As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Breast cancer is characterized in part by chronic inflammation and tissue remodelling in the mammary gland. Adipocyte enhancer binding protein 1 (AEBP1), a pro-inflammatory protein, is up-regulated in breast cancer and enhances cytokine secretion in the mammary tumour microenvironment. AEBP1 over-expression in cultured macrophages resulted in increased enzymatic activity of MMP-9, a matrix metalloproteinase implicated in processing cytokines and stimulating tumour cell growth and mobility. MMP-9 activates the cytokine tumour necrosis factor-alpha (TNF?), and is required for the transformation of epithelial cells by the cytokine interleukin 6 (IL6). Treatment of epithelial cells with TNF? and IL6, both of which promote tumourigenesis, induced AEBP1 expression. Chromatin immunoprecipitation results suggested AEBP1 induction is directly mediated by pro-inflammatory transcription factors NF-?B and STAT3, downstream effectors of TNF? and IL6, respectively. AEBP1 induction may enhance inflammation, thereby contributing to cell proliferation and survival.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit” hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with increased activation of the proinflammatory transcription factor NF-κB, and augmented expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which resides in plasma membrane lipid rafts. The objective of this work is to define cellular mechanisms of macrophage priming by oxidative stress following shock resuscitation. The main hypothesis investigated is that altered cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation strategies can diminish these effects. AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface levels of TLR4, which in turn results in augmented NF-κB translocation in response to small doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy iii transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress can be diminished by early exposure to resuscitation regimens with direct or indirect systemic antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline. Hyperosmolarity was found to modulate AM TLR4 gene and protein expression. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling pathway early during shock resuscitation may represent an anti-inflammatory strategy able to ameliorate late morbidity and mortality following S/R.

No description available.

Synthetic biology is a fairly recent field that aims to engineer novel functions in biological systems. In a broad sense synthetic biology encompasses the development of tools that makes the engineering of biology easier. In this thesis I develop a collection of standard DNA parts (Biobricks) that consists of a tool to build custom eukaryotic plasmids. This is not just intended for biology researchers in the field of synthetic biology, but also for more general use. Besides the development of molecular biology tools that facilitate the engineering of biology, synthetic biology researchers have implemented devices that are electronics-like in behavior and have demonstrated the potential of the field for the production of biofuels, pharmaceutics and biosensors. Here I present a sensor of iron regulatory protein activity, based on Biobricks. To demonstrate its use I apply it to the study of a novel reconstituted two cell-type co-culture (BNL CL.2 and RAW 264.7), surrogate for hepatocyte-macrophage communication / La biología sintética es un campo recientemente desarrollado con el objectivo de implementar nuevas funciones en sistemas biológicos. De forma global, la biología sintética incluye el desarrollo de herramientas para facilitar la ingeniería de sistemas biológicos. En diversas publicaciones, investigadores en el campo de la biología sintética han implementado dispositivos que funcionan de forma similar a circuitos electrónicos y han demonstrado el potencial del campo para la producción de biocarburantes, farmaceuticos y biosensores. Para la presente tesis he creado una colección de plasmidos estandarizados (Biobricks) que pueden ser de interés para biólogos fuera del campo da la biología sintética. Además, utilizando estos Biobricks, he creado un sensor de la actividad de las proteínas reguladas por el hierro. Para demonstrar su aplicación, he utilizado el sensor para estudiar un nuevo sistema de co-cultura de dos tipos celulares (BNL CL.2 y RAW 264.7), substituto para la comunicación entre hepatocitos y macrófagos

Wound healing is governed by a complex cascade of related processes, involving cells, extracellular matrix and cytokines. In adults this always results in a scar whilst embryonic wound healing is scarless and extensive research worldwide is aimed at reducing scarring in adults. A mathematical framework for problems in dermal wound healing is developed that incorporates models of the individual processes involved. Cells are modelled as discrete individuals. Cytokines and other biologically active factors are modelled as continua. A novel tensorial approach is taken to modelling the extracellular matrix. The numeric and computational challenges associated with combining models for the individual processes are identified and investigated. These include the development of data structures and numeric methods for the continuous and discrete species. Effective visualisation methods for the large amounts of data generated by the model are also discussed. The possibilities offered by high performance computing in mathematical biology are highlighted in this work. The final part of this thesis gives an example of a combined model of the inflammatory and proliferative phases of dermal wound healing using the new computational framework. Both quantitative and qualitative methods are used to analyse the information-rich data sets generated by the model, offering insight into the dynamic systems that can be modelled using the new approach.