Characterisation of novel mitochondrial proteins in \kur{Trypanosoma brucei}
RAŠKOVÁ, Vendula
2023 - English
This study analyses and characterises novel mitochondrial proteins in the parasitic protist Trypanosoma brucei. Applying phylogenetic analysis was described the evolutionary origin of ZapE protein in eukaryotes, using a newly developed proximity-dependent biotinylation approach (BioID2) we identified ZapE interaction partners like Oxa1. We also discovered a relationship when distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. TbPams were detected by phylogenetic analyses as orthologs of corresponding proteins in Opistokonts. We analyse the function of TbPam18 and TbPam16 in the replication of the mitochondrial DNA and determine, how the TMDs of TbPam18 and TbPam16 are essential for their functions. Finally, we evaluated a set of putative mitochondrial proteins of the heterolobosean N. gruberi defined by Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and analyse the origin of mtFfh and mtFtsY. This study analyses and characterises novel mitochondrial proteins in the parasitic protist Trypanosoma brucei. Applying phylogenetic analysis was described the evolutionary origin of ZapE protein in eukaryotes, using a newly developed proximity-dependent biotinylation approach (BioID2) we identified ZapE interaction partners like Oxa1. We also discovered a relationship when distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. TbPams were detected by phylogenetic analyses as orthologs of corresponding proteins in Opistokonts. We analyse the function of TbPam18 and TbPam16 in the replication of the mitochondrial DNA and determine, how the TMDs of TbPam18 and TbPam16 are essential for their functions. Finally, we evaluated a set of putative mitochondrial proteins of the heterolobosean N. gruberi defined by Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and analyse the origin of mtFfh and mtFtsY.
Keywords:
Trypanosoma brucei; kDNA; mitochondrion; TbPam protein; ZapE; kinetoplast
Available in the Digital Repository of University of South Bohemia.
Characterisation of novel mitochondrial proteins in \kur{Trypanosoma brucei}
This study analyses and characterises novel mitochondrial proteins in the parasitic protist Trypanosoma brucei. Applying phylogenetic analysis was described the evolutionary origin of ZapE protein in ...
Úloha izoenzymů v regulaci metabolismu sinic
BACHHAR, Anushree
2023 - English
Modelová sinice Synechocystis PCC 6803 je jedním z nejstudovanějších druhů sinic, který má významný podíl v biotechnologických aplikacích, což vysvětluje, proč se jí také říká "zelená E. coli". Experimentální studie mohou poskytnout in vitro charakterizaci isozymů, ale nikdo neví, zda a jak přesná je taková parametrizace ve srovnání se stavem in vivo. Také navzdory studiím s jedním/mnoha mutanty, stále plně neznáme rozsah rolí izoenzymů. Důvody, které stojí za tímto problémem, jsou: i) vyřazení jednoho izozymu je pravděpodobně kompenzováno zbývajícími izozymy, ii) deaktivace všech izozymu nepočítá s neznámou multifunkčností jiných enzymů nebo iii) metabolická plasticita Synechocystis umožňuje přesměrování metabolického toku na alternativní dráhy v rámci centrálního metabolismu uhlíku, např. prostřednictvím glykolytických drah (Embden-Meyerhof-Parnasova dráha, Entner-Doudorova dráha, fosfoketolázová dráha a oxidativní pentózofosfátová dráha). V této studii jsem se zaměřila na rozluštění některých regulačních mechanismů v rámci centrálního metabolismu uhlíku Synechocystis s pomocí metabolického modelu, který integruje fluxomické, metabolické a transkriptomické údaje z literatury a různých databází, např. Uniprot, KEGG. V první polovině svého doktorského výzkumu jsem se zaměřila na vliv většinou opomíjené glykolytické fosfoketolázové dráhy. Zejména jsme ukázali, že tok uhlíku cestou fosfoketolázy může dosáhnout 250 % toku přes Embden-Meyerhof-Parnas glykolýzu za autotrofních podmínek a úrovně CO2 okolního prostředí. Tedy, fosfoketolázová dráha hraje klíčovou roli tím, že zmírňuje dekarboxylaci, tj. ztráty uhlíku, ke kterým dochází v ostatních glykolytických cestách. Dále jsme předpověděli, že role fosfoketolázové dráhy za mixotrofních podmínek je u Synechocystis spíše zanedbatelná, přestože je tato dráha klíčová u většiny bakterií za heterotrofních podmínek. Kromě toho jsme potvrdili existenci předpokládaného izoenzymu fosfoketolázy (PKET 2) a kvantifikovali následující výsledky za autotrofních podmínek a úrovně CO2 okolního prostředí: 1) 17% snížení toku přes RuBisCO pro delta pket1 a 2) 11,2-14,3% snížení růstu pro delta pket2 v turbulentním prostředí a 3) preference substrátů pro fosfoketolázovou dráhu za daných růstových podmínek. V druhé polovině své výzkumné práce jsem se zaměřila na nedávno potvrzenou glykolytickou Entner-Doudoroffovu dráhu u Synechocystis. Entner-Doudoroffova dráha byla dříve považována za velmi běžnou (~92 %) mezi sinicemi, ale moje bioinformatická analýza na základě dostupných zdrojů dat předpověděla, že výskyt Entner-Doudoroffovy dráhy je nižší než 50 %. Zajímavé je, že jsme také identifikovali pravděpodobné izozymy v rámci Entner-Doudoroffovy dráhy u některých sinic. V případě Synechocystis jsme poskytli první odhad metabolického toku přes Entner-Doudoroffovu dráhu na základě údajů změn růstu při zablokování této dráhy; je zatím obtížné provést experimentální ověření s pomocí izotopu 13C vzhledem k současným limitům detekce velmi nízkých koncentrací metabolitů v přístrojích hmotnostní spektrometrie. Nicméně jsme zjistili příliš mnoho nejasností (multifunkčnost enzymů a problémy s identitou enzymů Entner-Doudoroffovy dráhy), takže je obtížné anotovat nebo předpovědět rozsah možných metabolických a regulačních funkcí Entner-Doudoroffovy dráhy u Synechocystis. Model cyanobacterium Synechocystis PCC 6803 is one of the most studied species of cyanobacteria, having a significant impact in biotechnology, which explains why it is also known as "green E. coli". Experimental studies could deliver in vitro characterization of isozymes, but nobody knows if and how accurate such parametrization is in comparison to in vivo conditions. Also, despite the single/multi-mutant studies, the extent of isoenzymes roles is still lacking. The reasons behind this issue are: i) single knock-out is likely to be compensated by remaining isozyme(s), ii) deactivation of all isozymes does not count for unknown multi functionalities or iii) metabolic plasticity of Synechocystis allowing the redirection of metabolic flux towards alternative pathways within the central carbon metabolism, e.g., via glycolytic pathways (Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, phosphoketolase pathway and oxidative pentose phosphate pathway). In this study, I aimed to decipher some of the regulatory mechanisms within the central carbon metabolism of Synechocystis with the aid of metabolic model, integrating fluxomic, metabolic and transcriptomic data from literature and various databases e.g., Uniprot, KEGG. In the first half of my PhD research, I have focused on the impact of the mostly ignored glycolytic phosphoketolase pathway. In particular, we have shown that carbon flux via phosphoketolase pathway could be above 250% of the flux via Embden-Meyerhof-Parnas glycolysis under autotrophic conditions ambient CO2, thus playing a crucial role by mitigating the decarboxylation, i.e., carbon loss, occurring in other glycolytic pathways. We predicted that role of phosphoketolase pathway under mixotrophic conditions is rather negligible in Synechocystis, despite phosphoketolase pathway is crucial for heterotrophic conditions in most bacteria. In addition, we supported the existence of putative phosphoketolase isoenzyme (PKET 2) and quantified the following results under autotrophic conditions ambient CO2: 1) 17% flux reduction via RuBisCO for delta pket1 and 2) 11.2-14.3% growth decrease for delta pket2 in turbulent environment and 3) the substrate preference for phosphoketolase pathway under a given growth conditions. In the second half of my research work, I have focused on the recently confirmed glycolytic route in Synechocystis, the Entner-Doudoroff pathway (ED-P). Entner-Doudoroff pathway was previously concluded to be a very common (~92%) pathway among cyanobacteria, but my bioinformatic analysis based on available data sources predicted, Entner-Doudoroff pathway occurrence is below 50%. Interestingly, we have also identified plausible isozymes within Entner-Doudoroff pathway for some cyanobacteria. In the case of Synechocystis, we provided the first estimation of flux via Entner-Doudoroff pathway based on the growth impairment data which cannot be currently detected by 13C labelling experiments due to current detection limit for lower concentrations metabolite levels in mass-spectrometry devices. Nevertheless, we have identified too many uncertainties (enzyme multifunctionalities and identity issues) that it become difficult to annotate or predict the extent of possible metabolic and regulatory functions of Entner-Doudoroff pathway in Synechocystis.
Keywords:
Synechocystis PCC 6803; centrální metabolismus uhlíku; isoenzymy; kinetické modelování; multiomika.
Available in the Digital Repository of University of South Bohemia.
Úloha izoenzymů v regulaci metabolismu sinic
Modelová sinice Synechocystis PCC 6803 je jedním z nejstudovanějších druhů sinic, který má významný podíl v biotechnologických aplikacích, což vysvětluje, proč se jí také říká "zelená E. coli". ...
Genome evolution of endosymbiotic bacteria in blood-sucking insects
ŘÍHOVÁ, Jana
2023 - English
The research on insect-bacteria symbiosis is experiencing a rapid burst leading to the identification of much higher complexity and dynamics of the insect-bacteria associations than ever before envisaged. The generally accepted concept of obligate bacterial symbionts as providers of essential compounds (vitamins, aminoacids) has been further developed into the framework of microbiomes, i.e. communities of microorganisms associated with the host. The majority of the knowledge has been derived from plant sap-feeders, while data on blood-feeding insects remain fragmented and even inconsistent. In this thesis, we employ amplicon, genomic and in-vivo approaches to investigate several symbiotic systems of obligate blood-feeders and address the following questions: What is the diversity of symbiotic bacteria in our studied systems? What are the metabolic capacities of these symbionts? What is the role of symbionts in the biology of their hosts? What are the possible sources of bacteria prone to establish symbiosis with obligate blood-feeders? Where these symbionts reside in the host´s body? What is the coevolutionary pattern of these bacteria with their hosts? As an answer to these questions, I present the results detailed in the course of this thesis, which are preceded by a comprehensive introduction to the studied topics and wraps up with a summary of the main results.
Keywords:
symbiotic bacteria; obligate blood-feeders; microbiome; amplicon sequencing; whole-genome sequencing; metaboic capacities
Available in the Digital Repository of University of South Bohemia.
Genome evolution of endosymbiotic bacteria in blood-sucking insects
The research on insect-bacteria symbiosis is experiencing a rapid burst leading to the identification of much higher complexity and dynamics of the insect-bacteria associations than ever before ...
Biomolecular simulations in non-aqueous media
FADAEI, Fatemeh
2023 - English
Available in the Digital Repository of University of South Bohemia.
Biomolecular simulations in non-aqueous media
Dung beetles of Central Europe: Diversity and conservation
AMBROŽOVÁ, Lucie
2023 - English
Tato disertační práce se skládá ze tří ochranářsky orientovaných studií, které se zabývají zejména faktory určujícími diverzitu koprofágních brouků ve střední Evropě. První studie hodnotila vliv rutinního odčervování hospodářských zvířat ivermektinem na druhovou diverzitu a funkční výkonnost společenstva koprofágních brouků. Druhá studie zkoumala vliv alternativních lesních managementů pařazení a odstranění svrchní vrstvy půdy na druhovou a funkční diverzitu lesních koprofágních brouků a drabčíků. Poslední studie se zabývala environmentálními a managementovými faktory, které určují -diverzitu koprofágních brouků, s cílem identifikovat prioritní oblasti druhové ochrany středoevropských Koprofágů. The thesis consists of three conservation-oriented studies, mainly focusing on the factors determining dung beetle diversity in Central Europe. We assessed the role of routine ivermectin treatment on diversity and functional performance of dung beetle community. Alternative forest managements of coppicing and additional topsoil removal were evaluated in terms of their suitability for increasing diversity of dung-inhabiting dung and rove beetles. Further, we examined environmental and management-related factors driving dung beetle beta-diversity to facilitate conservation prioritization of Central European dung beetles.
Keywords:
ivermektin; beta-diverzita; ochrana přírody; chráněné druhy; koprofágní brouci; Scarabaeidae; Geotrupidae; pastevní management
Available in the Digital Repository of University of South Bohemia.
Dung beetles of Central Europe: Diversity and conservation
Tato disertační práce se skládá ze tří ochranářsky orientovaných studií, které se zabývají zejména faktory určujícími diverzitu koprofágních brouků ve střední Evropě. První studie hodnotila vliv ...
Isolation and characterisation of extracellular vesicles of parasitic helminths
MAZANEC, Hynek
2023 - English
Exkrečně sekreční produkty (ESP) slouží jako důležití mediátoři mezibuněčných a mezidruhových komunikací. Původně se předpokládalo, že jsou vylučovány hlavně v rozpustné formě, ale nedávné objevy naznačují i jejich vylučování za pomoci extracelulárních vezikul (EV). Tyto membránové částice poskytují svému obsahu ochranu před degradací a zároveň i cílený transport skrz specifických receptorů na svém povrchu. Díky tomu jsou studovány jako silné imunomodulátory vztahů mezi hostitelem a patogenem. S ohledem na parazitické helminty, EV jsou studovány jako prostředky pro diagnostiku, vakcinaci či terapii. Nicméně jejich obecná biologie, zejména pak jejich biogeneze, je stále špatně prostudována v porovnání s jejich potenciální funkců. Cílem této práce je tedy podrobněji prozkoumat sekreční aktivitu EV u různých zástupců tasemnic a jejich životních stádií. Životní cyklus tasemnice Schistocephalus solidus byl založen v laboratorních podmínkách za účelem studia sekrece EV napříč jednotlivými stádii. Zároveň jsme prozkoumali hlavní cesty biogeneze EV u tasemnice Hymenolepis diminuta za pomoci ultrastrukturních a proteomických analýz. Oba směry zároveň umožnily porovnání tvorby EV u tasemnic s různými typy hostitelů. Excretory-secretory products (ESP) serve as important mediators of intercellular and inter-species communication. They were originally thought to be secreted mainly in a soluble form, but recent discoveries indicate their delivery through extracellular vesicles (EVs). These membraneous particles provide protection from degradation for some of their components, as well as targeted delivery through various receptors on their surface. As such they have been studied as potent immunomodulators in host-pathogen relationships. With respect to parasitic helminths, EVs are being studied as agents for diagnosis, vaccination or therapeutics. However, their general biology, especially their biogenesis patterns, is still poorly understood compared to their functional role.Therefore, the goal of this thesis is to investigate in more detail the secretion activity of EVs in different tapeworms and their life-cycle stages. Under laboratory conditions, the life cycle of Schistocephalus solidus, a tapeworm with an aquatic life cycle, was established to explore EVs secretion at different developmental stages. Moreover, we used proteomic analyses and ultrastructural observations to identify the main biogenesis pathways behind EVs secretion in a terrestrial tapeworm Hymenolepis diminuta. This also allowed for the comparison of EVs generation in tapeworms with different host types.
Keywords:
Extracelulární vezikuly; parazit; tasemnice; Schistocephalus solidus; Hymenolepis diminuta; elektronová mikroskopie; proteomika
Available in the Digital Repository of University of South Bohemia.
Isolation and characterisation of extracellular vesicles of parasitic helminths
Exkrečně sekreční produkty (ESP) slouží jako důležití mediátoři mezibuněčných a mezidruhových komunikací. Původně se předpokládalo, že jsou vylučovány hlavně v rozpustné formě, ale nedávné objevy ...
Microbial and Viral Genomic Diversity in Freshwater Environments
SILVA KAVAGUTTI, Vinicius
2022 - English
Most abundant microbes from natural environments remain uncultured as traditional culture approaches fail to sufficiently simulate native conditions. Moreover, the efforts to obtain axenic cultures are also hampered due to inter-microbial dependencies. The absence of cultured representatives has several downstream effects, e.g., little chance of experimentation and creating genetic models or hypothesis testing, phage isolation, or even producing complete, closed reference genomes. Lack of all these maintains severe gaps in our abilities to understand microbes in appropriate phylogenetic and ecological contexts and also their habitat at large. This thesis tries to circumvent these issues through a meticulous investigation of freshwater microbes and their viruses using a metagenomic approach in the Římov reservoir. Most abundant microbes from natural environments remain uncultured as traditional culture approaches fail to sufficiently simulate native conditions. Moreover, the efforts to obtain axenic cultures are also hampered due to inter-microbial dependencies. The absence of cultured representatives has several downstream effects, e.g., little chance of experimentation and creating genetic models or hypothesis testing, phage isolation, or even producing complete, closed reference genomes. Lack of all these maintains severe gaps in our abilities to understand microbes in appropriate phylogenetic and ecological contexts and also their habitat at large. This thesis tries to circumvent these issues through a meticulous investigation of freshwater microbes and their viruses using a metagenomic approach in the Římov reservoir.
Available in the Digital Repository of University of South Bohemia.
Microbial and Viral Genomic Diversity in Freshwater Environments
Most abundant microbes from natural environments remain uncultured as traditional culture approaches fail to sufficiently simulate native conditions. Moreover, the efforts to obtain axenic cultures ...
W chromosomes in Lepidoptera: evolution, diversity and molecular features
HEJNÍČKOVÁ, Martina
2022 - English
Sex chromosome evolution is a fascinating and very dynamic process, which is best to be studied on diverse groups of organisms. Moths and butterflies (Lepidoptera) are known for their species diversity and female heterogamety, which makes them an ideal research model. Most species of Lepidoptera have a WZ/ZZ (female/male) system, although some species lack the W chromosome. The first part of this thesis discusses possible mechanisms and points of its origin in terms of phylogeny. Specifically, it focuses on the lack of W chromosome in the group of bagworms (Psychidae), supporting recent theory about the independent origin of W chromosomes in Lepidoptera. The second part of the thesis provides valuable information about the W chromosome variability and molecular content within the group of loopers (Geometridae). Finally, the third part describes the accumulation of retrotransposons on the W chromosome in Peribatodes rhomboidaria and emphasizes their importance in the process of sex chromosome differentiation.
Keywords:
W chromosome; sex chromosomes; Lepidoptera; moths and butterflies; sex chromosome evolution; sex determination; retrotransposons; Geometridae; Psychidae; sex chromatin; repetitive DNA
Available in the Digital Repository of University of South Bohemia.
W chromosomes in Lepidoptera: evolution, diversity and molecular features
Sex chromosome evolution is a fascinating and very dynamic process, which is best to be studied on diverse groups of organisms. Moths and butterflies (Lepidoptera) are known for their species ...
Conservation genetics of Galápagos mockingbirds: from immune genes to genomes
VLČEK, Jakub
2022 - English
In this thesis I have dealt with the population genetic processes of mockingbirds in the Galápagos Islands ( Mimus ) in relation to the limited island area, from the perspective of two types of immune genes and the genome-wide approach. The thesis starts with an introduction to population genetic concepts relevant to conservation genetics followed by description of immune genes: the major histocompatibility complex (MHC) and the Toll-like receptors (TLR). In the final part of the introduction, I address how genetic drift, selection and inbreeding shape genome-wide genetic patterns in small populations. The introduction is followed by four chapters, beginning with an examination of MHCII polymorphism in populations of mockingbirds in the Galápagos Islands. The study of the relationship between MHCII and the abundance of ectoparasites is the content of the second chapter. The third chapter shows how polymorphism of TLRs is shaped by interaction of genetic drift and natural selection. The last chapter deals with the effects of limited island size on inbreeding and genetic load, supported by inferences of the past demography of mockingbirds. The thesis concludes with a summary of the results and their potential implications in the field of conservation genetics. In this thesis I have dealt with the population genetic processes of mockingbirds in the Galápagos Islands ( Mimus ) in relation to the limited island area, from the perspective of two types of immune genes and the genome-wide approach. The thesis starts with an introduction to population genetic concepts relevant to conservation genetics followed by description of immune genes: the major histocompatibility complex (MHC) and the Toll-like receptors (TLR). In the final part of the introduction, I address how genetic drift, selection and inbreeding shape genome-wide genetic patterns in small populations. The introduction is followed by four chapters, beginning with an examination of MHCII polymorphism in populations of mockingbirds in the Galápagos Islands. The study of the relationship between MHCII and the abundance of ectoparasites is the content of the second chapter. The third chapter shows how polymorphism of TLRs is shaped by interaction of genetic drift and natural selection. The last chapter deals with the effects of limited island size on inbreeding and genetic load, supported by inferences of the past demography of mockingbirds. The thesis concludes with a summary of the results and their potential implications in the field of conservation genetics.
Keywords:
population genetics; birds; inbreeding; genetic drift; immunogenetics; conservation; MHC; TLR; major histocompatibility complex; Toll-like receptors; genome; genetic load; extinction; effective population size; island size
Available in the Digital Repository of University of South Bohemia.
Conservation genetics of Galápagos mockingbirds: from immune genes to genomes
In this thesis I have dealt with the population genetic processes of mockingbirds in the Galápagos Islands ( Mimus ) in relation to the limited island area, from the perspective of two types of ...
Drivers of karyotype evolution in Lepidoptera
PROVAZNÍKOVÁ, Irena
2022 - English
Research of lepidopteran karyotypes and their evolution has been challenging for decades due to their many peculiar characteristics. However, this field has advanced thanks to modern cytogenetic techniques and sequencing technologies. We combined explored possibilities how to detect chromosomal rearrangements, and cytogenetic and genomic approaches to explore evolutionary forces shaping karyotypes of non-model Lepidoptera including representatives of early diverging species. Results obtained in the present thesis point to a possible role of satellite DNA and sexual antagonistic selection in mobilisation of rDNA and sex chromosome turnover, respectively.
Keywords:
Lepidoptera; karyotype evolution; sex chromosomes; repetitive sequences; chromosomal rearrangements; gene families; FISH
Available in the Digital Repository of University of South Bohemia.
Drivers of karyotype evolution in Lepidoptera
Research of lepidopteran karyotypes and their evolution has been challenging for decades due to their many peculiar characteristics. However, this field has advanced thanks to modern cytogenetic ...
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