LU|ZONE|UL Community:https://zone.biblio.laurentian.ca/handle/10219/21622024-03-28T12:54:35Z2024-03-28T12:54:35ZSecretion and interaction of the heat-stable enterotoxin b (STb) with the gut hormone secreting STC-1 CellsKinkar, Eyadhttps://zone.biblio.laurentian.ca/handle/10219/39392024-03-27T07:35:59Z2021-08-31T00:00:00ZTitle: Secretion and interaction of the heat-stable enterotoxin b (STb) with the gut hormone secreting STC-1 Cells
Authors: Kinkar, Eyad
Abstract: Enterotoxigenic Escherichia coli (ETEC) employs a number of secretion systems for efficient
translocation of a number of virulence factors during infection. Studies of the relationship
between substrates and specific secretion pathways suggested that secretion substrates are
identified and are targeted to their proper secretion pathway via inherent amino acid sequences,
or secretion signals, within each substrate. ETEC utilizes the Sec-dependent pathway of the type
two secretion system (T2SS) to secrete the Heat-Stable Enterotoxin B (STb), in addition to
several other toxins, to the extracellular milieu. In this work, the nature of the nucleotide
sequence of STb’s mRNA, rather than the amino acid sequence of its signal sequence, was
investigated for its effect on STb secretion. Additionally, the interaction of STb with the gut
hormone secreting cells (STC-1 cells) was investigated to elucidate the nature and consequences
of such interaction. In the first study, the N-terminal signal sequence of premature STb (at the
mRNA level) was subjected to various mutations (a number of accumulative silent mutations and
a number of non-silent point mutations) to test whether or not its mRNA secondary structure
affects its targeting for secretion. In the second study, the two main key secretion factors of Secdependant pathway, ffh and secb, were separately knocked out and overexpressed to uncover
their potential involvement in pro-STb targeting and secretion processes. In the final study, an in
vitro approach was devised to identify and characterize the STb cell surface receptor on STC-1
cells (mouse intestinal enteroendocrine cell line). The results indicated that the nucleotide
sequence of the STb mRNA (within the N-terminal amino acid signal sequence of the toxin) can
affect toxin secretion. Reducing the AU richness within the 5′ end of the STb mRNA (without
altering the translated amino acid sequence of this region) significantly reduced toxin secretion.
From the second study, deletion of FFh, the protein component of the signal recognition particle in E. coli, resulted in significant effects on targeting and translocation of pro-STb and STb to the
cytoplasmic membrane and the extracellular milieu more so than SecB deletion. Overexpression
of FFh, not SecB, was shown to enhance STb synthesis and secretion. Finally, the results of the
third study confirm a direct disruption to the tight junction of STC-1 cells. The results strongly
suggest that STb interaction with these cells is mediated, at least in part, by the tight junction
associated protein occludin. This interaction ultimately led to Caspase 3-mediated apoptosis.2021-08-31T00:00:00ZUnraveling the metabolic networks involved in the utilization of L-glutamine in Pseudomonas fluorescens exposed to nutritional stressLegendre, Félixhttps://zone.biblio.laurentian.ca/handle/10219/39242024-03-28T07:05:13Z2022-06-28T00:00:00ZTitle: Unraveling the metabolic networks involved in the utilization of L-glutamine in Pseudomonas fluorescens exposed to nutritional stress
Authors: Legendre, Félix
Abstract: Sulfur plays an essential role in oxidative homeostasis due to its participation in sulfhydryl
groups (SH). A disruption of this vital nutrient is known to promote oxidative stress and activates
a plethora of anti-oxidative strategies. Phosphate, a micronutrient that is part of adenosine
triphosphate (ATP), the main molecule used as energy and other macromolecules in living cells.
The stress response to sulfur and phosphate deficiency in Pseudomonas fluorescens was
investigated with emphasis on ROS detoxification, and energy production. Metabolite profiling
was performed by High Performance Liquid Chromatography (HPLC), enzymatic analysis was
done using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and gene expression
assessment of targeted genes was performed with SYBR-Green real-time PCR (qPCR). When
cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the
microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant
and the limited production of NADH, a pro-oxidant. The up-regulation of isocitrate
dehydrogenase (ICDH)-NADP+ dependent in the soluble fraction of the cells obtained from the
S-deficient media results in enhanced NADPH synthesis. This reaction is aided by the
concomitant increase in NAD kinase (NADK) activity. The latter converts NAD+
into NADP+
in
the presence of ATP. Additionally, the microbe reprograms its metabolic pathways to produce
KG and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate
semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KDC) work in partnership to
synthesize KG. This process is further aided by the increased activity of the enzymes glutamate
decarboxylase (GDC) and γ-amino-butyrate transaminases (GABA-T). Taken together, the data
point to a metabolic network involving isocitrate, KG, and ICDH that converts NADH into
NADPH in P. fluorescens subjected to a S-deprived environment. Finally, when cultured in low
phosphate environments, the microbe can produce ATP via substrate level phosphorylation
(SLP), in a mechanism involving the reductive isocitrate dehydrogenase (ICDH-NADH),
isocitrate lyase (ICL), malate synthase (MS) as well as phosphoenol pyruvate carboxylase
(PEPC), phosphoenol pyruvate synthase (PEPS) and pyruvate phosphate dikinase (PPDK). This
metabolic reprogramming ensures the survival of the microbe and reveals the central role
metabolism plays in cellular adaptation to abiotic stress.2022-06-28T00:00:00ZTranscriptome and methylome analysis of trembling aspen (Populus tremuloides) under nickel stress.Czajka, Karolina M.https://zone.biblio.laurentian.ca/handle/10219/38812024-03-27T07:36:48Z2022-04-28T00:00:00ZTitle: Transcriptome and methylome analysis of trembling aspen (Populus tremuloides) under nickel stress.
Authors: Czajka, Karolina M.
Abstract: Nickel is an essential micronutrient required at low concentrations for adequate plant growth and
health. However, excessive amounts of bioavailable nickel ions in the surrounding soil can result
in plant toxicity symptoms. Plants have evolved heavy metal tolerance mechanisms to adapt and
cope with this abiotic stressor. The main objectives of the present research were to 1) further
characterize the P. tremuloides transcriptome 2) compare gene expression dynamics between
nickel-resistant and nickel-susceptible P. tremuloides genotypes with Whole Transcriptome (WT)
sequencing, 3) determine the effects of different nickel concentrations on P. tremuloides gene
expression and, 4) assess global methylation levels in P. tremuloides under nickel stress.
Trembling aspen (Populus tremuloides) seedlings treated with varying concentrations of nickel
nitrates (150 mg Ni / 1 kg of dry soil, 800 mg / kg, and 1, 600 mg / kg) showed phenotypic
segregation of physical toxicity symptoms at the highest nickel dose of 1, 600 mg / kg. This study
revealed that a metal transport protein (Potrs038704g29436 – ATOX1-related copper transport)
was among the top upregulated genes in resistant genotypes when compared to susceptible plants.
Other upregulated genes associated with abiotic stress were identified including a Dirigent Protein
10, GATA transcription factor, Zinc finger protein, Auxin response factor, Bidirectional sugar
transporter, and thiamine thiazole synthase. Overall, an upregulation in ribosomal and translation
activities was identified as the main response to Ni toxicity in the resistant plants. The results of
the dosage analysis suggested that the 800 mg / kg nickel dose is the threshold at which an early
abiotic stress response may be triggered as seen by the highly upregulated LEA protein and two
calcium binding proteins when compared to water. The cluster of genes that had increased gene
expression with increasing nickel dose also had multiple enriched GO terms related to heavy metal and abiotic stress including metal ion transport, antioxidant activity, photosynthesis, and ribosomal
activity.
Lastly, the initial screen for potential global methylation differences between nickel-resistant
genotypes and water showed no significant difference in overall methylation levels. However, the
potassium nitrate control for the 1,600 mg / kg dose did show significantl hypomethylation in
comparison to the nickel-treated or water control samples. Future experiments could use targetspecific methylation and gene expression assays to investigate the biological significance of the
heavy metal stress candidate genes identified in this top-down study in trembling aspen.
Understanding the heavy metal tolerance mechanisms and responses used by hardy species like
trembling aspen is important for environment bioremediation and maintenance of healthy
ecosystems.2022-04-28T00:00:00ZCellular effects of Ferula Assafoetida on breast cancer cells and inflammatory responses in cultured monocytesAlharbi, Albatulhttps://zone.biblio.laurentian.ca/handle/10219/38262024-03-27T07:29:58Z2021-12-16T00:00:00ZTitle: Cellular effects of Ferula Assafoetida on breast cancer cells and inflammatory responses in cultured monocytes
Authors: Alharbi, Albatul
Abstract: In traditional medicine, Ferula assafoetida (F. assafoetida), has been used as an antiseptic,
anti-diabetic, anti-inflammatory, and anti-cancer agent. In recent years its anti-cancer and antiinflammatory activities have become a focus in drug research. We investigated the in vitro
cytotoxicity and anti-inflammatory effects of ethanolic extracts of F. assafoetida and five known
components (ferulic acid, vanillic acid, quercetin, ellagic acid, and p-coumaric acid) on a group of
malignant and non-malignant breast cell lines and the THP-1 monocyte-like cell line. Our results
showed that treatment with the ethanolic extract of F. assafoetida, and the components, had a
significant effect on cell viability and apoptosis induction for the human MCF-7, MDA-MB-231,
and murine 4T1 breast cancer cell lines compared to the non-malignant human HBL-100 breast
cells. This research also showed that THP-1 peripheral blood monocytic leukemia cells,
differentiated into macrophages, could be further polarized into the M1 inflammatory phenotype
by treatment with extracts of F. assafoetida and the components. There was a significant increase
in the expression of CD80, a marker associated with the M1 macrophage subtype, but no increase
in expression of the M2 subtype marker, CD163, in treated cells. Further, this polarization of the
THP-1-dependent macrophages showed an increased ability to damage MCF-7 or MDA-MB-231
cell monolayers in co-culture experiments. Therefore, treatment with F. assafoetida extracts can
also indirectly cause the death of cancer cells via activation of immune cells. These results confirm
that F. assafoetida is a potential source of anti-cancer and immune modulatory compounds and
that further investigation is needed to reveal the mechanisms of F. assafoetida’s effects on
apoptosis and immunomodulation.2021-12-16T00:00:00Z