Comparative Study of Adult Hippocampal Neurogenesis in Lurcher and Wild-Type Mice | Matěj Zvoneček
Running title: Neurogenesis in Adult Mice Hippocampus
Authors: Matěj Zvoneček (1), Parvathi Satheesh (1), Nilpawan Roy Choudhury (1), Jan Cendelín (1, 2),
Jan Tůma (1)
Supervisor: Jan Tůma (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Cerebellar degenerations are characterized by motor deficits including ataxia,
hypotonia, and nystagmus, but they are also associated with cognitive, affective, and sensory
impairments. Lurcher mice (Lc) represent a well-established model of cerebellar degeneration
caused by a mutation in the Grid2 gene encoding the GluRδ2 receptor subunit, leading to progressive
loss of Purkinje cells and other cerebellar neurons. Behavioral and molecular data also
suggest that the hippocampus may be altered in these mice. In this study, we investigate hippocampal
neurogenesis in Lc mice. Since adult hippocampal neurogenesis plays an important
role in learning, memory, and brain plasticity, alterations in this process may contribute to the
cognitive deficits associated with cerebellar degeneration.
Objective: The aim of this study is to quantify the rate of adult hippocampal neurogenesis in
wild-type (WT) and Lc mice. Considering the reduced learning capability in Lc mice, we hypothesize
that the rate of neurogenesis differs in adult hippocampus between Lc and WT mice.
Material and Methods: Adult Lc mice were used as a model of cerebellar degeneration and compared
with WT mice. Brains from Lc (n = 3) and WT (n = 3) mice were sectioned on a microtome
in 30 μm sections, mounted on slides and immunostained for doublecortin (DCX), a marker of
immature neurons, together with DAPI nuclear staining. Sections were imaged using confocal
microscopy, and z-stack images were analyzed in Fiji software. DCX-positive immature neurons
were systematically counted in the dentate gyrus of the hippocampus. All image analyses were
performed as a blind study. The data were evaluated using permutational t-test with 10,000
permutations.
Results & Discussion: Preliminary analysis of Lc and WT mice revealed no significant difference
between the groups. These findings suggest that the cognitive deficit observed in Lc mice in
behavioral tests, such as the Morris water maze, is unlikely to be caused by alterations in hippocampal
neurogenesis. Moreover, we have previously demonstrated that this cognitive deficit
is not associated with activation of the hypothalamic–pituitary–adrenal axis. Therefore, other
mechanisms may be involved, such as maladaptive strategies due to behavioral disinhibition or
motor impairment. The optional photo captures newly formed neurons (cells stained in red) in
dentate gyrus of adult mouse hippocampus. IHC staining and imaging author – Parvathi Satheesh.
Conclusion: The current study found no significant difference in hippocampal neurogenesis
between Lc and WT mice. However, additional brain samples will be analyzed to increase the
sample size and assesses other markers of brain plasticity.
Funding: This work was supported by the AZV grant No. NW26-08-00119, SVV262774, Cooperatio
(NEUR and MED/DIAG research areas), and GAUK project No. 70124.
Gallbladder cancer in bohemia exhibits a Distinct clinicopathologic profile enriched incertain tumor types compared to other populations | Marek Brousil
Bohemian Gallbladder Carcinoma Profile
Authors: Marek Brousil (1,2), Marián Švajdler (1,2), Irem Güvendir Bakkaloğlu (3), Tiziana Salviato (4),
Volkan Adsay (5)
Supervisor: Marián Švajdler (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Bioptical Laboratory, Ltd, Pilsen, Czech Republic (3) Kartal Dr Lütfi Kırdar
Education and Research Hospital, Istanbul, Turkey (4) Università degli Studi di Modena e Reggio
Emilia, Modena, Italy (5) Koç University Hospital, Istanbul, Turkey
State-of-the-Art: The incidence of gallbladder cancer (GBC) exhibits marked geographic het
erogeneity worldwide. High GBC rates are well-documented among Chilean indigenous popula
tions, where the disease is primarily linked to cholelithiasis and obesity. Additional hotspots are
recognised in Japan (associated with pancreatobiliary maljunction and biliary reflux) and India
(linked to S. typhi infection and heavy metal exposure).
However, an unusually high incidence is also observed in the Bohemian region of Central Eu
rope, where etiological factors remain poorly defined, and data on the incidence of GBC sub
types remain largely unavailable.
Objective: This study aimed to investigate the distinct clinicopathologic features of GBC cases
from Bohemia compared to other global cohorts.
Material and Methods: A total of 102 GBC cases diagnosed at Biopticka Laboratory (Plzeň,
Czech Republic) were analysed and compared with 606 cases from Chile, the USA, and Turkey.
The comparative analysis focused on a comprehensive clinicopathologic characterisation, in
cluding tumor types and precursor lesions.
Results & Discussion: 1. Hyalinizing Cholecystitis: More prevalent in the Bohemian cohort
(18.6%, 19/102) compared to others (5.9%, 36/606; p < 0.00001) (Fig 1a). These cases might be
associated with radon exposure.
Intracholecystic Papillary-Tubular Neoplasm (ICPN)-associated carcinoma: Observed in 14%
of Bohemian cases versus 6% in other populations (Fig 1c), suggesting an alternative chemical/
reflux-based etiology.
Conclusion: The Bohemian GBC cohort shows a distinct landscape enriched in hyalinizing chol
ecystitis, diffuse-type PCC, and ICPN-associated GBC. These findings suggest unique pathoge
netic pathways in this population, offering a novel model to explore underrecognized biliary
carcinogenesis.
Poorly Cohesive Carcinomas (PCCs): Represented 22.5% (23/102) of cases in Bohemia vs. 3.8%
(24/606) in other populations (p < 0.0001). Bohemian PCCs exhibited a striking „invisible car
cinoma“ pattern, resembling mammary lobular ca. with preserved mucosa and a linitis plasti
ca-like appearance, unlike the pleomorphic/destructive PCCs observed in other cohorts (Fig 1b).
Identification of sexually Dimorphic Anatomical features in tricentennial femora: An
osteometric study | Alesa Stepanichine
Whose femora were these?
Authors: Alesa Stepanichine(1), Styliani Spourdalaki (1), Devran Demir (1), Bacem Othman (1,2)
Supervisor: Omid Moztarzadeh (1)
(1) Department of Anatomy, Faculty of Medicine in Pilsen, Charles University and University Hos
pital, Pilsen (2) Bioptical Laboratory, Ltd., Pilsen, Czech Republic
State-of-the-Art: Forensic osteometry of mass burial assemblages provides a non-invasive
framework for reconstructing population composition, demographic structure. The Arthron R
package was used to provide a reference for population-calibrated discriminant functions that
translate continuous skeletal measurements into probabilistic sex, ethnicity and age estimates.
Objective: We aim to evaluate all discernible anatomical features of the 380-year-old femora in
order to estimate the sex and age category of the individuals to whom these bones belonged.
Material and Methods: Departmental femora purchased from Stahl.cz (stěhovací služba, spol.
s r.o.), with data enabling cross-referral to a Swedish mass grave assemblage in Bohemia (ante
1648), were assessed for sex, age, and skeletal morphometry using manual osteometric meas
urements and automated 3D landmark detection. The arthron R package reference dataset
provided population-level benchmarks. Variables recorded were femoral head diameter (FHD),
subtrochanteric diameter (SubD), vertical femoral head diameter (VD-FH), intertrochanteric
crest width (IT Crest), linea aspera prominence, neck-shaft angle (NSA), shaft length, and shaft
contour, to determine sex and approximate age of the remains.
Results & Discussion: Sex was estimated by two independent observers (Cohen‘s κ = 0.811;
18/20 agreement). Of the studied specimens, the cohort comprised males, females and juve
niles (60%, 30%, and 10%). Female femora showed femoral head diameter ratio of 0.91 (83.3 ±
9.8 mm in females versus 91.6 ± 8.5 mm in males; p = 0.083), shaft length ratio 0.93 (p = 0.034),
intertrochanteric crest ratio 0.89 (p = 0.050), NSA 105°–125° (mean 115° ± 7.1°). Only one femur
showed a distal shaft fracture consistent with low-energy axial loading. No specimen exhibited
cortical entry defects, stellate fracture patterns, or lead deformation traces of musket-ball inju
ries or other incapacitating injuries. The statistical significance seems underpowered owing to
the small samples obtained from historical context.
Conclusion: The heterogeneous sex composition, the representation of adult females, the in
clusion of juvenile individuals, and indications of weapon-related trauma collectively render the
assemblage inconsistent with an interpretation as a burial of military combatants or physically
depleted.
Genes with generally higher activity across multiple cell populations and tissues contain fewer upstream start and stop codons in their leader sequences | Dayana Nubayeva
Genes higher activity fewer upstream starts stops
Authors: Dayana Nubayeva (1)
Supervisor: Pavel Dvořák (1, 2)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: The 5′ untranslated region (5′ UTR), also called leader sequence, regulates
translation by shaping how ribosomes bind and scan mRNA. Its structures—such as hairpins,
stemloops, or Gquadruplexes—can slow or enhance initiation. Functional motifs like IRES, TOP
sequences, or Kozak elements further tune translation efficiency. Many 5′ UTRs also contain
upstream AUGs, uORFs, and stop codons that modulate ribosome scanning and influence expression
of the main coding sequence.
Objective: The aim was to test the hypothesis that genes that have different numbers of upstream
start and stop codons in their 5‘ UTR regions also have specifically different expression
at the protein level, either in general or in specific tissues.
Material and Methods: 1) Downloading sequences of 5‘UTR regions of all human protein-coding
genes from the Ensembl database in FASTA format. 2) Selection of Group 1 – genes in whose
sequences there are no upstream start or stop codons. 3) Selection of Groups 2 and 3 – genes
whose 5‘UTRs are approximately the same length as Group 1 (i.e. in the range of 120 to 400
bp) and contain fewer (i.e. 1 to 3) and more motifs of interest (i.e. 6 and more), respectively. 4)
Downloading data on protein expression in tissues from The Human Protein Atlas database. 5)
Assignment of protein expression data to genes from Groups 1, 2 and 3. 6) Statistical comparison
of selected data and interpretation of results.
Results & Discussion: Statistical comparison of the three tested gene groups showed that the
individual groups differ significantly in the level of expression assessed using the IHC technique
(categories „Not detected“, „Low“, „Medium“ and „High“) as well as in expression in different
cell types and tissues (χ² = 217.4, p ≈ 3.7×10⁻⁴¹). The most striking difference is the behavior of
Group 2, which differs from the others mainly in the higher representation of „Medium“ and
„High“ expression levels. At the „High“ level, all three pairwise differences between the groups
were statistically significant. In contrast, Group 1 has a dominant level of „Not detected“ in most
tissues and cell types. Group 3 can be characterized as in between Groups 1 and 2 with more
extremities.
Conclusion: Group 1 (no upstream start and/or stop codons) represents genes with low or no
expression; Group 2 (1 to 3 upstream starts and/or stops) genes with higher activity and significant
variability; Group 3 (6 or more upstream starts and/or stops) is in between, but with clear
tissue specializations.
Funding: Supported by the project „Integration of biomedical research and health care in the
Pilsen metropolitan area“ (reg. no. CZ.02.01.01/00/23_021/0008828) – co-funded by the European
Regional Development Fund, European Union, and State Budget of the Czech Republic.
The Interplay Between YAP, Sox2, and the Hippo Signaling Pathway in Human Fibrosarcoma Stem-Like Cells | Adam Gryč
YAP and Sox2 in Fibrosarcoma Stem Cells
Authors: Adam Gryč (1), Matěj Laub (1), Martin Prchal (1)
Supervisor: Jiří Hatina (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Fibrosarcoma is an uncommon but highly aggresive malignant tumor.
HT1080 is a permanent human fibrosarcoma cell line frequently used to model complex fibrosarcoma
biology. According to the cancer stem cell (CSC) hypothesis, tumor aggressiveness and
drug resistance are sustained by a minority subpopulation of undifferentiated stem-like cells.
There are many approaches how to identify putative CSCs, such as side popuation or aldehyde
dehydrogenase – based cell sorting, selection of anchorage-independently growing cells, using
of specific cell surface markers or fluorescent reporter constructs, such as the YAP-activated
mCherry used in our study.
Objective: This study investigates YAP oncogenic activity in HT1080 fibrosarcoma cells in relation
to the expression of a battery of stemness-related genes and anchorage independent
clonogenicity as a canonical stemness-related trait.
Material and Methods: We used HT1080 cells expressing an mCherry reporter driven by a
YAP-responsive promoter. Cells were cultured adherently with serum. Loss of contact inhibition
was assessed via fluorescence microscopy in confluent cultures. Cells were then sorted (FACS)
into mCherry-high (YAP-active) and mCherry-low subpopulations for two purposes: 1) evaluating
clonogenicity in methylcellulose with serum (anchorage-independent conditions), and 2)
isolating RNA for subsequent qRT-PCR of stemness-related genes. These include Sox2, ABCG2,
ZFP57, IGF2 and ALDH1A1.
Results & Discussion: Fluorescence microscopy revealed that YAP activity in HT1080 cells does
not undergo contact inhibition. Even in fully confluent cultures, a significant proportion of cells
remained strongly mCherry-positive, indicating sustained YAP activation. In anchorage-independent
assays, the YAP-active (mCherry-high) subpopulation exhibited a significantly higher
capacity to form spherical colonies compared to mCherry-low cells. Although final quantification
of specific genes is ongoing, the overall qPCR gene expression profile strongly indicates that
mCherry-high cells possess a pronounced stem-like character.
Conclusion: The Hippo pathway acts as a tumor suppressor, degrading YAP upon cell contact
(contact inhibition). When dysregulated, YAP accumulates and drives downstream targets like
CTGF, promoting tumor aggressiveness, and the CSC phenotype.
Cerebellar and hippocampal morphology in a mouse model of spinocerebellar ataxia
type 1 | Patricie Klosse
Cerebellar and hippocampal morphology in SCA1 mice
Authors: Patricie Klosse (1), Jan Látal (1), Štěpán Kápl (1), Nilpawan Roy Choudhury (1), Parvathi
Satheesh (1)
Supervisor: Jan Cendelín (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant cerebellar
ataxia caused by expanded CAG repeat in Ataxin 1 gene. Besides adult-onset progressive cerebellar
degeneration resulting in cerebellar motor and cognitive affective syndromes, there are
also changes in the hippocampus contributing to pathological cognitive and behavioral phenotypes.
The disease leads to severe disability of the patients and even premature death. SCA1
mice have been used as a knock-in model of this disease for about 20 years. They have been
considered replicating development and symptoms of SCA1 with well-described severe neurological
and neuropathological manifestations in advanced age. However, in the last two years,
the phenotype appears to be alleviating.
Objective: The aim of the study was to verify presence of cerebellar and hippocampal neuropathology
SCA1 mice.
Material and Methods: Paraformaldehyde-fixed brains of heterozygous SCA1 mice aged 7-8
months and strain- and age-matched healthy control mice were cryosliced and stained with
hematoxylin-eosin. The volumes of the hippocampus (dentate gyrus, pyramidal layer and stratum
oriens) and the cerebellum (total volume and molecular layer separately) were estimated
using Cavalieri principle. Heterozygous mice and mutation non-carriers were distinguished by
genotyping identifying presence of normal length allele as well as extended allele, or normal
length allele only, respectively.
Results & Discussion: We did not find any significant reduction in the volume of individual parts
of the hippocampus or the cerebellum as a whole or its molecular layer selectively in SCA1 mice.
It is in accordance with our previous observation of non-increased density of glial markers in
the hippocampus of SCA1 mice. However, it is in contrast with marked cerebellar and hippocampal
neuropathology described earlier in our laboratory in mice from the same colony even at a
younger age. It suggests that reduction of pathological functional phenotypes during several
years is accompanied by alleviation of neuropathology, although the mutation itself persists.
Conclusion: The results suggest instability of phenotype of SCA1 mice. Elucidation of the mechanisms
of this phenomenon requires sequencing of the allele, examination of its expression,
searching for potential disease-modifying gene mutations, and more detailed analysis of the
phenotype.
Funding: The work was supported by Cooperatio (NEUR, DIAG) and SVV 262774.
Enhancing cognitive performance in the first minutes after napping through gamma binaural beat stimulation | Natália Poláková
Gamma binaural beats and sleep inertia
Authors: Natália Poláková (1)
Supervisor: Karel Blahna (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Sleep inertia is a transitional state occurring immediately after awakening,
characterized by reduced alertness, slower reaction times, impaired cognitive performance,
and increased subjective sleepiness. These effects can persist for several minutes and may negatively
influence activities that require rapid decision-making and sustained attention. Various
approaches have been investigated to mitigate sleep inertia and accelerate the recovery of cognitive
functioning. Auditory stimulation using binaural beats has been shown to influence brain
activity and cognitive processes, with gamma-frequency stimulation being associated with increased
cortical activation and attention-related processes.
Objective: The aim of this pilot study was to investigate whether gamma-frequency (40 Hz)
binaural beat stimulation can influence vigilance and cognitive performance during the early
post-nap period characterized by sleep inertia.
Material and Methods: Ten healthy participants aged 18–30 took part in the study. The experiment
was conducted over two testing days in a within-subject design: one day without auditory
stimulation (control) and one day with 40 Hz binaural beat stimulation. Participants completed a
30-minute nap, followed immediately by a 10-minute Psychomotor Vigilance Task (PVT) assessing
reaction time and sustained attention. After the task, subjective sleepiness was evaluated
using the Stanford Sleepiness Scale (SSS) and the Karolinska Sleepiness Scale (KSS). Reaction
time data were analyzed using vigilance-related metrics reflecting temporal performance dynamics,
including optimal-performance episodes and the AUC_good metric. Linear mixed-effects
models were used for statistical comparison.
Results & Discussion: Preliminary analyses revealed differences in the temporal dynamics
of vigilance between the stimulation and control conditions. The overall aggregated vigilance
energy metric (AUC_good) did not show a statistically significant effect of binaural beat stimulation.
However, exploratory analyses of performance across the task suggested differences
in the temporal structure of vigilance between conditions, indicating that the stimulation may
influence short-term vigilance dynamics during the post-nap period. These findings suggest that
while the overall level of vigilance did not significantly differ between conditions, binaural beat
stimulation may affect how vigilance fluctuates over time.
Conclusion: This pilot s tudy found no significant overall improvement in vigilance after gamma-
frequency binaural beat stimulation during the early post-nap period. However, differences
in vigilance dynamics suggest subtle effects that should be examined in future studies with
larger samples.
Quantitative Evaluation of Skeletal Muscle Structure in a TFAM-Deficient Mouse Model of Mitochondrial Myopathy | Timo Nedela
Tfam knock-out mice muscle morphometry
Authors: Timo Nedela (1), Jan Nevoral (1), Daniel Follprecht (2), Yaroslav Kolinko (1)
Supervisor: Yaroslav Kolinko (1)
(1) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic (2) Department of Sports Medicine and Active Health Sciences, Faculty of Medicine
in Pilsen, Charles University, Czech Republic
State-of-the-Art: Mitochondrial myopathies cause progressive skeletal-muscle weakness
and remodeling, but linking genotype to quantitative tissue changes remains challenging. Muscle-
specific disruption of mitochondrial transcription factor A (Tfam) is an established model of
mtDNA depletion and myopathy. Recent studies have proposed reproducible, TFAM- associated
remodeling patterns in skeletal muscle. Nevertheless, these reports rely largely on qualitative
evaluation of histological sections and lack quantitative assessment of the analyzed material.
This prompted us to verify these reported changes and determine whether the observed differences
are consistent in the available mice with mitochondrial disease or reflect inter-individual
biological variability.
Objective: The aim of this pilot study was to perform a quantitative analysis of histological sections
of the quadriceps muscle in TFAM-deficient and wild-type control mice. For this purpose,
design-based stereological approaches were applied to analyze images obtained from the tissue
sections.
Material and Methods: Fifteen male mice were included in the study. Six animals served as
healthy controls, while nine had mitochondrial myopathy induced by skeletal muscle–specific
disruption of the Tfam-gene. At six months, animals were euthanized and the quadriceps
femoris muscle was dissected for histological evaluation. Samples were fixed in formaldehyde,
routinely processed, and sectioned in random orientation. Sections were stained with hematoxylin
and eosin (H&E) and green trichrome. Quantitative analysis was performed using the
Cavalieri method in Steriologer 11 software. The following parameters were assessed: fascicle
area, myofibril number and volume fractions, and the volume fractions of connective tissue and
myonuclei.
Results & Discussion: Histological examination of Tfam mice revealed a higher number of
blood vessels of varying diameters compared with WT controls. Quantitative analysis showed
a mild 3.5% increase in myofibril area fraction (WT 79.9 ± 12.7%; KO 82.7 ± 1.5%). The total connective
tissue area increased by 22% (WT 2.5 ± 0.875 mm²; KO 3.12 ± 1.599 mm²), although this
difference was likely influenced by variation in sample size distribution. In contrast, the connective
tissue area fraction per 1 mm² slightly decreased (WT 0.041 ± 0.009 mm²/1 mm²; KO 0.038
± 0.009 mm²/1 mm²). The overall myofibril amount remained within biological variability, while
the myonuclear volume fraction decreased by 16% (WT 0.019 ± 0.006 mm²/1 mm²; KO 0.016 ±
0.003 mm²/1 mm²).
Conclusion: Tfam mice showed increased vascularity compared with W T. However, no significant
differences in major morphometric parameters were detected. These findings suggest that
more advanced methods and biochemical or organelle-level analyses are needed. Trichrome
staining may improve structural assessment.
Funding: Cooperatio Program, research areas MED/DIAG.
Assessing Muscle-to-Muscle Augmentation and Impairment in the Brachium Using
Anatomage Virtual Dissection and OpenSim Simulation | Siddharth Roy
Morphometric Virtual Dissection of the Brachium
Authors: Siddharth Roy (1), Antonia Johanna Stimmel (1)
Supervisor: Bacem Othman (1,2)
(1) Department of Anatomy, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Bioptical Laboratory, Ltd., Pilsen, Czech Republic
State-of-the-Art: Computational musculoskeletal modelling of the upper limb has relied
mainly on Delp’s upper extremity (1995) and Holzbaur’s (2005) models, and their cadaveric
derivatives, to assess many parameters. These parameters include force-generating capacity,
pennation geometry, and moment arm profiles. We propose an Anatomage-based model that
evaluates inter-individual and inter-sex variation in brachial muscle architecture.
Objective: We aim to determine which muscular neighbourhood configuration most can indicate
augmentation and functional impairment patterns within the anterior and posterior brachial
compartments.
Material and Methods: First, morphometric measurements of bilateral muscle belly radii and
lengths for biceps brachii, brachialis, triceps, coracobrachialis, and anconeus were retrieved
from Anatomage data,stratified by sex and laterality. Second, these measurements were exported
into OpenSim 4.5 software program to estimate maximum isometric force at a specific
tension, inverse kinematics, static optimisation, and musclature performance of the brachium.
Results & Discussion:Maximum isometric forces in the anterior compartment revealed a bilateral
asymmetry. The brachialis demonstrated an 8.31-fold right-to-left force asymmetry with a
right optimal fibre length. In the posterior compartment, the triceps produced identical forces
bilaterally, with a greater optimal fibre lengths of 0.241 m on the right. The coracobrachialis
demonstrated a 3.13-fold right-to-left force differential. The anconeus showed moderate bilateral
asymmetry. The biceps-brachialis pairing exhibited the greatest combined force asymmetry.
All tonicity readings characterise co-augmetation on the right side.
Conclusion: Anatomage virtual dissection provided morphometric fidelity sufficient to reveal
clinically and biomechanically meaningful inter-limb and inter-sex asymmetry in the brachial
muscle.