Commit e74fe8cf authored by mk11g11's avatar mk11g11
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corrections

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# General discussion {#discussion} # General discussion {#discussion}
## Environmental levels of neonicotinoids do not impact on the behavior or development of C. elegans ## Environmental levels of neonicotinoids do not impact on the behavior or development of *C. elegans*
Increased use of insecticides requires a better understanding of their environmental impact. Therefore, the initial aim of this project was to investigate the effects of neonicotinoid-insecticides on the Nematoda representative *C. elegans*. Neonicotinoids have been introduced to the marked in the 1990s and since have become the most commonly used insecticides worldwide [@jeschke2011]. They have many advantages, including a high potency against wide range pest insects (Section \@ref(potentpests)) and low mammalian toxicity. However, neonicotinoids can have significant field effects on non-target species. The adverse effects of environmental neonicotinoids on bees have been studied for decades (Section \@ref(sublethalbees)), whereas the effects on other ecologically important species is less understood but unlikely to have a problem. Increased use of insecticides requires a better understanding of their environmental impact. Therefore, the initial aim of this project was to investigate the effects of neonicotinoid-insecticides on the Nematoda representative *C. elegans*. Neonicotinoids have been introduced to the marked in the 1990s and since have become the most commonly used insecticides worldwide [@jeschke2011]. They have many advantages, including a high potency against wide range pest insects (Section \@ref(potentpests)) and low mammalian toxicity. However, neonicotinoids can have significant field effects on non-target species. The adverse effects of environmental neonicotinoids on bees have been studied for decades (Section \@ref(sublethalbees)), whereas the effects on other ecologically important species is less understood but unlikely to have a problem.
...@@ -49,7 +49,7 @@ Taken together, a small number of *C. elegans* nAChR subunits have amino acids c ...@@ -49,7 +49,7 @@ Taken together, a small number of *C. elegans* nAChR subunits have amino acids c
The differences between the insect and *C.elegans* binding pocket are mainly found in the complementary site, suggesting the contributions from the complementary site determine neonicotinoids-selectivity. This is supported by the literature [@marotta2014; @hansen2004]. For example, swapping of $\beta$ subunits in $\alpha4\beta2$ mammalian receptor diminished cytisine activity on this receptor [@harpsoe2013]. Receptor becomes responsive to cytisine at sub-$\mu$M concentrations by a mutation in a single amino acid in the complementary binding pocket [@marotta2014]. Thus the variation in the complementary binding pocket residues gives rise to ligand binding specifities and pharmacological differences between various compounds and receptors. The differences between the insect and *C.elegans* binding pocket are mainly found in the complementary site, suggesting the contributions from the complementary site determine neonicotinoids-selectivity. This is supported by the literature [@marotta2014; @hansen2004]. For example, swapping of $\beta$ subunits in $\alpha4\beta2$ mammalian receptor diminished cytisine activity on this receptor [@harpsoe2013]. Receptor becomes responsive to cytisine at sub-$\mu$M concentrations by a mutation in a single amino acid in the complementary binding pocket [@marotta2014]. Thus the variation in the complementary binding pocket residues gives rise to ligand binding specifities and pharmacological differences between various compounds and receptors.
(ref:gendiscussion-celegansinsectalagnment) **Sequence alignment of the pharmacophore of insect and *C. elegans* nAChR subunits.** Ligand binding pocket is formed from the loops originating from the principal (a) and complementary (b) receptor subunits. Amino acids important in forming drug-receptor interactions are color-coded as in Figure \@ref(fig:binding-pocket-label). Non-conserved residues are underlined. Numbering is according to the AChBP of Ls sequence. Mp = *M. persicae* (peach aphid), Dm = *D. melanogaster* (fruit fly), Ce = *C. elegans*. Sequence alignment generated by MUSCLE. (ref:gendiscussion-celegansinsectalagnment) **Sequence alignment of the pharmacophore of insect and *C. elegans* nAChR subunits.** Ligand binding pocket is formed from the loops originating from the principal (a) and complementary (b) receptor subunits. Amino acids important in forming drug-receptor interactions are color-coded as in Figure \@ref(fig:binding-pocket-label). Non-conserved residues are underlined. Numbering is according to the AChBP of Ls sequence. Mp = *M. persicae* (peach aphid), Dm = *D. melanogaster* (fruit fly), Ce = *C. elegans*. Sequence alignment generated by MUSCLE.
```{r gendiscussion-celegansinsectalagnment-label, fig.cap="(ref:gendiscussion-celegansinsectalagnment)", fig.scap= "Sequence alignment of the pharmacophore of insect and *C. elegans* nAChR subunits", fig.align='center', out.width= '80%', echo=FALSE} ```{r gendiscussion-celegansinsectalagnment-label, fig.cap="(ref:gendiscussion-celegansinsectalagnment)", fig.scap= "Sequence alignment of the pharmacophore of insect and \\textit{C. elegans} nAChR subunits", fig.align='center', out.width= '80%', echo=FALSE}
knitr::include_graphics("fig/gen_discussion/celegans_and_insect_added_loopG_and_B.png") knitr::include_graphics("fig/gen_discussion/celegans_and_insect_added_loopG_and_B.png")
``` ```
...@@ -58,7 +58,7 @@ The differences between the insect and *C.elegans* binding pocket are mainly fou ...@@ -58,7 +58,7 @@ The differences between the insect and *C.elegans* binding pocket are mainly fou
<!-- Conversy, anthelmintic levamisole has potent action on *C. elegans*, but is a weak agonist on insect receptors. Levamisole at conc induce depolarisation of ...*C. elegans* receptors (ref). In contrast, they weakly depolarise cocroach motor neuro [@pinnock1988]. These data suggest that there is a striking difference between the neonicotinoid affinity on insect neurons and on *C. elegans* pharynx. --> <!-- Conversy, anthelmintic levamisole has potent action on *C. elegans*, but is a weak agonist on insect receptors. Levamisole at conc induce depolarisation of ...*C. elegans* receptors (ref). In contrast, they weakly depolarise cocroach motor neuro [@pinnock1988]. These data suggest that there is a striking difference between the neonicotinoid affinity on insect neurons and on *C. elegans* pharynx. -->
<!-- Other pharma differences : read this papar: tornoe1994. --> <!-- Other pharma differences : read this papar: tornoe1994. -->
## *C. elegans* pharynx a platform for pharmacological characterisation of nAChRs ## *C. elegans* pharynx as a platform for the pharmacological characterisation of nAChRs
Low sensitivity of *C. elegans* to nitenpyram, clothianidin and thiacloprid in behavioural and cellular assays precludes its use as a model to study the mode of action of neonicotinoids per se, but highlights its potential use as a suitable background for the heterologous expression of insect nAChRs. New insecticides are needed to prevent the negative environmental impact of neonicotinoids and combat emerging resistance. The first step towards the synthesis of neonicotinoids with improved selective toxicity profile is the heterologous expression of nAchRs from pests and non-target species in a suitable host. Low sensitivity of *C. elegans* to nitenpyram, clothianidin and thiacloprid in behavioural and cellular assays precludes its use as a model to study the mode of action of neonicotinoids per se, but highlights its potential use as a suitable background for the heterologous expression of insect nAChRs. New insecticides are needed to prevent the negative environmental impact of neonicotinoids and combat emerging resistance. The first step towards the synthesis of neonicotinoids with improved selective toxicity profile is the heterologous expression of nAchRs from pests and non-target species in a suitable host.
...@@ -68,7 +68,7 @@ Acetylcholine is a major neurotransmitter of the pharynx, released by at least 7 ...@@ -68,7 +68,7 @@ Acetylcholine is a major neurotransmitter of the pharynx, released by at least 7
EAT-2 is a single nAChR subunit that confers pharyngeal 5-HT sensitivity and feeding response in *C. elegans* [@mckay2004]. We show that *eat-2* mutant is a suitable genetic background, in which the expression of heterologous nAChRs could be scored in behavioural assays. Transgenic line in which *eat-2* is heterologously expressed, rescued the blunted pharyngeal response to food and restored 5-HT resistance of the *eat-2* mutant (Section \@ref(behaviourofeat2rescue)). However, the expression of human $\alpha7$ nAChR in the *C. elegans* mutant pharynx had no observable phenotypical consequences (Section \@ref(feedingalpha7celegans) and Section \@ref(htandtransgenicalpha7)). Similarly, expression of this receptor in the wild-type worm revealed no differences in pharyngeal responses to nAChR agonists nicotine or choline (Section \@ref(pharmaalpha7transegnicworms)). Staining with fluorescently labelled human $\alpha7$ nAChR antagonist $\alpha-Bgtx$, reveled increased fluorescence in the pharyngeal muscle of transgenic, when compared to control worms (Section \@ref(bgtxstaining)). $\alpha$-bgtx binds to the extracellular, domain of the receptor [@dellisanti2007]. This suggests that $\alpha7$ receptor is expressed on the cell surface of the pharyngeal muscle, however due to the lack of phenotype its functionality is unclear. Further pharmacological experiments of transgenic strains (as described in the Discussion of Chapter 6) should be carried out to determine whether $\alpha7$ retains its function upon expression in the pharynx of *C. elegans*. EAT-2 is a single nAChR subunit that confers pharyngeal 5-HT sensitivity and feeding response in *C. elegans* [@mckay2004]. We show that *eat-2* mutant is a suitable genetic background, in which the expression of heterologous nAChRs could be scored in behavioural assays. Transgenic line in which *eat-2* is heterologously expressed, rescued the blunted pharyngeal response to food and restored 5-HT resistance of the *eat-2* mutant (Section \@ref(behaviourofeat2rescue)). However, the expression of human $\alpha7$ nAChR in the *C. elegans* mutant pharynx had no observable phenotypical consequences (Section \@ref(feedingalpha7celegans) and Section \@ref(htandtransgenicalpha7)). Similarly, expression of this receptor in the wild-type worm revealed no differences in pharyngeal responses to nAChR agonists nicotine or choline (Section \@ref(pharmaalpha7transegnicworms)). Staining with fluorescently labelled human $\alpha7$ nAChR antagonist $\alpha-Bgtx$, reveled increased fluorescence in the pharyngeal muscle of transgenic, when compared to control worms (Section \@ref(bgtxstaining)). $\alpha$-bgtx binds to the extracellular, domain of the receptor [@dellisanti2007]. This suggests that $\alpha7$ receptor is expressed on the cell surface of the pharyngeal muscle, however due to the lack of phenotype its functionality is unclear. Further pharmacological experiments of transgenic strains (as described in the Discussion of Chapter 6) should be carried out to determine whether $\alpha7$ retains its function upon expression in the pharynx of *C. elegans*.
The expression of $\alpha7$ was driven a myo-2 promoter, which should drive the expression in all cells of the pharyngeal musculature [@@altun2009a]. However, $\alpha-Bgtx$ staining of transgenic worms was concentrated in pm7 and pm8 muscle cells, suggesting $\alpha7$ is expressed in the terminal bulb, which does not overlap with the localisation of native EAT-2, native protein. Thus, native EAT-2 promoter should be used to ensure correct localisation of the expressed protein. The expression of $\alpha7$ was driven a myo-2 promoter, which should drive the expression in all cells of the pharyngeal musculature [@altun2009a]. However, $\alpha-Bgtx$ staining of transgenic worms was concentrated in pm7 and pm8 muscle cells, suggesting $\alpha7$ is expressed in the terminal bulb, which does not overlap with the localisation of native EAT-2, native protein. Thus, native EAT-2 promoter should be used to ensure correct localisation of the expressed protein.
<!-- In dissected animal, in the absence of food pharynx continues to pump. It is myogenic or neurogenic? --> <!-- In dissected animal, in the absence of food pharynx continues to pump. It is myogenic or neurogenic? -->
...@@ -163,7 +163,7 @@ knitr::include_graphics("fig/gen_discussion/celeganseat2andacr7andhumanalpha7.pn ...@@ -163,7 +163,7 @@ knitr::include_graphics("fig/gen_discussion/celeganseat2andacr7andhumanalpha7.pn
Comparison of human $\alpha7$ and pharyngeal nAChR subunits EAT-2 and ACR-7 revealed high sequence similarity. Almost all residues forming ligand binding pocket are conserved between these subunits, suggesting pharyngeal nAChR subunits are homologous to human $\alpha7$. Besides pharyngeal nAChR subunits, homologs of over two thirds of human proteins can be found in *C. elegans* [@sonnhammer1997; @lai2000]. The similarities between mammals and *C. elegans* extends beyond genetics. *C. elegans* has conserved synaptic function, due to the presence of almost all vertebrate neurotransmitters and conserved neuronal signalling pathways [@bargmann1998; @kaletta2006]. There are however some differences. *C. elegans* presents expresses inhibitory glutamate-gated chloride channel absent in mammals [@cully1994], but lacks voltage-gated sodium channels which is present in humans [@bargmann1998]. Despite these limitations, *C. elegans* emerges as an attractive model for toxicity studies [@hunt2017]. Comparison of human $\alpha7$ and pharyngeal nAChR subunits EAT-2 and ACR-7 revealed high sequence similarity. Almost all residues forming ligand binding pocket are conserved between these subunits, suggesting pharyngeal nAChR subunits are homologous to human $\alpha7$. Besides pharyngeal nAChR subunits, homologs of over two thirds of human proteins can be found in *C. elegans* [@sonnhammer1997; @lai2000]. The similarities between mammals and *C. elegans* extends beyond genetics. *C. elegans* has conserved synaptic function, due to the presence of almost all vertebrate neurotransmitters and conserved neuronal signalling pathways [@bargmann1998; @kaletta2006]. There are however some differences. *C. elegans* presents expresses inhibitory glutamate-gated chloride channel absent in mammals [@cully1994], but lacks voltage-gated sodium channels which is present in humans [@bargmann1998]. Despite these limitations, *C. elegans* emerges as an attractive model for toxicity studies [@hunt2017].
Methods to use *C. elegans* as a model to study acute toxicity as well as developmental and reproductive toxicology have been developed [@boyd2010; @xiong2017]. A good correlation between the rank order of toxicity of many compounds on *C. elegans* and mammals for acute toxicity, growth and reproduction endpoints have been found [@williams1988; @boyd2010]. This includes organophosphates, which act by disrupting cholinergic neurotransmission at the synapse [@chadwick1947]. The rank order of acute toxicity, as measured by LC50, correlated well with the LD50 ranking of these agents in rats and mice [@cole2004]. This highlight the potential suitability of *C. elegans* as a model for toxicity testing of cholinergic and other neurotoxins. Introduction of *C. elegans* into toxicity testing has a potential to reduce the use of conventional mammalian models, resulting in the reduction of cost and duration of such studies [@williams1988]. Methods to use *C. elegans* as a model to study acute toxicity as well as developmental and reproductive toxicology have been developed [@boyd2010; @xiong2017]. A good correlation between the rank order of toxicity of many compounds on *C. elegans* and mammals for acute toxicity, growth and reproduction endpoints have been found [@williams1988b; @boyd2010]. This includes organophosphates, which act by disrupting cholinergic neurotransmission at the synapse [@chadwick1947]. The rank order of acute toxicity, as measured by LC50, correlated well with the LD50 ranking of these agents in rats and mice [@cole2004]. This highlight the potential suitability of *C. elegans* as a model for toxicity testing of cholinergic and other neurotoxins. Introduction of *C. elegans* into toxicity testing has a potential to reduce the use of conventional mammalian models, resulting in the reduction of cost and duration of such studies [@williams1988].
<!-- ## Cholinergic drive of the *C. elegans* pharynx --> <!-- ## Cholinergic drive of the *C. elegans* pharynx -->
......
...@@ -6,7 +6,7 @@ ...@@ -6,7 +6,7 @@
knitr::include_graphics("fig/appendix/seq_align_1a.png") knitr::include_graphics("fig/appendix/seq_align_1a.png")
``` ```
(ref:pharacophore-seq) Sequence alignment of the ligand binding pocket of the AChBPs and nAChRs. Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numering corresponds to the sequence of Ls AChBP. Alignment was generated with MUSCLE [@edgar2004]. Abbreviations used: Ls - *Lymnaela stagnalis* (great pond snail), Am- *Apis mellifera* (honeybee), Mz- *Myzus persicae* (peach aphid), Hs- *Homo sapiens* (human), Gg- *Gallus gallus* (chicken), Ce - *C. elegans*. (ref:pharacophore-seq) Sequence alignment of the binding pocket of the ligand binding protein and nicotinic acetylcholine receptors. Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for agonist binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numbering corresponds to the sequence of the great pond snail acetylcholine binding protein (AChBP). Alignment was generated with MUSCLE [@edgar2004]. Abbreviations used: Ls - *Lymnaela stagnalis* (great pond snail), Am- *Apis mellifera* (honeybee), Mz- *Myzus persicae* (peach aphid), Hs- *Homo sapiens* (human), Gg- *Gallus gallus* (chicken), Ce - *C. elegans*.
```{r pharacophore-seq-label, fig.cap="(ref:pharacophore-seq)", fig.scap="Sequence alignment of the ligand binding pocket of the AchBPs and nAChRs.", fig.align='center', echo=FALSE, fig.pos='H'} ```{r pharacophore-seq-label, fig.cap="(ref:pharacophore-seq)", fig.scap="Sequence alignment of the ligand binding pocket of the AchBPs and nAChRs.", fig.align='center', echo=FALSE, fig.pos='H'}
knitr::include_graphics("fig/appendix/seq_align_1b.png") knitr::include_graphics("fig/appendix/seq_align_1b.png")
......
# DNA sequence used for the expression of *eat-2* nAChR subunit in the pharyngeal muscle of *C. elegans*. # DNA sequence used for the expression of *eat-2* in the pharyngeal muscle of *C. elegans*.
\newpage \newpage
(ref:app2) Sequencing of myo-2-eat-2 from the pDEST vector. Myo-2::eat-2 nucleotide fragment from the expression vector used to generate *C. elegans* transgenic strains was sequenced following cloning. 3 forward (Fw) and a reverse (Rev) primer were used to generate overlaping sequencing fragments spaning the entire sequence of interets (a). Sequencing results authenticated the identity of the construct (b) and confirmed the amino acid sequence of the eat-2 gene. (ref:app2) Sequencing of *myo-2-eat-2* from the *pDEST* vector. Myo-2::eat-2 nucleotide fragment from the expression vector used to generate *C. elegans* transgenic strains was sequenced following cloning. 3 forward (Fw) and a reverse (Rev) primer were used to generate overlaping sequencing fragments spaning the entire sequence of interets (a). Sequencing results authenticated the identity of the construct (b) and confirmed the amino acid sequence of the *eat-2* gene.
```{r echo=FALSE, out.height = '80%'} ```{r echo=FALSE, out.height = '80%'}
knitr::include_graphics("fig/results4/PNG/1-myo2-eat-2.png") knitr::include_graphics("fig/results4/PNG/1-myo2-eat-2.png")
...@@ -20,8 +20,8 @@ knitr::include_graphics("fig/results4/PNG/3-myo2-eat2.png") ...@@ -20,8 +20,8 @@ knitr::include_graphics("fig/results4/PNG/3-myo2-eat2.png")
knitr::include_graphics("fig/results4/PNG/4-myo2-eat2.png") knitr::include_graphics("fig/results4/PNG/4-myo2-eat2.png")
``` ```
```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of myo-2-eat-2 from the pDEST vector", fig.align='center', include="TRUE", results="show"} \newpage
```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of \\textit{myo-2-eat-2} from the \\textit{pDEST} vector", include="TRUE", results="show", echo=FALSE}
knitr::include_graphics("fig/results4/PNG/5-myo2-eat2.png") knitr::include_graphics("fig/results4/PNG/5-myo2-eat2.png")
``` ```
# DNA sequence used for the expression of human $\alpha$ 7 nAChR subunit in the pharyngeal muscle of *C. elegans*. # DNA sequence used for the expression of human $\alpha7$ in the pharyngeal muscle of *C. elegans*.
\newpage \newpage
(ref:app1) Sequencing of pmyo2-CHRNA7 from the pDEST expression vector. (ref:app1) Sequencing of *pmyo2-CHRNA7* from the *pDEST* expression vector.
```{r out.height = '80%', echo=FALSE} ```{r out.height = '80%', echo=FALSE}
knitr::include_graphics("fig/results4/PNG/1-myo2-chrna7.png") knitr::include_graphics("fig/results4/PNG/1-myo2-chrna7.png")
...@@ -20,6 +20,6 @@ knitr::include_graphics("fig/results4/PNG/3-myo2-chrna7.png") ...@@ -20,6 +20,6 @@ knitr::include_graphics("fig/results4/PNG/3-myo2-chrna7.png")
knitr::include_graphics("fig/results4/PNG/4-myo2-chrna7.png") knitr::include_graphics("fig/results4/PNG/4-myo2-chrna7.png")
``` ```
```{r app1-lbl, fig.cap="(ref:app1)",, fig.scap = "Sequencing of myo-2-$\\alpha$-7 from the pDEST vector", fig.align='center', out.height = '80%', echo=FALSE} ```{r app1-lbl, fig.cap="(ref:app1)",, fig.scap = "Sequencing of \\textit{myo-2-$\\alpha$-7} from the pDEST vector", fig.align='center', out.height = '80%', echo=FALSE}
knitr::include_graphics("fig/results4/PNG/5-myo2-chrna7.png") knitr::include_graphics("fig/results4/PNG/5-myo2-chrna7.png")
``` ```
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# Sequence of the DNA sequence used for the expression of human $\alpha$ 7 ECD in E. coli # DNA sequence used for the expression of human $\alpha7$ extracellular domain in *E. coli*
(ref:appe) **Sequencing of pelB-3C cloned into pET27 expression vector.** Inserted into pET27 pelB-3C sequence was sequenced using universal T7 forward and T7 terminator primers (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). Single nucleotide mutation from A to C occured, highlighted in red, changing the codon from GCC to GCA, both of which encode for alanine. The cloned nucleotide sequence was translated (c) and major functional domains, as well highlighted. @nauen1996
\newpage @neher1995
```{r out.height = '80%', echo=FALSE} @nguyen1995
knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png")
```
```{r echo=FALSE} @niacaris2003
knitr::include_graphics("fig/results5/png/pelb-3c_seq_2.png")
```
```{r pelb-3c-lbl, fig.cap = "(ref:appe)", fig.scap= "Sequencing of pelB-3C cloned into pET27 expression vector", fig.align='center', echo=FALSE} @noda1982
knitr::include_graphics("fig/results5/png/pelb-3c_seq_3.png")
``` @noda1983
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@okkema1993
@orr1990
@partridge2008
@pereira2015
@perry2008
@petzold2011
@planson2003
@putrenko2005
@raizen1994
@raizen1995
@rand1984
@rand1985
@rand1989
@reynolds1978
@richmond1999
@rogers2006
@rosano2014
@ruan2009
@ruaud2006
@salom2012
@sattelle1981
@sattelle1983
@sattelle2005
<!-- (ref:appe) **Sequencing of *pelB-3C* cloned into *pET27* expression vector.** Inserted into *pET27 pelB-3C* sequence was sequenced using universal T7 forward and T7 terminator primers (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). Single nucleotide mutation from A to C occured, highlighted in red, changing the codon from GCC to GCA, both of which encode for alanine. The cloned nucleotide sequence was translated (c) and major functional domains, as well highlighted. -->
<!-- \newpage -->
<!-- ```{r out.height = '90%', fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png") -->
<!-- ``` -->
<!-- ```{r fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_2.png") -->
<!-- ``` -->
<!-- ```{r pelb-3c-lbl, fig.cap = "(ref:appe)", fig.scap= "Sequencing of \\textit{pelB-3C} cloned into \\textit{pET27} expression vector", fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_3.png") -->
<!-- ``` -->
\ No newline at end of file
---
output:
pdf_document: default
html_document: default
---
# Sequencing of the DNA sequence used for the expression of of the $\alpha7$ ECD-2GSC # Sequencing of the DNA sequence used for the expression of of the $\alpha7$ ECD-2GSC
\newpage \newpage
......
book_filename: "thesis-dissertaion" # Change this to the actual title book_filename: "thesis-dissertaion" # Change this to the actual title
delete_merged_file: true delete_merged_file: true
rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro_2.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.Rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd", "21-appendix-a.Rmd", "22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"] rmd_files: ["index.Rmd", "00-preface.Rmd", "26-appendix-f.Rmd",]
#rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd","21-appendix-a.Rmd","22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"] #rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro_2.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.Rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd", "21-appendix-a.Rmd", "22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"]
language: language:
ui: ui:
chapter_name: "Chapter " chapter_name: "Chapter "
\ No newline at end of file
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