Automated Stabilization, Enhancement and Capillaries Segmentation in Videocapillaroscopy

Oral capillaroscopy is a critical and non-invasive technique used to evaluate microcirculation. Its ability to observe small vessels in vivo has generated significant interest in the field. Capillaroscopy serves as an essential tool for diagnosing and prognosing various pathologies, with anatomic–pathological lesions playing a crucial role in their progression. Despite its importance, the utilization of videocapillaroscopy in the oral cavity encounters limitations due to the acquisition setup, encompassing spatial and temporal resolutions of the video camera, objective magnification, and physical probe dimensions. Moreover, the operator’s influence during the acquisition process, particularly how the probe is maneuvered, further affects its effectiveness. This study aims to address these challenges and improve data reliability by developing a computerized support system for microcirculation analysis. The designed system performs stabilization, enhancement and automatic segmentation of capillaries in oral mucosal video sequences. The stabilization phase was performed by means of a method based on the coupling of seed points in a classification process. The enhancement process implemented was based on the temporal analysis of the capillaroscopic frames. Finally, an automatic segmentation phase of the capillaries was implemented with the additional objective of quantitatively assessing the signal improvement achieved through the developed techniques. Specifically, transfer learning of the renowned U-net deep network was implemented for this purpose. The proposed method underwent testing on a database with ground truth obtained from expert manual segmentation. The obtained results demonstrate an achieved Jaccard index of 90.1% and an accuracy of 96.2%, highlighting the effectiveness of the developed techniques in oral capillaroscopy. In conclusion, these promising outcomes encourage the utilization of this method to assist in the diagnosis and monitoring of conditions that impact microcirculation, such as rheumatologic or cardiovascular disorders

Non-invasive detection of anemia using lip mucosa images transfer learning convolutional neural networks

Anemia is defined as a drop in the number of erythrocytes or hemoglobin concentration below normal levels in healthy people. The increase in paleness of the skin might vary based on the color of the skin, although there is currently no quantifiable measurement. The pallor of the skin is best visible in locations where the cuticle is thin, such as the interior of the mouth, lips, or conjunctiva. This work focuses on anemia-related pallors and their relationship to blood count values and artificial intelligence. In this study, a deep learning approach using transfer learning and Convolutional Neural Networks (CNN) was implemented in which VGG16, Xception, MobileNet, and ResNet50 architectures, were pre-trained to predict anemia using lip mucous images. A total of 138 volunteers (100 women and 38 men) participated in the work to develop the dataset that contains two image classes: healthy and anemic. Image processing was first performed on a single frame with only the mouth area visible, data argumentation was preformed, and then CNN models were applied to classify the dataset lip images. Statistical metrics were employed to discriminate the performance of the models in terms of Accuracy, Precision, Recal, and F1 Score. Among the CNN algorithms used, Xception was found to categorize the lip images with 99.28% accuracy, providing the best results. The other CNN architectures had accuracies of 96.38% for MobileNet, 95.65% for ResNet %, and 92.39% for VGG16. Our findings show that anemia may be diagnosed using deep learning approaches from a single lip image. This data set will be enhanced in the future to allow for real-time classification

EXPANDING VORTEX TRAPPING AND ELECTROPORATION CAPABILITIES TO SMALL CELLS

Microfluidic Vortex devices have been commercialized for purifying rare circulating tumor cells from patient samples, enriching these rare cells by up to a 100,000-fold. These purified, viable cells can then be interrogated through live-cell assays and genetic sequencing, giving clinicians valuable insight into patient disease state and potential treatment directions. Vortex devices can also be integrated with solution exchange and electropermeabilization for on-chip susceptibility assays and biomolecular delivery. Currently these devices are limited to particles larger than 15 μm in diameter. In the biological field, cell separation and integrated electroporation is valuable for the biomolecular delivery to lymphocytes (7-9 μm), spermocytes (7-8 μm), and stem cells (~10 μm). In the pharmaceutical industry, separation integrated with solution exchange for modifying surface properties of drug microparticles (< 10 μm). These exemplify the need to expand Vortex device capabilities particles in the 7-10 μm range. In Chapter 1, we provided background information on Vortex separation devices and integration with other functionalities. In Chapter 2, we expanded Vortex separation capabilities, to microparticles 7-10 μm by manipulating device geometry and operational conditions. We tested device performance with polystyrene microparticles and living cells. We show that our novel trapezoidal chamber with a narrowing channel leads to a 4-fold increase over tested rectangular chambers when trapping microparticles in the 7-10 μm size range. In Chapter 3, we confirmed that our new cell trapping device retains the capability of integrated electroporation. We designed serrated electrodes to electroporate smaller cell by amplifying the electric field strength, without significant electrolysis. Next, we use real-time fluorescent measurement and cell viability and electroporation efficiency analysis to identify electroporation conditions for biomolecular delivery of fluorescent dye to small 7-10 μm cells. The device discussed Chapters 2 and 3 expand the capabilities of Vortex trapping with integrated electroporation functionality. If optimized, the presented platform could potentially perform electroporation-mediated biomolecular delivery to cells in the 7-10 μm size range, with therapeutic applications to lymphocyte, sperm, and stem cell engineering. Chapters 4-6 discuss contributions to the commercialization of electrode-integrated microfluidic devices, the development of tunable microfluidic phantoms, and inertial microfluidic devices for sorting retinal ganglion cells

INFLUENCE OF MEDITERRANEAN DIET ON INCIDENCE AND COURSE OF INFLAMMATORY RHEUMATIC DISEASES

Th e Greek “Father of Medicine” and physician Hippocrates, said around 400 B.C. “Let thy food be thy medicine and thy medicine be thy food” (Nikiphorou et al. 2018) Therefore, over the last decades we become increasingly aware and concerned about how nutrition affects our health and the field of nutrition have meet unprecedented interest and expansion. On the other hands, a number of dietary factors might act as environmental triggers in rheumatic and muskuloskeletal diseases (RMDs) development. Overall, a ‘Western’ type diet rich in energy intake, total and saturated fat, an unbalanced ratio of n-3 to n-6 fatty acids, high in sugar and low in fiber and antioxidants might increase the risk of RMDs both directly through increasing inflammation (Minihane et al. 2015) and indirectly through increasing insulin resistance, obesity and associated co-morbidities, with obesity being a known risk factor for RMDs (Qin et al. 2015). In detail, high consumption of foods characteristic of the ‘Western-type’ diet such as red meat, meat and meat products combined, or total protein have been shown to increase the risk of inflammatory polyarthritis suggesting a role of advanced glycation end products (AGEs) (Pattison et al. 2004). This is supported by findings of regular consumption of sugar-sweetened soda, but not diet soda, being associated with an increased risk of seropositive rheumatoid arthritis (RA) in women (Hu et al. 2014), and of high fructose corn-syrup sweetened soft drinks, fruit drinks and apple juice being associated with arthritis in young US adults (DeChristopher et al. 2016). It is hypothesized that regular consumption of excess free fructose and HFCS contributes to fructose reactivity in the gastrointestinal tract and intestinal in situ formation of enFruAGEs, which once absorbed, travel beyond the intestinal boundaries to other tissues and promote inflammation (DeChristopher et al. 2016). Individual biomarkers of antioxidant intake have also been previously investigated in relation to RA with some evidence that low serum levels of selenium and alpha tocopherol (Knekt et al. 2000) and beta carotene (Comstock et al. 1997) are associated with an increased disease risk. Interestingly, a meta-analysis also suggests that coffee consumption of ≥ four cups per day is associated with an elevated risk of seropositive RA but not seronegative RA (Lee et al. 2014). However, the results should be interpreted with caution due to other potential confounders. The same meta-analysis found no association between tea consumption and risk of RA (Lee et al. 2014). On the contrary, consumption of longchain omega-3 polyunsaturated fatty acids, derived from fish and fish oil, is associated with a reduced risk of inflammatory RMD like RA (Di et al. 2014) probably due to their anti-inflammatory properties. The Mediterranean diet (MD), rich in plant-based foods such as wholegrains, legumes, fruit, vegetables, extravirgin olive oil and low in red meat consumption, might have the potential to reduce the risk of RA. It has been shown that greater adherence to the MD is associated with lower concentrations of inflammatory biomarkers (Fung et al. 2005), while daily consumption of monounsaturated fatty acids from olive oil is thought to be the key factor in suppressing RA disease activity (Matsumoto et al. 2017). Other nutritional approaches like vegan, elemental or elimination diets did not showed any superiority to the MD (Ciccia et al 2018, Philippou et al. 2018) regarding the interference on RMDs. In addition, recent evidences suggest the diet pattern, by modifying the composition of intestinal microbiome, might influence the activation of innate immune pathways such as inflammasome and autophagy directly involved in the production of pro-inflammatory cytokines such as IL-1b and IL-18 with effects on RMDs Based on current research evidence, it is concluded that adherence to the MD with an increased consumption of fatty fish, reduced consumption of sugar-sweetened drinks and maintenance of a normal body weight, contributes to reducing the risk of RA. Interestingly, looking at the “chrononutrition” following the body circadian rhythms (Nobel Prize for Medicine 2017) it has been assessed that circadian misalignment, behavioral processes such as food intake or sleep occurring at inappropriate endogenous circadian times, commonly occurs during shift work (i.e. night shift workers) are associated with serious health problems over the time including RMDs (Cutolo 2018). In conclusion, both correct quality and timing in nutrition, are essential in prevention and/or co-management of RMD-

MIRNAS AS BIOMARKERS IN AUTOIMMUNE RHEUMATIC DISEASES

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs of about 22 nucleotides in length that act as post-transcriptional regulators of gene expression. Depending on the complementarity between miRNA and target mRNA, cleavage or destabilization of mRNA or translational suppression occurs within the RISC complex. As gene expression regulators, miRNAs are involved in a variety of biological functions. Dysregulation of miRNAs and their target genes contribute to pathophysiology of many disorders including autoimmune rheumatic diseases. For example, dysregulation of miR-155, miR-146a or miR-203 have been known for a long time to contribute to aggressive behavior of synovial fibroblasts and inflammatory milieu in rheumatoid arthritis. Dysregulation of miR-155 or miR-130b infl uence inflammatory or resident renal tubular cells in systemic lupus erythematosus. MiR-29 appears a key regulator of collagen expression in systemic sclerosis. Many miRNAs have been shown to be of therapeutic potential in in vivo animal models. MiRNAs are also present extracellularly in body fluids. Their incorporation into membrane vesicles or protein complexes with Ago2, HDL or nucleophosmin 1 protects them against RNases. Cell-free miRNAs can be delivered to another cell in vitro and maintain their functional potential. Therefore, miRNAs can be considered mediators of intercellular communication. Remarkable stability of cell-free miRNAs makes them accessible in body fluids. However, their origin, target tissue/organ or mechanism of action at the targeted site remains to be elucidated. We aim to summarize growing pieces of evidence supporting diagnostic and prognostic potential of cell-free miRNAs in autoimmune rheumatic diseases such as rheumatoid arthritis, axial spondyloarthritis, systemic lupus erythematosus, systemic sclerosis, idiopathic inflammatory myopathies or Sjögren´s syndrome. Acknowledgement: Projects MHCR 023728. References: 1. Filková M, Jüngel A, Gay RE, Gay S. MicroRNAs in rheumatoid arthritis: potential role in diagnosis and therapy. BioDrugs. 2012 Jun 1;26(3):131–41

The development of an anti-scarring burn dressing

Introduction: Scarring has a significant impact on the function and quality of life in burn patients. This thesis describes selected stages of the development of an anti-scarring burn dressing and an objective scar assessment panel. Methods: This thesis is divided into two sections. Section 1 covers the development of an objective scar measurement tool based panel and score via a systematic review and subsequent reliability testing and validation of selected devices. Section 2 covers different aspects of dressing formulation. The cytotoxicity effects of decorin were investigated in dermal fibroblast cultures to provide guidance to safe and effective decorin dosing. Manufacturing, sterilisation and clinical use exposes decorin to elevated temperatures and the effects of this on the structure of decorin and bio-activity of decorin is investigated with circular dichroism and in-vitro cell cultures respectively. Lastly, a skin contact study in healthy volunteers was performed to establish the safety of two gellan formulations (sheet and fluid gel). Results: Objective scar measurement tools were found to be more reliable than subjective scar scores and an objective scar score was created consisting of high frequency ultrasound and pliability measures. Decorin had no measurable cytotoxicity on dermal fibroblasts even at high concentrations. Conformational change in decorin structure was seen at relatively low temperatures however results suggest that heating may enhance its bio-activity. Both gellan formulations were found to be safe for use on intact skin. Conclusion: The new objective scar scale can be used to accurately measure the effects of antiscarring treatments. Decorin and gellan are safe to be used in patients but the dressings may need to be protected against high temperatures

EARLY DIAGNOSIS OF GIANT CELL ARTERITIS – DOES IT MATTER?

Purpose: If untreated, giant cell arteritis can lead to blindness and stroke. The study objectives were to assess diagnostic procedures and treatment in early interventional clinic in University Clinical Centre Maribor in patients with temporal arteritis. Methods: Retrospective study (from 2012 to 2017) of patients diagnosed with temporal arteritis. We assessed epidemiological data, delay of diagnosis, and diagnostic procedures. Results were assessed with statistical methods (SPSS 22.0). The main goal was to determinate the delay in days between symptom onset and admission to the interventional rheumatology clinic and to assess the causes of delay. Results: Fift y-three GCA (66 % female) patients with mean age 76.25 (from 63 – 89 years) years were included. Mean time duration of symptoms before admission to our early interventional clinic was 33.74 (0–180) days. The diagnostic procedure was completed in mean time of 2.04 days from the presentation at our interventional rheumatology clinic. Th e median time to the temporal artery biopsy (TAB) performed in 52 /53 patients was 2 days, with the median 2 days to the preliminary histological results from admission. TAB was positive in 43 (81.1%) of cases. The median time from admittance to colour Doppler sonography (CDS) of aortic arch branches was 2 days and it was positive in all 19 (35.8%) performed cases. 16 (30.2%) patients had polymyalgia rheumatica, 35 (66%) patients had visual disturbances, permanent one eye blindness occurred in 12 (22.64%) patients, and 2 (2.8%) patients experienced permanent blindness on both eyes. Seventeen patients (32.1%) were initially treated with intravenous methylprednisolone pulse. Th e mean initial dose of oral methylprednisolone was 45.55 (+/– 15.54) mg. All patients received low dose Aspirin. Conclusions: Early diagnosis and treatment of giant cell arteritis are very important as miss- or non-diagnose GCA can lead to permanent blindness of the patient. With better education and public awareness, better access and better professional education of primary care physicians, and early admission to secondary interventional clinics we might spare these patients from the devastating consequences of the GCA

COLOUR DOPPLER SONOGRAPHY OF FACIAL AND OCCIPITAL ARTERIES IN PATIENTS WITH GIANT CELL ARTERITIS

Background: Giant cell arteritis (GCA) is the most common systemic large and medium size artery vasculitis in Western countries. Colour Doppler Sonography (CDS) allows the study of involvement of cranial arteries other than the temporal arteries, which are inconvenient to biopsy, such as the facial (FaA), and occipital (OcA) arteries. Objectives: We aimed to estimate the frequency of the FaA, and OcA involvement in GCA; and to explore the clinical characteristics of these subgroups of patients. Methods: From 1 January 2014 to 31 December 2016, we prospectively performed a CDS of the FaA, and OcA in addition to the temporal (TA), and the extracranial supra-aortic arteries in all newly diagnosed patients suspected of having GCA. We used a Philips IU22 with a 5–17.5 MHz multi-frequency linear probe from January 2014 to August 2016 and a Philips Epiq 7 with a 5–18 MHz multi-frequency linear probe from September 2016 to December 2016. All the arteries were evaluated in two planes for the highly specific halo-sign. Results: During the 36-month observation period we performed a CDS of the cranial and extra-cranial arteries in 93 GCA (66.7% female) patients. The patients’ median (IQR) age was 73.7 (66.1–79.1) years, and they had a median (IQR) symptom duration of 30 (21–90) days. We observed the halo-sign on the FaA, and OcA in 38 (40.9%), and 29 (31.2%) cases, respectively. The FaA, and OcA were simultaneously affected in 18/93 (19.4%) cases. Either FaA, or OcA were affected in 4/22 (18.2%) patients with a negative TA CDS. FaA involvement significantly correlated with jaw claudication and with severe visual manifestations, including permanent visual loss. Patients with OcA involvement least commonly had extracranial large vessel disease. Conclusions: A fifth of patients with a negative CDS of the TAs had signs of vasculitis on the CDS of the FaA, or OcA. Th e addition of FaA and OcA CDS to the routine CDS of the TAs could identify 4.3% more patients and thus further improve the sensitivity of the CDS in the suspected GCA. References: 1. Ješe R, Rotar Ž, Tomšič M, Hočevar A. Th e role of colour doppler ultrasonography of facial and occipital arteries in patients with giant cell arteritis: A prospective study. European Journal of Radiology. 2017;95:9–12