<br><b>Introduction:</b> Extracellular vesicles (EVs) are membrane-bound structures that playa crucial role in intercellular communication and molecular transport. Due to theirbiological functions, EVs hold great potential as a novel diagnostic tool or as the targeteddrug carriers in anticancer therapies. Elevated glucose concentrations affect cellularmetabolism, thereby modifying the composition of the EVs and their lipid bilayer. This, inturn, can influence EVs' properties and morphology, which is important in their function.</br><br><b>Hypothesis:</b> Hyperglycemic conditions, by altering the composition and metabolism ofEVs, influence their size distribution and physicochemical properties.</br><br><b>Objective:</b> Visualisation and size distribution comparison of EVs isolated fromnormoglycemic (NG) and hyperglycemic (HG) conditions.</br><br><b>Methods:</b> Two complementary methods based on different physical principles were appliedand compared in terms of their applicability: nanoparticle tracking analysis (NTA) and cryo--transmission electron microscopy (Cryo-TEM). In this study, 1.1B4 cells were cultured innormo- (NG, 5 mM glucose) and hyperglycemic (HG, 25 mM glucose) conditions.</br><br><b>Results:</b> The mean hydrodynamic EV size under hyperglycemic conditions (236 28 nm)was slightly higher than the control (209 43 nm; Paired Sample Wilcoxon Signed Test,p = 0.05). Physical EV sizes (Cryo-TEM), as well as mode hydrodynamic sizes (NTA) wereconsistent and not affected by glucose concentration. In contrast to NTA, Cryo-TEM isa more precise technique for real-size distribution analysis, whereas NTA provides a rapidand cost-effective assessment of hydrodynamic radii. The theoretical level of detection ofNTA for EVs was close to what was observed in results.</br><br><b>Conclusions:</b> Hyperglycemic conditions slightly influenced only the mean hydrodynamic radius of EVs, indicating effects on EV corona size and the biogenesis of large EVs (lEVs).</br>

This study proposes a methodology for the automated localisation of lower limb bones on T1 weighted MRI scans, employing a deep learning (DL) approach. The primary objective is to facilitate precise identification of skeletal structures, thereby supporting radiomics based diagnostics of neuromuscular disorders. The developed framework is not confined to the recognition of lower limb bones. A dataset of 1,243 MRI scans was used, with a subset of 29 manually labelled bone segmentations of six key lower limb bone classes. Axial slices were divided into training (2,283), validation (300), and hold-out (378) sets. A two part segmentation pipeline was developed using a combination of U-Net and ResNet architectures, with a custom cost function to handle variable label presence across slices. A novel method for obtaining precise bone-related slice location on the MRI volume was developed. Segmentation quality for Tibia and Femur was high, achieving 86.04% and 86.97% Dice score on the hold-out subset. The ResNet classifier correctly identified the defined regions on the volume, achieving AUCs over 97% on the hold-out subset for most leg fragments except for the knee label. This work introduces a new method for anatomical spatial localisation estimation in MRI scans. Unlike previous studies, which could only recognise body parts, the proposed method also estimates the precise bone-related slice location within the scanned volume, providing an added layer of anatomical context. The developed pipeline can be retrained for other body fragments. This solution exhibits a strong potential for use in clinical workflows, especially for studies involving musculoskeletal diseases.

Objective: Positron Emission Tomography enables non-invasive imaging of metabolic processes. Standard PET reconstructs radiotracer distribution using two back-to-back photons from electron-positron annihilation. However, about 1% of annihilations results in creation of three photons. Three photons may be created in direct annihilation and in annihilation via formation of metastable ortho-positronium carrying additional information not used by conventional PET. The Jagiellonian-PET detector allows us to use this information by applying positronium imaging based on ortho-positronium lifetime or the 3γ/2γ decay ratio. Accurate tomographic images require attenuation correction. This study investigates photon absorption in phantom models to form a basis for future attenuation maps for three-gamma decays. Methods: Monte Carlo simulations in ROOT and GATE were performed for water sphere, cylinder, a simplified head model, and the mesh50_XCAT phantom. Both p-Ps and o-Ps decays were simulated as uniformly distributed sources. Absorption probabilities were calculated by checking whether any photon in a multiplet interacted within the phantom. Emission-point-specific absorption maps were generated for all models. Toy Monte Carlo and GATE simulations showed good overall agreement. Results: Photon triplets from o-Ps decays experienced higher absorption than photon pairs from p-Ps due to lower individual energies and higher attenuation. Absorption maps showed dependence of photon survival probability on the decay location. In the mesh50_XCAT phantom, 24.9% of p-Ps pairs and 10.3% of o-Ps triplets escaped without interaction. Conclusions: Gamma absorption depends strongly on positronium decay mode and location, with o-Ps events experiencing higher attenuation. The generated absorption maps provide the first step toward dedicated attenuation correction for three-gamma positronium imaging, enabling accurate reconstruction in novel 3γ/2γ positronium imaging technique. A Study presented indicates that the absorption of 3γ in the head is only about 2.5 times higher than for the 2γ, which is encouraging for further development of the 3γ/2γ rate ratio imaging.

<b>Objectives:</b> The HER2 receptor is often overexpressed in various cancers, particularly in breast and ovarian cancers, and this overexpression significantly contributes to the growth and spread of these tumors. Trastuzumab (Herceptin) and pertuzumab (Perjeta) are widely used humanized monoclonal antibodies (mAbs) that have shown promise in treating patients with HER2-positive breast cancer. To enhance their effectiveness, mAbs have recently been combined with chemotherapeutic agents and radionuclides. The aim of our studies was to investigate the potential therapeutic use of trastuzumab and pertuzumab labeled with the Auger electron emitter – <sup>125</sup>I. <br><b>Methods:</b> The radioimmunoconjugates synthesized using <sup>125</sup>I and <sup>131</sup>I were tested in various in vitro studies on SKOV-3 cells. These studies included tests for specificity, binding affinity, internalization, cytotoxicity (MTS assay) and confocal imaging. <br><b>Results:</b> The results confirmed that radio-iodinated mAbs have high affinity and internalization properties in an HER2+ cell line. In contrast to trastuzumab, significant localization of iodinated pertuzumab on the cell membrane was observed. MTS assay and spheroid studies demonstrated minor toxic effects from both radio-conjugates resulting from the combination of the mAbs' immuno-toxic effect and the interaction of Auger electrons. However, [<sup>125</sup>I]I-pertuzumab exhibited higher cytotoxicity. <br><b>Conclusions:</b> Despite high internalization, both radio-bioconjugates showed low cytotoxicity due to the lack of radionuclide localization in the cell nucleus. However, [<sup>125</sup>I]I-pertuzumab accumulated in the cell membrane, resulting in slightly higher cytotoxicity. To improve therapeutic efficacy, modifying [<sup>125</sup>I]I-trastuzumab and [<sup>125</sup>I] I-pertuzumab to transport them to the cell nucleus, e.g., using nuclear localization signal (NLS) peptides, is crucial.

<b>Objective:</b> In this study, the gap was addressed in effective rehabilitation methodologies for preventing sepsis sequelae utilising a novel, two-way, diagnostically-monitored personalised rehabilitation model implemented to enhance quality of life (QoL) among sepsis survivors. <br><b>Methods:</b> Of 66 sepsis survivors post-hospital discharge, and those who met the inclusion criteria, 30 were eventually allocated into three groups: a control group (Group C, n = 10, no rehabilitation) and two intervention groups undergoing three-month, diagnostically-monitored rehabilitation programmes. Group E (n = 10) received personalised aerobic and functional training, while Group H (n = 10) underwent intermittent hyperbaric oxygen therapy as a novel rehabilitative treatment alternative. QoL was evaluated at hospital discharge and again after 3 months using the newly developed Life After Sepsis Survey.<br><b>Results:</b> After three months, statistically significant (<i>p</i> < 0.05) improvement was observed in QoL among sepsis survivors from both intervention groups concerning the significant parts of the assessed physiological, physical and psychological functions. <br><b>Conclusions:</b> The personalised, patient-centric and supportive approach significantly improved QoL among sepsis survivors, highlighting the potential effectiveness of this rehabilitation model. The Life After Sepsis proved to be a practical assessment tool and currently stands as a uniquely suitable measure for evaluating sepsis rehabilitation outcomes.

<b>Objectives:</b> Proton therapy, while highly effective and successful, still lacks a key feature: the ability to assess, in-vivo, the dose and end-point location of irradiations. Known as proton range verification, this capability can be realized by incorporating positron emission tomography (PET) systems in both conventional and emerging modalities, such as FLASH proton therapy. FLASH itself may revolutionize radiation oncology with its purported ability to better spare healthy tissues, but only if the underlying mechanisms can be understood. We summarize our work towards establishing in-beam PET modalities and elucidating the mystery of the FLASH effect.<br> <b>Materials:</b> We've developed a PET scanner designed for live, in-beam imaging during therapeutic proton irradiations that can use short-lived positron emitting species (PES) activated by the beam to validate the range and dose of proton depositions. This scanner is made up of PET modules consisting of arrays of LYSO (lutetium-yttrium oxyorthosilicate) scintillating crystals coupled one-to-one to silicon photomultiplier (SiPM) arrays. These modules are readout by electronics based on the TOFPET2 ASIC platform from PETsys Electronics.<br> <b>Methods:</b> Our collaboration with MD Anderson Cancer Center has given us opportunities to take real in-beam data using a non-clinical beamline capable of delivering FLASH proton irradiations into target phantoms made of polymethyl methacrylate (PMMA), high density polyethylene (HDPE), and water. Data collected both during and afterirradiations were used to perform novel analyses and to reconstruct images of PES activity due to the beam.<br> <b>Results:</b> Exploratory studies, using a subset of our PET scanner, have demonstrated successful data acquisition during and after FLASH beam spills including quantitative imaging and dosimetry of activated phantoms. The full results, explored in this work, are highly promising and prove that in-beam PET can deliver on its goals. Upcoming experiments conducted using both FLASH and conventional beams will employ the full PET scanner and involve a rich experimental program with novel ideas for irradiation targets, beam characterization, and in-depth comparisons of the two irradiation modalities.<br> <b>Conclusions:</b> This work demonstrates the unprecedented proof-of-principle for the capabilities of an in-beam PET scanner for imaging and dosimetry of both conventional and FLASH proton beams. These results open a new PET modality with proton beams which is particularly attractive for FLASH therapy but can serve effectively all proton irradiations, leading to improved treatment monitoring and image-guided therapy.

Ultra-high dose rate (FLASH) proton radiotherapy is a promising treatment method for cancer patients. In our research, we want to compare the FLASH method with a conventional radiation method to show what effect they have on the biochemical structure of the tumour (3D model – spheroids) and the secretion of extracellular vesicles (EVs) and their cargo. The use of a modern method of creating spheroids will enable us to create conditions that are better able to mimic the tumour microenvironment.

Positronium imaging is a new technique complementary to positron emission tomography (PET) based on the histogramming of time delay between the emission of a de-excitation photon, and a consequent electron-positron annihilation, to estimate the mean lifetime of orthopositronium (o-Ps), which depends on the local size of the voids, concentration of oxygen and bioactive molecules. We improve the resolution and reduce noise in positronium imaging by building time-delay spectra from the PET activity reconstructed by a 3-photon time-of-flight maximum likelihood expectation maximisation. The method was tested on the data measured for four human-tissue samples injected by <sup>22</sup>Na and put in the Jagiellonian PET “Big barrel” scanner. Due to an ill-posed problem of fitting time-delay histograms, a multistage optimisation procedure was explored along with inferential analysis of the solution space. Run in parallel for multiple sets of initial guesses, we compared the second-order Levenberg- Marquardt algorithm (LMA) and the direct search Nelder-Mead simplex (NMS) method. The LMA proved to be faster and more precise, but the NMS was more stable with a higher convergence rate. The estimated mean o-Ps lifetimes in the 1.9 ns – 2.6 ns range were consistent with the reference results, while other fitting parameters allowed differentiation between the two patients who provided the tissue samples.

We present an algorithm for Monte Carlo simulations of positron tracks in biological materials. The algorithm takes into account the cross-section data for elastic and inelastic collisions between positrons and molecules and processes like direct annihilation, ionization and positronium formation. In the case of positronium formation, the algorithm considers the interactions of positronium with molecules. The algorithm can be used to identify the processes that are responsible to determine the lifetime of the positrons and their annihilation mechanism (direct or through positronium formation).

Selecting the proper dosimeter and methodology is crucial for accurate dose measurement, especially since the requirements are different for clinical and retrospective dosimetry. Regardless of the field-radiotherapy, X-ray diagnostic radiology or nuclear medicineefforts are continuously being made to improve radiation measurement accuracy through the development of new dosimeters, accurate calibration of instrumentation, training of staff, proper quality control and enhancement of radiation safety procedures. For instance, for retrospective dose estimation during radiation accidents, the selection of the appropriate material and knowledge of the intrinsic background signal of the selected material are crucial. In both clinical and retrospective dosimetry it is important to have adequate protocols as well as expertise in possible uncertainties, discussed here based on the authors own research.