing an explanation for the observed lack of efficacy

Docetaxel-loaded liposomes: The effect of lipid composition and purification on drug encapsulation and in vitro toxicity

Docetaxel (DTX)-loaded liposomes have been formulated to overcome DTX solubility issue, improve its efficacy and reduce its toxicity. This study investigated the effect of steric stabilisation, varying liposome composition, and lipid:drug molar ratio on drug loading and on the physicochemical properties of the DTX-loaded liposomes. Size exclusion chromatography (SEC) was used to remove free DTX from the liposomal formulation, and its impact on drug loading and in vitro cytotoxicity was also evaluated. Liposomes composed of fluid, unsaturated lipid (DOPC:Chol:DSPE-PEG2000) showed the highest DTX loading compared to rigid, saturated lipids (DPPC:Chol:DSPE-PEG2000 and DSPC:Chol:DSPE-PEG2000). The inclusion of PEG showed a minimum effect on DTX encapsulation. Decreasing lipid:drug molar ratio from 40:1 to 5:1 led to an improvement in the loading capacities of DOPC-based liposomes only. Up to 3.6-fold decrease in drug loading was observed after liposome purification, likely due to the loss of adsorbed and loosely entrapped DTX in the SEC column. Our in vitro toxicity results in PC3 monolayer showed that non-purified, DTX-loaded DOPC:Chol liposomes were initially (24h) more potent than the purified ones, due to the fast action of the surface- adsorbed drug. However, we hypothesize that over time (48 and 72h) the purified, DTX-loaded DOPC:Chol liposomes became more toxic due to high intracellular release of encapsulated DTX. Finally, our cytotoxicity results in PC3 spheroids showed the superior activity of DTX-loaded liposomes compared to free DTX, which could overcome the DTX poor tissue penetration, drug resistance, and improve its therapeutic efficacy following systemic administration

Auxin enters the matrix - assembly of response machineries for specific outputs

The basic mechanism of auxin as a modulator of gene expression is now well understood. Interactions among three components are required for this process. Auxin is first perceived by its receptor, which then promotes degradation of inhibitors of auxin response transcription factors. These in turn are released from inhibition and modify expression of target genes. How this simple signaling pathway is able to regulate a diverse range of auxin responses is not as well understood, however a clue lies in the existence of large gene families for all components. Recent data indicates that diversification of gene expression patterns, protein activity, and protein-protein interactions among components establishes a matrix of response machineries that generates specific outputs from the generic auxin signa