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Ciljevi doktorske disertacije bili su istražiti izražaj adhezijskih molekula na granulocitima i monocitima štakora koji su pred infarkt miokarda konzumirali bijelo vino te izražaj istih molekula na matičnim stanicama karcinoma dojke i prostate nakon tretmana novosintetiziranim inhibitorom fosfolipaze C, 3-amino-5-okso-N-naftil-5,6,7,8-tetrahidrotieno [2,3-b] kinolin-2-karboksamidom. Adhezijske molekule CD15s i CD11b na plazma membranama neutrofila i monocita uključene su u započinjanje, a CD44 i u zaustavljanje upalnog odgovora te cijeljenje nakon infarkta miokarda. Krv Sprague Dawley štakora, koji su konzumirali bijelo vino 4 tjedna prije izazivanja infarkta miokarda, uzorkovana je 24 sata nakon infarkta te analizirana metodom protočne citometrije. Dobiveni rezultati uspoređeni su s rezultatima kontrolne skupine, prividno operirane skupine te skupine kod koje nije bilo intervencija. Prividno operirana i skupina u kojoj nije bilo intervencija nisu se razlikovale ni u postotku pojedinih subpopulacija niti u izražaju pojedinih biljega po jednoj stanici. Konzumacija vina posredovala je višestruko povećanje izražaja CD44 na velikim monocitima i na monocitima uobičajene veličine. Budući da CD44 potiče protuupalno djelovanje te sudjeluje u transformaciji fibroblasta u miofibroblaste, pojačan izražaj CD44 otkriva mogući povoljan učinak vina na te važne mehanizme cijeljenja srčanog tkiva. Djelovanje inhibitora fosfolipaze C analizirano je kroz parametre rasta i preživljenja (MTT test), apoptoze (analiza PI i aneksina-V), postotka matičnih stanica (CD44+CD24-) te glikofenotipa (CD15s i GM3 (NeuAc)) tumorskih linija dojke (MDA-MB-231) i prostate (Du-145). Svi parametri, osim kod MTT testa, određivani su protočnom citometrijom nakon 48-satnog tretmana 2 μM inhibitorom. Učinak pet različitih koncentracija inhbitora tijekom četiri vremenska intervala analiziran je MTT testom. Citotoksični učinak, zapažen kod obiju staničnih linija, kod trostruko negativnog raka dojke je bio posredovan apoptozom. Inhibitor je uzrokovao blago, ali statistički značajno smanjenje postotka subpopulacije matičnih stanica u ukupnoj populaciji raka dojke uz istovremeno značajno povećanje njihova izražaja GM3(NeuAc), važnog inhibitora prijenosa signala čimbenika rasta. Postotak potencijalno metastatske subpopulacije matičnih stanica (CD15s+) bio je snižen kod obiju tumorskih staničnih linija nakon tretmana inhibitorom. MDA-MB-231 stanična linija predstavlja trostruko negativni rak dojke. Uzimajući u obzir činjenicu da je takav tip raka karakteriziran povećanim postotkom matičnih stanica raka te poznavajući njihovu povezanost s povećanim rizikom metastaziranja i smrtnosti, novi inhibitor iz skupine tieno [2,3-b] piridina bi mogao biti djelotvoran za tretiranje ovakvog tipa raka., The aims of this doctoral thesis were to determine the expression of adhesion molecules on granulocytes and monocytes in rats that consumed white wine before myocardial infarction and on the breast and prostate cancer stem cells after treatment with newly synthesised inhibitor of phospholipase C, 3-amino-5-oxo-N-naphthyl-5,6,7,8-tetrahydrotieno[2,3-b]quinoline-2-carboxamide. Neutrophil and monocyte CD15s and CD11b adhesion molecules are involved in the initiation of inflammation while CD44 molecule is implicated in the resolution of cardiac inflammation as well as in healing processes after the myocardial infarction. Blood of male Sprague Dawley rats, that consumed white wine during 4 weeks before infarction, was sampled 24 hours after infarction and analysed using flow cytometry. This group was compared with water only drinking controls, sham animals (subject to surgery without myocardial infarction) and baseline group (intact animals that received no intervention prior to being sacrificed). Sham animals did not differ from baseline animals nor in the percentage of subpopulations neither in the adhesion molecule expression. Wine drinking was associated with multiple increase in CD44 expression on large monocyte and monocytes. Knowing modulation ability of CD44 to suppress post-infarction inflammation and to transform fibroblast into myofibroblasts, the CD44 finding implicates benefitial role of white wine in the cardiac healing. The effects of the newly synthesized phospholipase C inhibitor were analyzed through the parameters of growth and survival (MTT test), apoptosis (PI and annexin-V analysis), cancer stem cell percentage (CD44+CD24-), and stem cell glycophenotypes (CD15s and GM3 (NeuAc)) of breast (MDA-MB-231) and prostate (Du-145) tumor lines. All parameters, except for the MTT assay, were determined using flow cytometry, 48 hours after treatment with 2 μM inhibitor. The MTT test analyzed the effect of five different concentrations of inhibitor over four time intervals. Inhibitor produces cytotoxicity in both cell lines, mediated by apoptosis in breast cancer cells, and slightly but statistically significantly lowering breast stem cell subpopulation while GM3(NeuAc) expression per one breast stem cell was increased compared with controls. GM3(NeuAc) is the important inhibitor of growth factor signal transduction. The percentage of potentially metastatic stem cell subpopulation (CD15s+) was lower in both cell lines after treatment with the inhibitor. Cell line MDA-MB-231 represents triple-negative breast cancer. Considering that such cancers are characterized by an increased percentage of breast cancer stem cells and knowing their association with an increased risk of metastasis and mortality, the new phospholipase C inhibitor (belonging to thieno[2,3-b]pyridine inhibitors) may be a potentially effective drug for triple-negative breast cancer treatment.

Background: The primary pathological event in multiple sclerosis (MS) is demyelination with degeneration and loss of axons, which correlates with a permanent functional deficit. Monocytes, together with Th1 and Th17 cells, play a key role in CNS inflammation in MS. Gjelstrup et al. [1] showed a decreased expression of CD40 and CD192 markers in the total monocyte population in people with MS compared to the control group. Further, recent findings suggest an association between the pathophysiological mechanisms of MS (demyelination and loss of axons) and TMS neurophysiological measures [2-4]. Objective: To investigate CD40+ and CD192+ blood monocyte subpopulations in relapsing-remitting MS subjects undergoing TMS assessment of corticospinal tract integrity by recording MEPs from upper and lower extremity muscles. The CD40+ and CD192+ blood monocyte subpopulations were compared to healthy controls and MS subjects having prolonged latency of MEPs and subjects having neat MEP findings. Methods: Blood samples needed for flow cytometry were collected from 23 MS subjects and 10 healthy controls and incubated with anti-human-CD14 FITC antibodies, of phycoerythrin-conjugated antibodies reactive to human CD16, mouse antibodies reactive to human CD192 conjugated with BB700 and Alexa Flour 647 conjugated antibodies reactive to human CD40. Results: All MS subjects and MS subjects with prolonged MEP latency differed from the healthy controls in the percentage of CD192 in non- classical anti-inflammatory CD14+CD16++ monocytes, expression of CD40 in CD14++CD16-, percentage of total monocytes positive for CD40, and expression of CD192 in classical CD14++CD16-. MS subjects with prolonged MEP latency differed from MS subjects with neat MEP finding in the expression of CD40 in CD14++CD16- and the percentage of total monocytes positive for CD40.The percentage of CD192 in anti-inflammatory CD14+CD16++ monocytes correlated with the intensity of stimulation used to elicit MEP responses from lower extremity muscles. Conclusions: Distinct results were found concerning the percentage of CD192+ non-classical monocytes and expression of CD192 in classical monocytes when comparing MS subjects with altered MEP latency and those with neat MEP findings. Study results point to the subclinical relevance of CD192+ monocyte subpopulations in MS. The study represents the first attempt to apply TMS in evaluating corticospinal tract integrity with the immunological investigation of MS. References [1] Gjelstrup MC, Stilund M, Petersen T, i sur. Subsets of activated monocytes and markers of inflammation in incipient and progressed multiple sclerosis. Immunology & Cell Biology 2018 ; 96(2):160-174. doi: 10.1111/imcb.1025 [2] Chalah MA, Palm U, Ayache SS. Editorial: Corticospinal Excitability in Patients With Multiple Sclerosis. Frontiers in Neurology 2021 ; 11: 635612. doi: 10.3389/fneur.2020.635612 [3] Neva JL, Lakhani B, Brown KE, i sur. Multiple measures of corticospinal excitability are associated with clinical features of multiple sclerosis. Behavioral Brain Reserach 2016 ; 297:187-95. doi: 10.1016/j.bbr.2015.10.015 [4] Stampanoni Bassi M, Buttari F, Gilio L, i sur. Inflammation and Corticospinal Functioning in Multiple Sclerosis: A TMS Perspective. Front Neurol 2020 ; 11:566. doi: 10.3389/fneur.2020.00566

Background: Examining the integrity of the corticospinal tract by navigated TMS could help in understanding the neurophysiological correlates of multiple sclerosis (MS) symptoms. Objective: To investigate MEP measures of corticospinal tract integrity with navigated TMS and its correspondence with neurological (EDSS) and neuro- radiological (MRI) classifications in people with relapsing-remitting MS (pwMS). Methods: In a cross-sectional study, MEP measures of corticospinal excitability (RMT, MEP latency, MEP amplitude), clinical disease-related (EDSS), and MRI lesion data were collected in 23 pwMS receiving teriflunomide and compared to non- clinical samples. Results: PwMS subjects differed from non-clinical samples in MEP latency for upper and lower extremity muscles. PwMS with altered TMS finding (prolonged MEP latency or absent MEP), compared to pwMS with neat TMS finding, had higher EDSS score [Median = 3.5(range 0-4) vs. 0.5(0- 2.5)] and EDSS functional pyramidal score [Median = 3.0(0-3.5) vs. 0.5(0-2)]. RMT intensity for mapping representations for lower extremity muscles was predictive for EDSS functional pyramidal scores. Overall, TMS findings classified pwMS as the same as EDSS in 70-83% of cases and were similar or more successful than MRI, which corresponded to EDSS in 57- 65% of cases. PwMS with altered TMS findings differed from those with neat TMS finding on the total number of lesions in the brain corticospinal and in cervical corticospinal tract. Conclusions: The correspondence of TMS with MRI, and EDSS classifications implies the clinical utility of navigated TMS in assessing corticospinal tract integrity in pwMS.

Multiple sclerosis (MS) is currently an incurable autoimmune demyelinating and inflammatory disease affecting more than two million people worldwide. The neurophysiologic studies of the central nervous system (CNS) function comprising motor evoked potentials (MEP) elicited by transcranial magnetic stimulation (TMS) reported consistent and substantial impairments in CNS generally correlating with disability. Studies of CNS showed prolonged central motor conduction times, asymmetry of nerve conduction motor pathways, and prolonged latencies, while resting motor threshold, MEP amplitude, and cortical silent periods showed conflicting results. Studies of peripheral nervous system (PNS) function comprising electroneuronography (ENG) reported impairments of the PNS in MS that were less pronounced and inconsistent. However, neurophysiological investigations applying both TMS and ENG are not frequently used in routine diagnostic procedures to elucidate neurophysiological changes in CNS and PNS in patients with MS. We describe a case of a patient with MS examined with TMS and ENG. The ENG findings did not show clinically meaningful deviations in the PNS of upper and lower extremity muscles. Simultaneously, TMS proved pathological findings in MEPs of upper and lower extremity muscles.

Introduction: There is strong epidemiological evidence that moderate consumption of wine has beneficial effects on human health, particularly on cardiovascular system. Increase in antioxidant defence is one of the proposed biological pathways that may contribute to the observed health benefits. The aim of our study was to determine the effect of moderate white wine consumption on the expression of GPx (glutathione peroxidase), CAT (catalase), NQO1 (NAD(P)H Quinone Oxidoreductase 1) and HSP-70 (heat shock protein) in cardiomyocytes. Materials and methods: Male Sprague Dawley rats were given either a combination of white wine (Graševina, Krauthaker, 13% alcohol vol.) and water (n= 6) or only water (n=7) for 28 days. After that, the animals were sacrificed. From the harvested hearts, cardiomyocytes were isolated, fixated and stored at -20 ⁰C. When all the samples were collected, cells were permeabilized, incubated with antibodies and analysed by flow cytometry. Results: Consumption of white wine increased HSP- 70 expression by 1.46 ± 0.29-fold (p=0.0002) when compared to control group. Furthermore, expressions of CAT, GPx and NQO1 were increased in wine consuming rats by 1.3 ± 0.21 (p=0.008), 1.45 ± 0.33 (p=0.0006), and 1.36 ± 0.12 (p=0.003) fold, respectively. Conclusion: Moderate white wine consumption for 4 weeks resulted in increased expression of the enzymes that are important in the cell protection from the oxidative stress.

Cells in every epithelium can be roughly divided in three compartments: stem cell (SC) compartment, transient amplifying cell (TA) compartment and mature or functional cell (FC) compartment. Maturation of stem cells is characterized epithelial stromal interaction and sequential maturational movement of stem cell’s progeny through those compartments. In this work we hypothesize that providing an artificial stroma, which murine breast cancer metastatic cells can infiltrate, will induce their differentiation. BALB/c female mice were injected with 106 isogenic 4T1 breast cancer cells labeled with GFP. After 20 days primary tumors were removed, and artificial ε-PCL implants were implanted on the contralateral side. After 10 more days mice were sacrificed and implants along with lung tissue were harvested. Mice were divided in four groups: tumor removal with sham implantation surgery (n=5), tumor removal with ε-PCL implant (n=5), tumor removal with VEGF enriched ε-PCL implant (n=7) and mice without tumor with VEGF enriched ε-PCL implant (n=3). Differentiational status of GFP+ cells was assessed by Ki67 and activated caspase 3 expression, thus dividing the population in SC like cells (Ki67+ aCasp3-), TA like cells (Ki67+ aCasp3+) and FC like cells (Ki67- aCasp3+/-) on flow cytometry. Lung metastatic load was reduced by 20% in mice with simple ε-PCL implant when compared to tumor bearing group with no implant (p

Small subpopulation of cancer stem cells (characterized by CD44+CD24− phenotype) expresses GM3(NeuAc) and CD15s glycoconjugates. Acidic glycosphingolipid GM3(NeuAc), containing neuraminic acid substituted with acetyl residue, is known to inhibit tyrosine kinase associated with fibroblast growth factor receptor. CD15s glycoconjugate is the major ligand of endothelial (E) selectin, responsible for cancer cell metastasizing. We performed preliminary studies of viability, and GM3(NeuAc) and CD15s expression after treatment of MDA-MB-231 triple-negative breast cancer cells with a newly developed thienopyridine anticancer compound (1). The MTT assay was used for the cellular metabolic activity determination. Cells expressing highly GM3(NeuAc) and CD15s were analysed for their CD44+CD24- subpopulation percentage, and GM3(NeuAc) and CD15s median fluorescence intensity in CD44+CD24- gate using flow cytometry. The percentages of highly expressing GM3(NeuAc) and CD15s cancer stem cells were lower, and their GM3(NeuAc) median fluorescence intensity was higher after treatment with compound 1 in comparison to non-treated cells. There was no difference in CD15s expression after treatment with the same compound. Knowing the inhibitory role of GM3(NeuAc) in fibroblast growth factor signal transduction, these findings render compound 1 potentially useful for triple- negative breast cancer treatment.

Introduction: The aim of our study was to investigate the effect of white wine consumption on the expression of early inflammatory markers (MMP-2, MMP-9, NF-B) in the myocardial tissue following experimentally induced permanent myocardial ischemia. Materials and methods: Male Sprague-dawley rats (n=10) were randomized into two groups: drinking a combination of wine and water and those drinking water only for 28 days. After performing the coronary ligation, animals were left to survive for 24 hours and then sacrificed. Three representative zones for each group of animals: infarct/ischemic, periinfarct/ border zone and control/nonischemic zones were analysed for the expression of immunoreactivity for the above markers. The threshold area % of signal density for each marker was measured and compared. Results: For MMP-9, a significantly smaller expression was found in all three zones of wine drinking animals group (p

Introduction: After myocardial infarction (MI), CD44 is critical for healing and left ventricle remodelling. Monocyte CD44 antigen is hyaluronan ligand. Hyaluronan is responsible for extracellular matrix structural maintenance and inflammation regulation. CD11b+ monocytes have a role in slowing down angiogenesis and stimulation of inflammation after MI. The aim of this study was to estimate the effect of wine consumption at CD44 and CD11b monocyte expression after MI. In addition, CD15s glycoantigen, known as CD44 and CD11 branch, was determined. Materials and Methods: CD44, CD11b and CD15s positive monocytes in peripheral blood were measured by flow cytometery 24h after MI in male Sprague–Dawley rats (n=9) that consumed white wine for 4 weeks, and compared with control (C), water drinking (C6h and C24h after MI) and sham group. Results: Relative to C24h group, wine-consuming rats differed as follows: percentages of CD15s+CD11b+ monocyte and large monocyte subpopulations were decreased, % of CD44+ monocytes was increased ; expression of CD44 per one cell was two and five fold increased at CD11b+ monocytes and large monocytes, respectively ; CD44 was increased at CD15s+CD11b- monocytes ; CD11b was decreased at CD15s+CD44+ monocytes. In comparison to wine- consuming rats, C6h group showed additional significant difference in the increase of % of CD44+ monocytes and CD44 expression at CD11b+ and CD15s+CD11b- monocytes and CD11b+ large monocytes. Conclusions: Considering that CD44+ monocytes favour left ventricle remodelling and CD11b+ monocytes slow down angiogenesis and can promote injurious inflammation, our findings could indicate beneficial effects of wine pre-treatment on cardiac inflammatory response to MI.

Metastasis, tumor relapse and resistance to therapy remain the principal causes of death for breast cancer patients. The emerging paradigm posits that tumor progression is driven by a small subpopulation of cancer cancer stem cells that exhibit the ability to self-renew and the ability to regenerate the phenotypic heterogeneity of the parental tumor. Many cancer cellular functions have been discovered to be regulated by phospholipase C-gamma 2 (PLC) activation, suggesting that it represents an important therapeutic target for development of anticancer drugs. Here, we investigate the influence of a newly developed, small molecule PLC gamma inhibitor, with or without glucosylceramide synthase inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, D-PDMP) therapy, on the growth, survival and glycophenotype (CD15s and GM3) of breast cancer stem cells (CSCs: CD44+CD24-). MDA-MB-231 breast cancer cells were incubated with glucosylceramide synthase inhibitor (D-PDMP) and/or PLC gamma inhibitor. The viable cells were determined by the MTT assay. Flow cytometric analysis of cells positive to anti-CD44 and glycoantigens, and negative to CD24 was performed 48h after inhibitor treatment. Treatment of the MDA-MB-231 cells with the PLC gamma inhibitor decreased the number of total viable cells. Additional decrease was achieved after combined inhibitor treatment. Percentage of CSCs was significantly decreased only after PLC inhibitor alone treatment. CSC GM3 geometric mean fluorescence intensity (GMI) was increased after PLC inhibitor alone and combined inhibitor treatment. CSC CD15s GMI was slightly decreased after PLC inhibitor alone treatment but significantly after combined inhibitor treatment. PLC gamma inhibitor alone was the most effective against CSCs, but observed decrease of CSC CD15s GMI after combined inhibitor treatment indicates possible lower metastatic ability of dually treated cells.

INTRODUCTION: Prostate cancer remains the second most common cause of cancer related death among men, highlighting the need for new therapies. Many cancer cellular functions have been discovered to be regulated by phospholipase C (PLC) gamma activation, suggesting that it represents an important therapeutic target for development of anticancer drugs. Here, we investigate the influence of a newly developed, small molecule PLC gamma inhibitor, with or without taxane therapy, on the growth and survival of sub-populations of a prostate cancer cell line. MATERIALS AND METHODS: Cells were incubated 48h with Paclitaxel (5 nM) and PLC gamma inhibitor (1 microM) alone or in their combination.The viable cells were determined by the MTT assay. Flow cytometric analysis of cells positive to anti- CD44, anti-CD54, and propidium iodide staining was performed to characterise apoptotic Du145 sub- populations 48h after inhibitor treatment. RESULTS: Treatment of the DU145 prostate cancer cell line with the PLC gamma inhibitor resulted in cell cycle arrest with minimal increase in apoptosis. Sub-populations of prostate cancer cell lines have unique phenotypes (with CD44+ cells being more proliferative and CD54+ cells serving as better CD8+ T cell targets). We examined the effects of the PLC gamma inhibitor on these subpopulations and found that exposure decreased the percentage of both CD44+ (p=0.00007) and CD54+ (p=0.009) sub-populations. In contrast, treatment with Paclitaxel only effected CD44+ cells (p=0.0002). Combination treatment of the PLC gamma inhibitor and Paclitaxel however had synergistic effects on both CD44+ and CD54+ DU145 cells (p=0.005 and p=0.0002, respectively). CONCLUSIONS: These results suggest that a combination of PLC gamma inhibitor and Paclitaxel could be a novel strategy for the treatment of prostate cancer.

Globotetraosylceramide (Gb4Cer, GalNAc alpha1-3Gal alpha1-4Gal alpha1-4 Glc alpha1-1Cer) has been identified among the major renal neutral glycosphingolipids. Gb4Cer molecules are found in membrane lipid rafts where they can influence the function of receptors and transporters embedded in these domains. Apical membranes of the kidney proximal tubule epithelial cells contain lipid rafts that support absorptive strategy for nutrient reabsorption. Considering diabetic nephropathy and changed glycosphingolipid metabolism in diabetes, the aim of our study was to determine renal Gb4Cer expression in rat model of diabetes type 1. Diabetes was induced with streptozotocin (55 mg kg−1) injection two weeks before Gb4Cer analysis in the kidney of Sprague-Dawley rats weighing 140-160 g (8 males and 5 females) and compared to respective control animals (3 males and 3 females). Gb4Cer fractions in the tissues of diabetic and control rats were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific anti-Gb4Cer polyclonal antibody. Diabetic male rats showed increased expression of Gb4Cer. In addition, two bands of Gb4Cer derivative (corresponding to sulphated Gb4Cer), chromatographed bellow Gb4Cer, were also increased in diabetic male rats compared to control. In contrast to control male rats, control female rats had lower Gb4Cer and only one Gb4Cer derivative band. Diabetic female rats showed lower expression of Gb4Cer and Gb4Cer derivative compared to control female rats. Results of this study are significant in the view of the risk to develop end-stage renal disease that is doubled in men compared with women when age at diabetes type 1 onset is ≥15 years. Knowing that estrogen treatment decreases the content of Gb4Cer in rat kidney, our results point at the role of Gb4Cer in nephropathy development particularly in males.

Transcription and export of competent mRNPs are coupled by the CTD (Carboxy Terminal Domain) of RNA polymerase II that serves as a platform for seyuential recruitment of different protein factors. Defective mRNPs are recognised, retainde, and degraded by nuclear quality contro. Our team have developed a system to artificially disrupt mRNP biogenesis in the cell to better understand the molecular mechanisms involved in the recognition of defective mRNPs. We have shown that one of the major components of quality control is the NRD complex whose association with the CTD allows the recognition of aberrant transcripts and their elimination by the nuclear exosome. This quality control may be destroyed if certain proteins involved in transcription termination are over-expressed, thus preventing the detection/destruction of defective mRNPs. To understand how this delicate balance between transcription termination proteins and surveillance system operates in the cell, we have over-expressed several transcription termination factors in vivo and analysed the consequences in the cells where defective mRNPs were synthesised. This research has shown that proteins involved in transcription termination have distinct roles: the over-expression of some proteins disrupts the process of recognition of defective mRNPs while the others do not show this effect.

U radu je istraživano preživljavanje i sposobnost agregacije suspendiranih stanica pekarskog kvasca Saccharomyces cerevisiae tretiranih niskofrekventnim pulsnim ultrazvučnim valovima (20 kHz, 40 % snage ultrazvuka, intervali pulsa 10 sekundi) tijekom različitog vremena izloženosti (1-10 minuta). Koncentracija stanica u suspenziji bila je 1 mg/mL (na suhu tvar), a pH vrijednost 9, 5. Morfološke promjene stanica izazvane ultrazvučnim tretmanom praćene su pod svjetlosnim mikroskopom (bojanjem metilenskim modrilom), te snimane na računalo. Dobiveni rezultati pokazali su da se s povećanjem vremena izloženosti pulsnim ultrazvučnim valovima vidljivo povećavao udjel oštećenih stanica, što je rezultiralo povećanjem veličina aglomerata. Pri različitim vremenima izloženosti stanica, mikroskopski je potvrđeno stvaranje nakupina (7 min) i velikih aglomerata (10 min). Fiziološka aktivnost stanica pekarskog kvasca nakon tretmana pulsnim ultrazvukom bila je relativno nezadovoljavajuća, jer je broj mrtvih stanica nakon 10 minuta bio 67 %, a analitičkim određivanjem (UV- spektrofotometrijski), udjel proteina je smanjen za 80 % od početne vrijednosti, 45, 39%).