Direct current electrical resistance measurement techniques for assessment of colorectal cancer during laparoscopic surgery

The next generation of surgical tools will employ intraoperative sensing technologies to provide real-time information to the surgeon. Sensing in this way may facilitate personalised tissue resections during cancer surgery, thereby reducing radicality and improving outcomes for the patient. This thesis details the development and testing of electrochemical based sensing techniques aimed at integration into the next generation of laparoscopic surgical tools. Literature reviewed as part of the work highlights the broad nature of surgically appropriate sensing technologies. Based on the features of simplicity and scalability, the biogalvanic tissue characterisation technique was explored as the most practically suitable candidate. Development and systematic testing of a biogalvanic measurement system with porcine tissues showed variation that is not explained using the current system model. Correlation with electrochemical measurements verified this unaccounted system complexity. Electrode polarisation and diffusion controlled reduction at the cathode limit the tissue specificity of the output metrics. An improved analytic model fitting technique was developed to reduce the influence of the electrodes. Through collaborative development of a numerical model of the system, the practical limitations of the biogalvanic techniques as a surgical sensor were realised. To mitigate these limitations, a novel galvanostatic technique for improved resistance characterisation was developed. Testing was conducted on ex vivo tissues, showing stability for relevant parametric variation. Surgical applicability was found from a practical perspective, with results showing low sensitivity to switching rate, current range and tissue contact conditions. Testing was also conducted on a number of freshly excised cancerous human colon samples. Measurements were centralised on each tumour and compared to a corresponding healthy region. Every case showed a highly significant difference between tissue types with cancerous tissues having a consistently lower resistance. These findings suggest that the proposed technique of multi-reference galvanostatic resistance characterisation may be a suitable candidate for integration into surgical tools for colorectal cancer surgery

Novel approaches to MRI of glioma

Gliomas are extremely heterogeneous, both morphologically and biologically, which contributes to a very poor prognosis. Current imaging of glioma is insufficient for a thorough diagnosis, therapy assessment and prognosis prediction. Moreover, refined and more sophisticated imaging technique could help in furthering our knowledge of gliomas. In order to facilitate proliferation, cancer cells undergo a change in structure and an increase in metabolism that results in distortion and disruption of tissue architecture. Gliomas are characterised by an increase in cells of variable sizes, as well as changes in the tissue microstructure. Diffusion-Weighted Imaging (DWI) and the apparent diffusion coefficient (ADC), have been extensively studied as potential imaging biomarkers for cellularity and tissue architecture. However, several studies have shown partial overlap in the measured values between tumour subtypes. Moreover, ADC is influenced by several factors and does not provide detailed information on the tissue microstructure. The Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumours (VERDICT) is a novel diffusion model that infers tissue microstructure compartment from conventional DWI measurements. This model derives metrics for the intracellular, intravascular and extracellular– extravascular spaces providing a more detailed interpretation of the tissue microstructure. To date, VERDICT has been applied to xenograft models of colorectal cancer, patient studies of prostate cancer and recently its feasibility in glioma has been shown. In this PhD I have applied a shortened version of the VERDICT method to image intratumoral and intertumoral heterogeneity in glioma. The results have also been validated with histology as part of a prospective study. Gliomas also exhibit a significant increase in mitotic activity within the tumour. The increased number of mitosis alters cell density which, in turn, affects the total concentration of tissue sodium as the concentration of tissue sodium is approximately ten-fold higher in the extracellular compared to the intracellular space. In addition, there is a decrease in Na+/K+-ATPase activity in tumours due to ATP depletion, which contributes to disturb sodium homeostasis. Non-invasive detection of 23Na with MRI has the potential to quantify sodium concentration and therefore could be an imaging probe of cell morphology and membrane function within the tumour microenvironment, as well as a method of probing tissue heterogeneity. During my PhD, a novel 23Na-MRI technique has been used to evaluate sodium distribution within glioma and in the surrounding tissue. Metabolic reprogramming is one of the major driving forces for determining glioma growth and invasion. Therefore, the non-invasive characterization of metabolic intratumoral, peritumoral and intertumoral heterogeneity in vivo could help to better stratify patients and to develop novel therapeutic strategies targeting cancer-specific metabolic pathways. 13C magnetic resonance imaging (MRI) using dynamic nuclear polarization (DNP) is a novel technique that allows non-invasive assessment of the metabolism of hyperpolarized (HP) 13C-labelled molecules in vivo, such as the exchange of [1-13C]pyruvate to [1-13C]lactate in tumours (Warburg effect). Part of my PhD has focused on developing and translating HP [1-13C]pyruvate MRI to explore metabolic reprogramming in glioma and the surrounding microenvironment. The overall aim of my PhD has been to develop novel approaches to imaging glioma with MRI to probe both the architectural and metabolic changes of Glioma. The preliminary evidence suggests that these tools can more deeply phenotype tumours than conventional imaging approaches. Although the main focus of this work has been gliomas, the techniques developed and presented here may be applied to study other pathological conditions within the brain, which raises the possibility of other potential clinical applications for this work

Tuning and targeting semiconducting polymer nanoparticles to enhance in vivo photoacoustic imaging

Photoacoustic imaging (PAI) is a promising imaging modality which combines high spatial resolution with excellent contrast generation. To fulfil the potential of using PAI in clinical settings for cancer detection, the development of novel contrast agents with strong absorptions in the near infrared and high tumour specificity in vivo is required. Semiconducting polymer nanoparticles (SPNs) encapsulating organic semiconducting polymers in lipid nanoparticles have emerged as excellent candidates for photoacoustic (PA) contrast generation: they retain the polymer’s ability to generate high PA contrast, and the lipid formulation grants SPNs excellent physiological properties. However, these SPNs rely so far on the enhanced retention and permeability (EPR) effect for tumour accumulation. The reliability of this passive mode of accumulation in clinical settings has been recently called into question. To address this, the formulation of targeted SPNs using EGFR-targeting peptides was explored. SPNs based on novel semiconducting polymers as well as commercially available polymers were formulated via the mini-emulsion and nanoprecipitation methods. Lipid formulations included PEGylated lipids as well as functional PEG lipids on which N-terminal Cysteine-modified EGFR-targeting peptides were conjugated via the thiol-maleimide Michael addition. The synthetic accessibility of both the pre- and post-formulation functionalisation strategies was assessed in Chapter 3. To quantify surface functionalisation, a novel NMR characterisation strategy for the routine quantification of maleimide moieties tethered to the surface of nanoparticles was proposed. To compare the targeting efficiency of EGFR-targeting peptides A-R, D4 and GE11, a library of peptide-dye conjugates was constructed using fluorescein-5-maleimide. This library included scrambled controls and short PEG spacers introduced between the targeting sequence and the cysteine residue. The synthesis of these conjugates is described in Chapter 4. While synthesising these peptides, preliminary data supporting the intramolecular transcyclisation of the thiol-maleimide adducts was obtained. The intramolecular transcyclisation of thiol-maleimide adducts is underreported in the literature and clinically relevant. The thiazine rearrangement products are protected from thiol-exchange which, in physiological conditions, can lead to the partial loss of functionality of targeting ligands synthesised using the thiol-maleimide reaction

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Cancer Nanomedicine

This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field

The 1989-1990 NASA space biology accomplishments

Individual technical summaries of research projects on NASA's Space Biology Program for research conducted during the period May 1989 to April 1990 are presented. This program is concerned with using the unique characteristics of the space environment, particularly microgravity, as a tool to advance the following: (1) knowledge in the biological sciences; (2) understanding of how gravity has shaped and affected life on the Earth; and (3) understanding of how the space environment affects both plants and animals. The summaries for each project include a description of the research, a list of accomplishments, an explanation of the significance of the accomplishments, and a list of publications

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world