Functional genomics in the stroke-prone spontaneously hypertensive rat: genome wide and candidate gene analysis

The stroke-prone spontaneously hypertensive rat (SHRSP) is an inbred model of hypertension. Renal microarrays and functional genomic strategies investigated chromosome 2 candidate hypertension genes, focussing on the oxidative-stress defence gene, glutathione s-transferse mu type 1 (Gstm1). Ingenuity pathway analysis of renal microarrays in 5 and 16-week SHRSP, normotensive Wistar Kyoto (WKY) and chromosome 2 congenic rats identified differential expression of several glutathione cycling genes. The Gstm1 promoter was investigated by luciferase and Transfac bioinformatic analysis, implicating two polymorphism clusters and several transcription factors in reduced SHRSP Gstm1 expression. Recombinant adenoviruses expressing Gstm1 and short-hairpin RNA-interference sequences to reduce Gstm family expression were produced. In-vivo overexpression of Gstm1 did not improve endothelial nitric-oxide bioavailability in SHRSP carotid arteries. Bacterial artificial chromosome and linear expression constructs were purified for production of Gstm1 transgenic rats, putative transgenic rats were screened by PCR. The strategies developed in this project are an example of thorough functional genomic analysis in experimental hypertension research

The Role of Red Blood Cell Membrane Rigidity on Cellular and Drug Particle Carrier Dynamics in Blood Flow

Blood and heart-related diseases remain a significant challenge for modern-day medicine. Blood cell-related diseases have also proven to be challenging to understand and treat, specifically diseases involving the loss of deformability (rigid) in red blood cells (RBCs). Disease involving rigid RBCs are typically of genetic origin and thus limit treatment options and treatment efficacy. Rigid RBC disorders give rise to many medical complications, including vaso-occlusion, pulmonary hypertension, and cardiac dysfunction. Patients inflicted with Sickle Cell Disease (SCD), hereditary spherocytosis, iron-deficient anemia, pyruvate kinase deficiency, human immunodeficiency virus (HIV), malaria, sepsis, and even natural aging all have less deformable (rigid) RBCs than healthy patients. Rigid RBCs cause major physical damage when traveling through the body by occluding microvasculature, depriving tissues of nutrients, and damaging walls of the spleen, liver, and lungs. The core work presented in this dissertation aims to probe how decreases in RBC deformability affect hemodynamics and impact functionality of other blood cells, clarifying the pathology of RBC-related diseases. We initially present a model of artificially rigidified human RBCs which offers an experimental control over extent of membrane stiffness as well as the fractional composition of rigid RBCs in whole blood. Here, we find that the presence of rigid RBCs in blood flow significantly alters the ability of immune cells to adhere to inflammation on the vascular wall of a microfluidic model. In some cases, the presence of highly rigid RBCs reduces leukocyte adhesion to the vascular wall by up to ~80%. Following this initial investigation, we take a pivotal focus on SCD and further quantifying the whole blood characteristics of SCD pediatric patient blood and its behavior in flow. This thesis presents multiple investigations highlighting the outcome of RBC rigidity in SCD. An interesting clinical case study is highlighted in this work as well as additional in vitro work showing how the presence of RBC rigidity alters immune cell adhesion functionality. An artificial model of blood infusion therapy is also developed to test how leukocyte adhesion to inflammation is impacted upon alteration of whole blood composition. This knowledge is essential in understanding why people with diseases related to RBC deformability are susceptible to infection and have irregular immune responses. In addition, we also investigate how rigid RBCs in blood flow alter the adhesion efficacy of vascular-targeted carriers (VTCs). The field of drug delivery has taken an interest in combating numerous blood and heart diseases such as atherosclerosis via the use of VTCs. Ideally, VTC technology increases drug delivery efficacy and simultaneously reduces cytotoxic effects, precisely localizing drugs only to the disease site through receptor-ligand interactions. Cellular interactions are not yet fully understood. The dynamics of disease-inflicted cells (rigid RBCs) are even less understood, thus compounding the problem of efficient VTC design under diseased blood conditions. We investigate various particle design parameters and assess their vascular wall adhesion performance in the presence of rigid RBCs. We find the vascular adhesion of stiff microparticles is reduced by up to ~50% in the presence of rigid RBCs. Interestingly, deformable hydrogel microparticles can experience an increase in vascular adhesion of up to ~ 80%. This work explores an opportunity to develop new therapeutics with high efficacy in diseased blood.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169853/1/gutieman_1.pd

Identification of novel therapeutic targets in Osteosarcoma for the development of Nanoparticle based drug delivery systems

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonOsteosarcoma (OS) is the most common primary malignant tumour of the bone, with a high incidence rate in children and adolescents. Importantly, OS therapeutics has remained unimproved for the last 30 years and therefore, clinically unsatisfactory. It is understood that determining the mechanisms underlying OS tumorigenesis and progression, along with the identification of novel therapeutic targets can greatly aid in the clinical management of the disease. Thus, one important area in need of further exploration is the tumour microenvironment (TME), which consists of a plethora of extracellular matrix components, tumour associated fibroblasts and immune cells. To determine the underlying mechanism(s) and identify novel therapeutic targets, we searched an OS gene microarray dataset deposited within the Gene Expression Omnibus, which included 84 primary OS biopsies and 12 primary Mesenchymal stem cell control samples. An alternative to R coding (GEO2R) was utilised to identify differentially expressed genes (DEGs) in OS, with further R analysis conducted to quantify levels of genetic association to OS. 1161 DEGs in OS were identified, consisting of 535 upregulated and 626 downregulated genes at cut-offs of |log2FC|>1 and an adjusted P-value<0.01. Through functional annotations, we show that the DEGs are involved in immune system processes, including defence and general immune responses. This suggests that the immune system is strongly linked to the OS microenvironment with DEGs potentially contributing to OS development and metastasis. For the identification of immune based therapeutic targets in the OS TME, upregulated genes were additionally refined based on the gene ontology (GO) term “immune receptor activity”. Through GO based refinement, the immune receptor Formyl-Peptide Receptor 3 (FPR3) was found to be significantly upregulated in OS and to have a close genetic association. Through both in vitro and in vivo staining, we first show that FPR3 is specifically expressed on malignant bone cells of mesenchymal origin, with a lack of expression on normal bone cells. Thereafter, through functional in vitro cell migration assays, we determined its potential in becoming a therapeutic target in OS. Results showed that when targeted with a FPR3 specific peptide antagonist (WRW4), cell migration in two OS cell lines were significantly reduced. Observable differences in responsiveness to the peptide antagonist, between the two assayed OS cell lines, could be explained by differences in receptor expression levels as shown by flow cytometric analysis. An alternate role could relate to its use as a prognostic biomarker in OS. For the therapeutic targeting of FPR3 in OS, we propose the use of Nanoparticle based drug delivery systems (NP-DDS). Nanoparticles (NPs) are highly promising tools for both clinical and therapeutic purposes. The surfaces of NPs can be modified for targeted therapy and the NPs themselves are able to controllably release drugs when used as drug carriers in NP-DDS. However, NPs are known to exhibit varying levels of toxicity, therefore, their biosafety still remains a concern. Here, we put forward the Galleria Mellonella (GM) model organism as an efficient, low-cost, high-throughput tool for determining NP toxicity in vivo. We utilise, for the first time, techniques such as flow cytometry, immunohistochemistry and a 4HNE ELISA, on GM larvae for the determination of NP-induced toxicity, in vivo. Based on the various assays, NP toxicity was found to be heavily dependent on physicochemical properties such as surface composition and size. In addition, we investigate cellular ROS production, both in vitro and in vivo, to determine the mechanism’s likelihood in playing a major role in NP induced toxicity, observed in vivo. Results show that intracellular ROS production is the likely mechanism underlying NP toxicity in vivo, however, tissue aggregation is an important feature of CNTs that induces strong toxicity. Lastly, SPION induced immunotoxicity was observed in vivo with the use of GM larvae. Therefore, we aimed to determine if NP intracellular trafficking, intracellular fate and in particular, SPION related endosomal escape played a role in SPION induced immunotoxicity. Preliminary data is presented for this aspect in this study. Overall, certain NP variants that were identified to be biologically safe, can be put forward as potential drug delivery agents for the treatment of diseases such as OS. These NPs can be designed for tumour specific delivery of therapeutic compounds such as receptor peptide ligands, whereby, the tumour target can be a receptor of interest with therapeutic potential such as FPR3

Sustainability of Olive Oil System

Sustainability, defined as ‘meeting current needs without compromising the future’, is a widely accepted goal across many sectors of society. Sustainability’s criteria and indicators often only regard sustaining present conditions through increased resilience, intended as a system’s capacity to experience shocks while retaining essentially the same functions and structures. However, new sustainability concepts, sometimes referred to as “sustainagility”, also consider the properties and assets of a system that sustains the ability (agility) of agents to adapt and meet their needs in new ways, preparing for future unpredictability and unforeseen changes. Therefore, resilience must coexist with adaptive capacity for real, long-term sustainability. Consumers are paying increasing attention to the sustainability of the food supply chain; thus, sustainable development is necessary for all food processes. Since the olive oil sector has a well-established historical tradition, any change and innovation that aims to obtain a sustainable development not only needs to be analyzed in terms of environmental, economic, and social aspects, it should also be significantly improved and closely monitored. Thus, this Special Issue is a collection of papers that can increase sustainability knowledge in the olive-oil-processing chain, to take a significant step forward in future developments

Novel therapies for cancer treatment : designing high affinity selective ligands against SIRT1 enzyme

The word ‘Sirtuin’ or Sir2 proteins are a class of proteins that possess either mono-ADP-ribosyltransferase, or deacylase activity, including deacetylase. SIRT1 is the most studied mammalian Sirtuins and predominantly localised in the nucleus and cytoplasm. Many Sirtuins targets are involved in cancer and in many types of cancers, SIRT1 is found to be overexpressed. Recent observations support SIRT1 being both an oncogene and a tumour suppressor, depending on the cancer etiology and type of tissue. To answer the question “How can SIRT1 behave as a tumour suppressor?”, highly selective ligands (aptamers) were developed against SIRT1 enzyme as the first step towards the development of an alternative chemotherapy for cancer diseases. The objectives of current study are to: (i) produce by in vitro SELEX procedures, SIRT1 binding single-stranded DNA (ssDNA) aptamers; (ii) characterise the interactions between selected aptamers and SIRT1 in vitro and determine their equilibrium dissociation constant (KD) values and; (iii) investigate the effects of selected aptamers on cancer cell lines. To achieve these objectives, ssDNA aptamers capable of binding SIRT1 enzyme were generated in vitro using a sequential approach known as SELEX. A total of eight novel SIRT1 aptamers (circular and linear), four circular aptamers from 8 rounds of circularisation-SELEX procedure, and the other four linear aptamers from 12 rounds of a basic-SELEX procedure were generated.The initial screening using the Fluor de Lys-SIRT1 assay for SIRT1 enzymatic activity in vitro indicated that an activator SIRT1 enzyme (circular3, circular4, linear3 and linear4) were obtained, these aptamers showed acceptable values of Km and Vmax to SIRT1 enzyme in kinetic characterisation studies. After equilibrium binding characterisation study of both linear and circular aptamers by SPR, it was show that circular3 and linear3 aptamers are good binder to SIRT1 enzyme, with the low KD constant (27.07± 0.959 nM and 48.3± 0.986 nM) respectively with highly exhibited stability for circular3 in human plasma. To investigate the effects of aptamers in cancer cell lines, it has been found that the lung cancer epithelial model A549, the colorectal adenocarcinoma model Caco-2, the liver hepatic model HepG2 were very sensitive with an IC50 (0.32, 0.67, 0.2μM) respectively. Both breast cancer models (MCF-7 and MDA-MB-468) were highly sensitive with an IC50 (0.14 and 0.13 μM), the very difficult to treat MCF-7 and the extremely challenging oestrogen negative MDA-MB-468 proved to be substantially sensitive with the longer exposure. A special mention should be the osteosarcoma model U2OS, this cancer is very prevalent as bone cancer in children and adults over 60 years of age, with prognosis being related to the cancer stage and current treatment leaving extremely non-desirable side effects, the aptamer was very effective on this cell line with IC50 = 0.06 μM. Notably, pre-treatment of adult human keratinocyte HaCaT cells with aptamers resulted in markedly decreased cell viability and the IC50 = 0.123 μM. The most interesting point who that the aptamer was very safe on normal cell line Beas-2b, which indicated that it is safe to non-cancerous tissue. In conclusion, a pharmacological activation of SIRT1 enhanced cell death suggesting a tumour suppressive function of SIRT1 and the high-affinity SIRT1- aptamers identified in this study may be used in the future for cancer treatment

Differential Roles of PRDM16 Isoforms in Normal and Malignant Hematopoiesis

PRDM16 is a transcriptional co-regulator that is highly expressed in HSCs and required for their maintenance. It is also involved in translocations in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and T-cell acute lymphoblastic leukemia. Prdm16 is expressed as both full-length (f Prdm16) and short-length (s-Prdm16) isoforms, the latter lacking an N-terminal PR domain homologous to SET methyltransferase domains. The roles of both isoforms in normal and malignant hematopoiesis are unclear. In chromosomal rearrangements involving PRDM16, the PR domain is deleted. Furthermore, overexpression of s-Prdm16, but not f-Prdm16, can cause leukemia in a p53-/- background predisposed to malignancy. Based on this, s-Prdm16 has been proposed as an oncogene whereas f-Prdm16 has been suggested to possess tumor suppressor activity. The aim of this thesis was to more clearly elucidate the role of each Prdm16 isoform in normal and malignant hematopoiesis. We first showed that Prdm16 is essential for adult HSC maintenance using a conditional deletion mouse model specific for hematopoietic cells, as previous findings using an embryonic-lethal global Prdm16-/- mouse demonstrated this only in fetal liver. We then found, using a specific f-Prdm16-/- mouse model, that full-length Prdm16 is essential for HSC maintenance and induces multiple genes involved in GTPase signaling and represses inflammation. Based on a comparison of Prdm16-/- HSCs lacking both isoforms, and f-Prdm16-/- HSCs which express s-Prdm16, we were able to infer some hematopoietic properties of s-Prdm16 – namely that this isoform induces inflammatory gene expression and supports development of a Lineage-Sca1+cKit- lymphoid progenitor distinct from CLPs which predominantly differentiates into marginal zone B cells. s-Prdm16 expression alone, however, was not sufficient to maintain HSCs. We used a mouse model of human MLL-AF9 leukemia and found that leukemia derived from Prdm16-deficient HSCs had extended latency, although expression of Prdm16 decreases during MLL-AF9 transformation and is undetectable in ex vivo leukemic cells. Forced expression of f-Prdm16 in these cells further extended leukemic latency, while forced expression of s-Prdm16 shortened latency. Gene expression profiling using RNAseq indicated that forced expression of f-Prdm16 resulted in altered respiratory metabolism of MLL-AF9 cells, whereas expression of s-Prdm16 induced a strong inflammatory gene signature, comparable to that seen in HSCs expressing only s-Prdm16. Several inflammatory cytokines and chemokines induced by s-Prdm16 are associated with MDS and with a worse prognosis in human AML. Furthermore, leukemia expressing s-Prdm16 had an elevated number of cells with abnormal nuclei, characteristic of dysplasia. Finally, we performed an analysis of PRDM16 in human AML from the publically-available Cancer Genome Atlas dataset, containing clinical and gene expression data for 179 cases of AML. PRDM16 expression negatively correlated with overall survival, both in the entire dataset and in the NPM1 mutated and MLL¬-rearranged subsets, and s-PRDM16 exhibited a stronger correlation than f-PRDM16. HOX gene expression correlated with PRDM16 expression, suggesting that HOX genes may positively regulate PRDM16 expression in AML. In NPM1-mutant and MLL-rearranged subsets of AML, we also found that high PRDM16 expression correlated with an inflammatory gene signature, thus corroborating our findings in mouse MLL-AF9. Our findings demonstrate distinct roles for Prdm16 isoforms in both normal hematopoiesis and AML, and identify s-Prdm16 as one of the drivers of prognostically-adverse inflammatory gene expression in leukemia

Role of Stathmin-1 in Colorectal Cancer Metastasis and Chemo-resistance

Ph.DDOCTOR OF PHILOSOPH

Biokinematic analysis of human body

Thesis (Doctoral)--Izmir Institute of Technology, Mechanical Engineering, Izmir, 2011Includes bibliographical references (leaves: 118-123)Text in English; Abstract: Turkish and Englishxiii, 123 leavesThis thesis concentrates on the development of rigid body geometries by using method of intersections, where simple geometric shapes representing revolute (R) and prismatic (P) joint motions are intersected by means of desired space or subspace requirements to create specific rigid body geometries in predefined octahedral fixed frame. Using the methodical approach, space and subspace motions are clearly visualized by the help of resulting geometrical entities that have physical constraints with respect to the fixed working volume. Also, this work focuses on one of the main areas of the fundamental mechanism and machine science, which is the structural synthesis of robot manipulators by inserting recurrent screws into the theory. After the transformation unit screw equations are presented, physical representations and kinematic representations of kinematic pairs with recurrent screws are given and the new universal mobility formulations for mechanisms and manipulators are introduced. Moreover the study deals with the synthesis of mechanisms by using quaternion and dual quaternion algebra to derive the objective function. Three different methods as interpolation approximation, least squares approximation and Chebyshev approximation is introduced in the function generation synthesis procedures of spherical four bar mechanism in six precision points. Separate examples are given for each section and the results are tabulated. Comparisons between the methods are also given. As an application part of the thesis, the most important elements of the human body and skeletal system is investigated by means of their kinematic structures and degrees of freedom. At the end of each section, an example is given as a mechanism or manipulator that can represent the behavior of the related element in the human body